Pubblicazione 7 PHD Thesis Marco Friuli Unisalento Xxxiii Ciclo Ing. Materiali

UNIVERSITY OF SALENTO
DEPARTMENT OF ENGINEERING FOR INNOVATION

PhD program
in
MATERIALS AND STRUCTURE ENGINEERING AND NANOTECHNOLOGY
Development of macromolecular hydrogels based on natural polymers as oviposition substrates and

matrices for the bioinsecticide Beauveria bassiana, aimed at the realization of an innovative device for
the control of the tiger mosquito (Aedes albopictus)
SUPERVISORS:
PROF. ALESSANDRO SANNINO
PROF. CHRISTIAN DEMITRI
PROF. CLAUDIA CAFARCHIA
PhD Candidate
Eng. MARCO FRIULI
Cycle XXXIII-2017/2020
Conta ciò che si può contare, misura ciò che
è misurabile e rendi misurabile ciò che non
lo è.
G. Galilei
A THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY IN MATERIALS AND STRUCTURE ENGINEERING AND NANOTECHNOLOGY
PhD Candidate
Eng. MARCO FRIULI
SUPERVISORS:
PROF. ALESSANDRO SANNINO
PROF. CHRISTIAN DEMITRI
PROF. CLAUDIA CAFARCHIA
Table of contents
Table of contents ........................................................................................................................................ I

List of figures............................................................................................................................................. IV
List of tables ............................................................................................................................................... V
Abstract .................................................................................................................................................... VII
Chapter 1
Introduction to the work ........................................................................................................................... 1
Chapter 2
From tissue engineering to mosquitoes: Biopolymers as a tool for the development of novel
biomimetic approaches to pest management ......................................................................................... 4
2.1 CHAPTER HIGHLIGHTS ........................................................................................................................... 4
2.2 INTRODUCTION .................................................................................................................................... 5
2.3 BIOMIMETIC LURE AND KILL APPROACH ...................................................................................................... 7
2.4 BIOMIMETIC LURE AND KILL APPROACH SPECIALIZED FOR AEDES ALBOPICTUS ...................................................... 9
Conclusions .............................................................................................................................................. 11
References ............................................................................................................................................... 12
Chapter 3
Preparation and evaluation of biopolymer-based hydrogels as biomimetic oviposition substrate for
trapping devices against Aedes albopictus ............................................................................................ 18
3.1 CHAPTER HIGHLIGHTS ......................................................................................................................... 18
3.2 INTRODUCTION .................................................................................................................................. 19
3.2.1 RATIONALE FOR OVIPOSITION PARAMETERS SELECTION ............................................................................... 19
3.2.2 RATIONALE IN BIOPOLYMER AND SUBSTRATE COMPOSITION SELECTION ......................................................... 20
3.2.3 AIM OF THE WORK ............................................................................................................................... 20
3.3 MATERIALS AND METHODS ................................................................................................................... 20
3.3.1 MATERIALS ......................................................................................................................................... 20
3.3.2 HYDROGELS PREPARATION..................................................................................................................... 21
 Physical hydrogels ........................................................................................................................................ 21
 Chemical hydrogels ...................................................................................................................................... 21
 Parameters variation .................................................................................................................................... 21
3.3.3 AEDES ALBOPICTUS OVIPOSITION ASSAY ................................................................................................... 22

 Aedes albopictus colony ............................................................................................................................... 22
 Lab-Oviposition assay................................................................................................................................... 22
I
3.3.4 OVIPOSITION SUBSTRATES CHARACTERIZATION ......................................................................................... 23
 Calorimetric analysis of free water content ................................................................................................. 23
 Water release............................................................................................................................................... 23
 Water release in plastic trap set-up at controlled conditions ...................................................................... 23
 Yield stress ................................................................................................................................................... 24
 Gel viscosity ................................................................................................................................................ 24
 Morphological analysis................................................................................................................................. 25
 On-field Oviposition assay and water release .............................................................................................. 25
3.4 RESULTS AND DISCUSSION .................................................................................................................... 25

3.4.1 OVIPOSITION 1 .................................................................................................................................... 25
3.4.2 OVIPOSITION 2 .................................................................................................................................... 26
3.4.3 OVIPOSITION 3 .................................................................................................................................... 27
3.4.4 WATER CONTENT AND WATER RELEASEAT CONTROLLED CONDITIONS ........................................................... 28
3.4.5 YIELD STRESS AND SHEAR VISCOSITY ....................................................................................................... 30
3.4.6 MORPHOLOGICAL ANALYSIS ................................................................................................................... 31
3.4.7 ON-FIELD WATER RELEASE AND OVIPOSITION ............................................................................................ 32
Conclusions .............................................................................................................................................. 32
References ............................................................................................................................................... 34
Chapter 4
Growth and effectiveness on Aedes albopictus eggs of Beauveria bassiana added to biomimetic
hydrogel oviposition substrate for ovitrap use. ..................................................................................... 38
4.1 CHAPTER HIGHLIGHTS ......................................................................................................................... 38
4.2 INTRODUCTION .................................................................................................................................. 39
4.3 MATERIALS AND METHODS ................................................................................................................... 40
4.3.1 BEAUVERIA BASSIANA ORIGIN AND INFECTION SUSPENSION PREPARATION ..................................................... 40
4.3.2 PREPARATION OF BEAUVERIA BASSIANA/GEL SYSTEMS ............................................................................... 41
4.3.3 ADES ALBOPICTUS EGGS ........................................................................................................................ 41
4.3.4 BIOASSAY 1: EVALUATION OF BEAUVERIA BASSIANA VIABILITY ..................................................................... 41
 Beauveria bassiana growth evaluation assay ............................................................................................... 41
 Beauveria bassiana conidial viability assay .................................................................................................. 42
 Beauveria bassiana vegetative growth assay ............................................................................................... 42
4.3.5 BIOASSAY 2: EFFECT OF BEAUVERIA BASSIANA/GEL SYSTEMS ON AEDES ALBOPICTUS EGGS .............................. 42
 Eggs hatching test ........................................................................................................................................ 42
4.3.6 SUBSTRATE CHARACTERIZATION ............................................................................................................. 43
 Morphological analysis................................................................................................................................. 43
 Gel viscosity measurement .......................................................................................................................... 43
4.4 RESULTS ........................................................................................................................................... 44
4.4.1 BIOASSAY 1: BEAUVERIA BASSIANA GROWTH EVALUATION IN BB/GEL SYSTEM ............................................... 44
4.4.2 CONIDIA VIABILITY IN BEAUVERIA BASSIANA/GEL SYSTEMS AND VEGETATIVE GROWTH ASSAY............................ 45
4.4.3 BIOASSAY 2: BEAUVERIA BASSIANA/GEL EFFECT ON AEDES ALBOPICTUS EGGS HATCHING ................................ 46
II
4.4.4 MORPHOLOGICAL ASSAYS ON BEAUVERIA BASSIANA/GEL SYSTEMS .............................................................. 46
4.4.5 GEL VISCOSITY MEASUREMENT ............................................................................................................... 47
4.5 DISCUSSION ...................................................................................................................................... 48
Conclusions .............................................................................................................................................. 49
References ............................................................................................................................................... 51
Chapter 5
Conclusions and future perspectives ..................................................................................................... 54
Appendix .................................................................................................................................................. 56
A.1 AEDES ALBOPICTUS ............................................................................................................................. 56
A.1.1 AEDES ALBOPICTUS: DESCRIPTION AND BEHAVIOUR ................................................................................... 56
A.1.2 AEDES ALBOPICTUS AS VECTOR: SPREAD AND MOSQUITO BORNE DISEASES .................................................... 59
A.2 MOSQUITO CONTROL METHODS ............................................................................................................ 61
A.2.1 IMPORTANCE OF VECTOR CONTROL ......................................................................................................... 61
A.2.2 CHEMICAL-BASED CONTROL METHODS: ADVANTAGES AND DRAWBACKS ....................................................... 61
A.2.3 ENVIRONMENTAL FRIENDLY MOSQUITO CONTROL METHODS ....................................................................... 63
A.2.4 LETHAL OVITRAPS AS POSSIBLE ALTERNATIVE TO PESTICIDE SPRAYING ........................................................... 64
A.3 ENTOMOPATHOGENIC FUNGI AS BIOINSECTICIDES ...................................................................................... 66
A.4.1 BIOLOGY OF ENTOMOPATHOGENIC FUNGI ............................................................................................... 66
A.4.2 FUNGI AS BIOPESTICIDE......................................................................................................................... 67
A.4.3 BEAUVERIA BASSIANA ........................................................................................................................... 68
A.4.4 BEAUVERIA BASSIANA MASS PRODUCTION ................................................................................................ 70
A.4 HYDROGEL........................................................................................................................................ 71
A.4.1 PRINCIPAL CONCEPTS ........................................................................................................................... 71
A.4.2 HYDROGELS CLASSIFICATION, USE AND PROPERTIES ................................................................................... 71
A.4.3 HYDROGEL FOR PRECISION PEST MANAGEMENT ........................................................................................ 72
A.4.4 CROSSLINKING MECHANISM IN HEC - PEGDE HYDROGELS ........................................................................ 74
A.4.5 CROSSLINKING MECHANISM IN SODIUM ALGINATE - CACL2 HYDROGELS ...................................................... 75
References ............................................................................................................................................... 77
III
List of figures
Chapter 2
Figure 2.1 Example of a fully biodegradable biomimetic lure and kill trap.. .................................................... 10
Chapter 3
Figure 3.1 Example of different turbidity gel.................................................................................................... 22
Figure 3.2 Traps setup in controlled conditions and on-field testing.. ............................................................. 24
Figure 3.3 Oviposition on physical hydrogel and chemically crosslinked ......................................................... 25
Figure 3.4 Effects of different parameters on oviposition................................................................................ 27
Figure 3.5 Lab oviposition test. ........................................................................................................................ 28
Figure 3.6 Free water % and water release as weight variations. .................................................................... 29
Figure 3.7 Shear viscosity and Yield stress values. ........................................................................................... 30
Figure 3.8 SEM micrographs. ........................................................................................................................... 31
Figure 3.9 On-field Water evaporation and oviposition .................................................................................. 32
Chapter 4
Figure 4.1 Bb growth for CS1 and CS2 preparations with and without polymer .............................................. 44
Figure 4.2 Comparison between CFU/ml values in Bb/Gel vs CS1-Bb and CS2-Bb. .......................................... 45
Figure 4.3 Conidia viability in Bb/Gel systems and vegetative growth assay.................................................... 45
Figure 4.4 Hatching test after 5, 10 and 15 contact days ................................................................................. 46
Figure 4.5 Morphological analysis on of sample CS2-HEC16 (10 days contact time) ....................................... 47
Figure 4.6 Shear viscosity after 24 days ........................................................................................................... 48
Chapter 5
Figure A.1 Diagrammatic representation of a female adult mosquito ............................................................ 56
Figure A.2 Chart of the principal characters of the stages in mosquitoes life cycle ......................................... 58
Figure A.3 Meta-analysis of natural larval breeding sites exploited by Aedes albopictus ................................ 59
Figure A.4 Aedes albopictus distribution in Europe .......................................................................................... 60
Figure A.5 Vector-borne disease annual number of cases and death .............................................................. 60
Figure A.6 Left: Common ovitraps used in recent mass trapping campaigns ................................................... 65
Figure A.7 Schematic life cycle of Beauveria bassiana...................................................................................... 66
Figure A.8 Mycelium and conidiophores of Beauveria bassiana on Rhipicephalus sanguineus ....................... 68
Figure A.9 Classification of hydrogels following different criteria .................................................................... 72
Figure A.10 Typical chemical reactions used cellulose‐based gels synthesis ................................................... 74
Figure A.11 Crosslinking mechanism in Sodium alginate using CaCl2 as crosslinker......................................... 75
Figure A.12 Egg-box shell model after SA crosslinking reaction ....................................................................... 75
IV
List of tables
Chapter 2
Table 2.1 Comparison between different control approaches ............................................................... 8
Chapter 3
Table 3.1 A panel of possible general oviposition influencing parameters a ....................................... 20
Table 3.2 Parameter variation ranges. ................................................................................................... 22
Table 3.3 Average values of Shear Viscosity and Yield stress ................................................................ 30
Table 3.4 A panel of range for oviposition parameters ......................................................................... 33
Chapter 4
Table 4.1 Overlapping parameters for the growth of Beauveria bassiana and oviposition. ............... 40
Table 4.2 Samples tested in hatching test for 5, 10, 15 days of contact .............................................. 43
V
VI
Abstract
Semiochemical based “lure and kill” Precision Pest Management (PPM) and Bioinsecticides have been
two approaches largely investigated by the pest control field as possible technically and economically
sustainable solutions to complement/replace traditional chemical methods in facing the emerging
new challenges such as control of invasive species, insecticide resistance and environmental concern.
However, both approaches present some weaknesses that could be overcome through materials
engineering approaches oriented to develop innovative solutions based, for example, on biomaterials
and bioinspired/biomimetic principles already exploited for other disciplines (e.g. medical sciences).
Difficulties of biopesticides in the on-field application and delivery and the lack of effectiveness
against some classes of insects not responsive to semiochemical stimuli are two of the unsolved
issues focused in this work.
The research activities are focused on an innovative, environmentally compatible approach called
“biomimetic lure and kill” based on biomimetics and biocompatible biopolymers used to attract
insects and simplify the target-selective delivery of biopesticides. The principle is to design a
biopolymer-based substrate that can lure a specific insect by reproducing some natural conditions
related to its behaviour (biomimetic lure) and host a natural living bioinsecticide (such as fungi,
bacteria, and many other active natural active substances) to obtain a lure and kill effect, bypassing
both pesticide and semiochemical use. Here the approach was specialized for the tiger mosquito
(Aedes albopictus) by developing an ovitrap oviposition substrate based on the biomimetic lure and
kill, i.e. a biomaterial-based hydrogel designed to reproduce the key natural parameters involved in
oviposition site selection and able to host the fungus Beauveria bassiana as the biopesticide.
Consistently to the structure of the biomimetic lure and kill, the research can be ideally divided into
three parts: i) research for the oviposition key parameters, ii) substrate design and preparation and
iii) evaluation of oviposition, substrate-bioinsecticide biocompatibility and efficacy against Aedes
albopictus eggs. The lure ability of some classes of hydrogels and the effect of the selected
parameters were verified through lab/semi-field and oviposition assays in comparison with standard
substrates. Hydrogels were then characterized to evaluate the feasibility to apply them into an
ovitrap. The best performing substrates were chosen for testing for Beauveria bassiana
biocompatibility, and then the systems Gel/Beauveria were lab-tested against Aedes albopictus eggs
to evaluate its lethality and mechanism of action. The trials have proven that it is possible to design
a hydrogel to be employed as a substrate for tiger mosquito oviposition. The results indicated that a
specific hydrogel composition effectively attracts tiger mosquitoes better than controls and, at the
same time, creates a suitable environment for Beauveria bassiana. It might be cost-competitive and
potentially applied to several pest species since all the technologies necessary to produce the raw
and finished products are already available on the market.
VII
1
Chapter 1
Introduction to the work
The theme of environmental sustainability has entered many aspects of everyday life. It now
influences both the choices of companies and consumers, directing them towards the promotion and
purchase of products and brands that are more sensitive to environmental issues. In some industries,
the cultural awareness of environmental issues is already so present that the green label is already a
tool to ensure superior product pricing and higher value on the market in terms of image for a brand.
Increasingly regulations favouring eco-friendly products and innovation or banning environmentally
impacting goods (e.g., diesel engines, disposable plastics) have tried to accelerate the green
transition even in the more resistant sectors to change. In some cases, they have pushed companies
to focus in advance on renewal to obtain a competitive advantage (by investing in research and
guiding consumers through environmental awareness campaigns). In other cases, they have
nevertheless been a lever to bring out the issue.
However, in this green promotion scenario, it is essential to consider that not always eco-sustainable
alternatives can replace standard products without practical consequences. Some classes of
products, developed to solve specific technical issues, must be replaced by technically equivalent (as
well as commercially competitive) products. One possible example is the difficulty in replacing
synthetic food packaging with biodegradable one. Although the former has a high ecological
footprint, it has played and still plays a fundamental role in increasing the shelf life of food, combating
food waste, and making specific categories of food more available at a low cost. Differently,
bioplastic-based packaging is more sustainable but often does not guarantee (at the same cost) the
same performance as synthetic packaging in terms of workability and/or food preservation. In other
words, replacing a highly efficient product with another one more environmentally sustainable but
less performing could produce the risks of reopening a problem to which a solution had already been
found where not replaced with a technically equivalent one.
Chemicals based pest management products seem to be as difficult to replace as packaging, if not
more so, and their replacement should be done by carefully evaluating the relationship between risks
and benefits. Their introduction and success (as for packaging) have deeper roots linked to their
effectiveness and historical role in the evolution of social well-being, for example, in the development
of intensive agriculture necessary to meet the growing food needs of the last 50 years or in
eradicating Malaria in west countries. Consequently, although their high environmental impact is in
open contrast with consumers’ new ecological awareness and regulatory bodies’ guidelines and their
replacement with greener products should be easy, they are still the principal pest control tool. The
reason could be the lack of a technically valid alternative or, in any case, not sufficiently effective to
justify higher production and sales costs to stimulate companies and consumers to shift to a new
generation of products.
However, some traditional solutions are gradually becoming less convenient/effective over time or,
in some cases, no longer available, forcing new technologies' entry. For example, in pest
2
management, and in particular in the control of the tiger mosquito, the action of external factors
such as resistance to insecticides, restrictive regulations, insects massive presence and behaviour in
urban areas have made chemical pesticides less cost/effectiveness by reducing the barriers to the
development and market of new products. These conditions, undermining the effectiveness of
chemical-based methods, could generally make costly (and sometimes labour intensive)
products/controls methods such as green ones commercially more acceptable and favour the
research and experimentation of new effective solutions until now ignored because with no market.
Consequently, in this context, the development of new tiger mosquito control solutions could be
facilitated.
The most logical alternative for tiger mosquito control might be the development of new green
insecticides formulations or the employment of bioinsecticides. However, new formulations are
technically complex to develop, and biopesticides are generally costly and environmental conditions
limit their use. Furthermore, for both of them (as well as already for chemical insecticides), there is
not an adequate targeted delivery strategy suited to the characteristics of the tiger mosquito (e.g.
precision pest management like solutions).
The precision pest management systems already developed for other pests are based on
semiochemical attractants (e.g. pheromones and baits) to deliver chemical insecticides more
selectively and allow lower lethal insecticide doses (semiochemical based lure and kill). For many
classes of insects, this approach is highly valid and can also be used to deliver green insecticides.
However, it is ineffective when its semiochemical based lure system fails, as it happens for insects
low-responsive to semiochemical or feeding stimuli such as tiger mosquitoes.
Therefore, the transition towards eco-sustainable solutions for the control of the tiger mosquito,
even before the development of new insecticides, must necessarily be based on the creation of new
effective lure and kill delivery strategies and, consequently, on the development of new insect-
specific attracting systems.
Consequently, the research aims to develop an innovative multifunctional attractive system that lures
and delivers natural insecticidal substances simultaneously to obtain an effective and commercially
competitive green product without pesticides and semiochemicals.
In particular, for tiger mosquitoes, the research aims to develop biopolymer-based macromolecular
hydrogels as oviposition substrates and matrices for the growth of the bioinsecticide Beauveria
bassiana finalized at the realization of a targeted and cost/effective lure and kill ovitrap. In other
words, to create a precision pest management product (developing an attractive system) that
integrates ovitrap and bioinsecticide control methods, two of the primary green and targeted
approaches against this pest.
The lack of specific synthetic or natural attractants has directed research to develop a biomimetic or
nature-inspired attractive system. In this approach, instead of using specific classes of molecules to
attract insects, some natural environmental conditions involved in insect-specific behaviours are
replicated using biocompatible biomaterials. For tiger mosquitoes, the reference behaviour was
oviposition. The lure and kill oviposition substrate has to host Beauveria bassiana and replicate the
most critical conditions related to the oviposition sites to promote oviposition and consequently the
insecticide delivery.
3
The biomimetic approach was developed by exploiting materials engineering to transfer pest
management the knowledge already consolidated in the medical field (e.g. study of cell-tissue
interaction and biocompatibility) to develop precision therapies, drug delivery, customized
regenerative systems, etc. For example, the biocompatibility was at the base of adding a
bioinsecticide to the preparation, and the process for medical scaffold production inspired the design
of the substrate.
Consequently, the research activity focused on more specific materials aspects (e.g. design,
characterization, and biocompatibility) and the analysis of the entomological behaviour of the tiger
mosquito and the mycological aspects of Beauveria bassiana. They must be evaluated to identify the
main parameters in oviposition and for bioinsecticide survival needed to design and produce the
biomimetic substrates.
In addition to material design, one of the difficulties encountered concerns identifying the key
features to be reproduced. Oviposition is a complex phenomenon influenced by countless drivers
then it is necessary to reduce the number of variables considering only the most influential.
Consequently, the multidisciplinary approach is a resource for the research. However, at the same
time, it represents a limiting factor due to the insufficient number of studies focused on analysing
deposition (and entomological behaviours) in a helpful quantitative way for the design of attractive
systems. The use of biomaterials and biomimetic approaches can provide a shared solution to the
technical and cost problems that prevent the spread of green methods for controlling the tiger
mosquito. The proposed solution cannot be separated from the market needs and must be
contextualized within the so-called “green revolution”, which is already underway in many fields, but
which has yet to prove that it can replace technically (at equal cost) traditional products but which is
now a major asset.
As guidance, a concise thesis overview is here presented:
 Chapter2 presents how biomaterials can be related to pest management and outlines the
fundamentals of the biomimetic approach and its specialization for tiger mosquitoes.
 Chapter 3 reports the rationale of selection for oviposition key parameters and substrate
design. Then are described preparation and evaluation of oviposition substrates to
indicate a specific attractive substrate formulation.
 Chapter 4 focuses on testing substrate-bioinsecticide biocompatibility and efficacy against
Aedes albopictus eggs.
4
Chapter 2
From tissue engineering to mosquitoes: Biopolymers as a tool for the development of

novel biomimetic approaches to pest management
2.1 Chapter Highlights
 Based on the analysis of pest management background and the expertise already exploited in
the medical field, an innovative and eco-sustainable precision pest management approach
has been conceptualized.
 Biomimetics and biomaterials biocompatibility are proposed as a tool to develop innovative
solutions to bypass the ineffective and harmful use of chemical semiochemicals and
insecticides.
 Biomaterials can be employed to produce a biomimetic substrate designed to reproduce
attractive environmental conditions for a specific insect and host and deliver biopesticides.
 The approach called biomimetic lure and kill is described and then is specialized for the tiger
mosquito and for the realization of a deposition substrate to be used inside ovitraps.
Abbreviations: Biomaterial BM, Biopolymer BP, Tissue Engineering TE, Beauveria bassiana, Bb; lethal
ovitrap, LOT.
5
2.2 Introduction
Biomaterials (BM) and biopolymers (BP) have already played a crucial role in the birth and
development of tissue engineering (TE), one of the most revolutionary approaches in regenerative
medicine of the last 30 years. TE combines and applies the principles of material engineering, life
sciences and biomimetic to produce scaffolds designed to mimicry the specific organ or tissue
features [1] to generate a new living and functional tissue that restore/maintain a whole organ or
improve its function [2]. TE rely on the finding that it is possible to manage and improve the outcome
of a biological target (e.g. cells seeding) by choosing and manipulating the scaffold’s composition and
features, based on the evidence that cells survival and production of physiologically functioning
structures are related to substrate properties (chemical and physical). Then, a biomimetic scaffolding
system, able to replicate physiologic tissue behaviour, better allowed seeded cells to survive and
proliferate to heal damages or produce new tissue. Consequently, the design and production of
biomimetic scaffolds are crucial for the success of TE [1]. BM and BP have been extensively studied
and resulted as the more suitable for biomimetic scaffold preparation. The continuous improvement
of innovative biocompatible materials processing and sources (e.g. polysaccharides and
polypeptides) allowed the production of complex 3D biomimetic structures and scaffolds more and
more able to replicate the physiologic mechanisms of transport and signalling [3] of human tissues.
Furthermore, BP’s constant knowledge improvement promoted TE’s advancement, such as in other
medical therapies (e.g. drug delivery or targeting), leading to precision and personalized medicine.
Consequently, there is significant expertise ready to use to produce biomimetic and biomaterial-
based substrates.
In recent decades, pest management, particularly the control of disease vectors, has been facing
numerous new challenges, dealing with them with technically and economically sustainable solutions
to complement or replace traditional approaches. Some of the main issues there are the appearance
and spreading of invasive species in new geographic areas and habitats (mainly due to climate change
and globalized movement of people and goods [4, 5] and the emergence of insecticide resistance
phenomena in pests with the consequent progressive loss of efficacy of principal molecules [6, 7]. In
addition, the impact of several insecticides on health and biodiversity has led to increasingly
restrictive regulations about legal molecules and practices [8, 9]. Some effective compounds are
considered too risky to be used in settings associated with humans, animals or foods (e.g. in the
livestock or food industry). Therefore, the reduction in control solutions strategies is pushing
companies, public health institutions and researchers to develop innovative sustainable solutions
such as biopesticides and novel pesticide-free control approaches.
Thus, pest management has slowly moved from a predominantly broad use of chemical insecticides
to a more sophisticated path of “precision” approaches. Pest management needs to be targeted
towards a specific insect group with solutions having an increasingly high environmental and health
safety profile by reducing (and eliminating, when possible) quantities and toxicity of insecticides
involved in applications. For example, strategies such as insecticide spraying, the former gold
standard of pest management, has finally been retained as a not very selective technique (and in
some cases not very effective given the lack of targeting and delivery) and, therefore, its use is limited
to strictly necessary (e.g. large agricultural areas or vector-borne disease transmission areas [10-13]).
6
The new generation of precision control tools points to increase pest targeting and insecticide
delivery efficacy mainly through insect attraction (the “lure and kill” approach) [14]. Among the most
common semiochemicals based strategies there is the use of sexual or food pheromones or
natural/synthetic baits to convey the insecticidal agent more directly and effectively by attracting a
specific insect on traps, toxic surfaces or favour ingestion of the toxic bait, as effectively shown for
malaria mosquitoes [15, 16]. Although based on the use of chemicals, the “precision” approach
represents a step forward to reduce the amounts of insecticides employed or reduce the risk of their
use indoors in the presence of people and/or animals or industrial disinfestation (e.g. food industry).
However, this approach fails when species-specific attractants or food preferences are not available
for the target pest. The semiochemical approach can be ineffective in these situations because the
insect ignores it or may even generate repellent reactions [17]. The lack of attractants is the condition
of the tiger mosquito.
Tiger mosquito Aedes albopictus (Diptera: Culicidae) is one of the major invasive species in the world
[18], vector of several arboviruses such as Dengue, Chikungunya and Zika [19, 20], causing heavy
public health and economic commitment, particularly in temperate areas [21-23]. This container-
breeding mosquito is a day-biting species primarily associated with anthropized contexts, typically
resting in shady/hidden places during inactivity [24-26]. The control of this mosquito is challenging
because of its strict association with artificial breeding habitats and green areas typically abundant in
peridomestic settings [27, 28]. These areas are difficult to treat through traditional approaches,
mainly because of the difficulties in spraying insecticides in private areas. Moreover, an increasing
insecticide resistance to different molecules is occurring in this species [29-31].
To compensate for the lack of lure and kill pest management due to the absence of attractants,
explore alternative strategies to reduce random insecticide use. Among them, lethal ovitraps (LOTs)
[32-34] and bioinsecticides such as bacteria, symbionts, entomopathogenic fungi [35-38], natural
essential oils etc. were mainly promoted for their low environmental impact.
Ovitraps have been one of the most used monitoring tools for tiger mosquitoes before it was
employed as control method by adding an active ingredient [39] to shift in lethal ovitraps (LOTs),
which have been largely investigated as potential large-scale control tools [40]. LOTs can be
numbered among lure and kill based “precision” control methods.
They are based on exploiting the tiger mosquitoes ovipositing behaviour to lay their eggs on humid
substrates placed inside a container [41, 42]. Then, the ovitrap provides a wooden oviposition
substrate inside a plastic pot filled with water enriched with insecticide and attractive substances. A
large number of attractive substances have been tested [43], but molecules as highly effective and
selective as pheromones have not been identified, resulting in less attractiveness than natural
breeding sites [44]. In addition, the need for periodic water and insecticide refill (short active period),
continuous monitoring, disposal, make traps still too much money and time-consuming to be
competitive with chemical insecticides as a large-scale control method. Furthermore, ovitraps are
still mainly chemical insecticides based devices [45] with consequent environmental and lower safety
profile than expected from a green product, even when biodegradable traps [46, 47].
On the other hand, bioinsecticides are safer than ovitraps but present storage difficulties [48], and
their efficacy and persistence are strongly conditioned by the substrate and the environmental
7
conditions of application. For example, the survival of Beauveria bassiana is reduced to a few days if
used in unsuitable operating conditions such as exposure to heat, UV radiation, and substrate drying,
thus affecting the persistence of the insecticide product [49].
Consequently, currently, there are no valid semiochemical based devices for tiger mosquito control,
and green strategies such as traps and biopesticides have low cost/effectiveness to replace/integrate
insecticide based control methods. It is, therefore, necessary to explore new precision control
methods and, in particular, the development of new attraction systems that do not exclusively use a
semiochemical or feeding signal to trigger a behavioural reaction of the insect. This approach could
be helpful to improve already ovitraps and biopesticides, improving their cost/effectiveness that
reduces their diffusion as commercially and technically competitive green alternatives.
2.3 Biomimetic lure and kill approach

Methods exploiting pest’s repetitive behavioural patterns related to essential life cycle activities such
as reproductive habits, search for suitable oviposition sites, etc. can be a potentially effective
alternative to semiochemicals.
For example, ovitraps are already based on this principle. They exploit the oviposition behavioural
pattern of tiger mosquitoes providing a breeding site (container, water, and oviposition substrate),
which is the only reason the insect interacts with them with the device and the insecticide. In this
strategy, a possible alternative to attractive substances to promote the trap among many other
natural breeding sites (improving its efficiency affected by competition with natural sites) could be
to create an ideal oviposition site by reproducing natural conditions.
Biomaterials and biopolymers, together with all the expertise in their processing derived from tissue
engineering, can be helpful for the success of a control approach like this for tiger mosquitoes and
other pests. As described for ovitraps, the key element is realising an artificial environment as close
as possible to the natural conditions requested to attract the insect showing a specific behaviour to
target (i.e. oviposition in tiger mosquitoes). At the same time, the artificial environment must be
lethal by releasing an active ingredient such as an insecticide or a natural bioactive
substance/pathogen or the material characteristic, e.g. mechanical trapping action.
This approach can be named “biomimetic lure and kill”, consisting in the realization of stand-alone
substrates or devices replicating or mimicking habitat features (physicochemical, morphological,
mechanical) identified as the most promising in attracting the target pest species and associate them
to intrinsic lethal activity or a killing agent. It can be divided into three phases: attraction, contact
between insect and the device and lethal action.
A possible suitable technical solution to develop the approach is the use of physical or crosslinked
macromolecular hydrogels. Hydrogels are a class of materials mainly composed of water that allows
to easily adjust some of their parameters within a wide range of values, guaranteeing the possibility
of imitating several different natural environments by varying the composition or processing of the
same polymer minimally (e.g. by varying polymer or crosslinker concentration). Synthetic hydrogels
are already used in pest management, mainly for chemical insecticides delivery through baits or for
controlled release in water or spraying of microencapsulated active substances [50, 51]. Alternatively,
they are inserted in traps [52, 53] to slowly release an insect growth regulator or other synthetic
8
insecticides [54, 55]. Synthetic hydrogels, although insecticide release agrees with the rationale of
precision pest management, are incompatible for biomimetic lure and kill approach development for
the lower biocompatibility with bioinsecticides, the potential repellent effect of chemical insecticides,
and for the use of synthetic polymers generally far from the composition of the natural to replicate.
In addition, the release of dangerous environmental toxic substances the risk of dispersion of
synthetic polymers contrasts with the sustainable environmental spirit of the new control methods.
The biomimetic lure and kill approach is based on a different problem-solution approach. The
hydrogel is not employed only for controlled release and/or pesticide encapsulation but represents
a multifunctional material able to bridge some of the most promising green control methods
compensating for their weakness and improve their efficacy.
Differently, hydrogels are based on low cost, naturally abundant biodegradable and biocompatible
biopolymers such as celluloses, alginates, starch, chitosan, or other polysaccharides [56].
Consequently, they can match the technical needs and the green spirit of the approach. For example,
as already reported in regenerative medicine applications [57], when biopolymer-based hydrogels
are appropriately configured, can create a favourable microenvironment for cell proliferation and,
therefore, similarly for the growth of insect-pathogenic microorganisms such as bacteria and fungi
[58], with the further advantage of using lower starting bioinsecticide concentrations to reduce costs.
Another possible advantage is the presence of the so-called auto-dissemination mechanism of the
insecticide spread by insects after oviposition, enhancing the effect of the insecticidal device.
Biopolymers and biopesticides employment, particularly hydrogel, guarantee further advantages
such as indoor use, biodegradability, durability (e.g. no need for water refill in traps) and, therefore,
the elimination of maintenance and disposal costs (Table 2.1). Finally, hydrogels can also be stored
after lyophilisation (e.g. through freeze-drying) and subsequently rehydrated (without properties
loss), ensuring ease of use, storage and transport [59]. Low-cost products allow mass applications of
green approaches.
Table 2.1 Comparison between different approaches. Costs refer to the final product and (reported on a scale
from one to four).
Lure and kill approach Insecticide based approach
Biomimetic Semiochemical Insecticide Bioinsecticide
Properties
lure and kill lure and kill application applications
Ecocompatibility ✓ x x ✓
Sustainability ✓ x x ✓
Safety profile High Low Low High
Efficacy on non-semiochemical
✓ x x x
sensitive pests
Targeted delivery ✓ ✓ x x
Resistance risk No Yes Yes Low
Easiness of application ✓ ✓ ✓ ✓
Cost 3 2 1 4
9
2.4 Biomimetic lure and kill approach specialized for Aedes albopictus
The exploited specific behavioural pattern to apply the biomimetic approach to tiger mosquitoes is
its oviposition. It is a complex and multifactorial event during which the gravid female, guided by
several stimuli, identifies the most suitable sites for the offspring survival [60]. Nevertheless, the main
components of an oviposition site can be resumed in the container, water and oviposition surface.
There are only uncertain specific features about the containers; consequently, the approach can be
specialized for tiger mosquitoes focusing on the two remaining components.
Given the extreme adaptability of Aedes albopictus [61], both in natural and in anthropized contexts
of different geographical areas [41], many specific features could play a role in identifying micro-
environments for site selection [62-66] and their choice for substrate design is crucial. The rationale
in oviposition driver selection will be described in detail in chapter 3.
Once the factor to reproduce is selected, the second step is to reproduce them by designing an
artificial oviposition substrate selecting a material and a process compatible with the selected
biopesticide presence, survival, and growth.
Among them, the group of hydrogel chemically crosslinked super-absorbing hydrogel [56] and
physical (non-crosslinked) solutions could be suitable to be employed in tiger mosquito control. The
peculiarity of this hydrogel class is the possibility of releasing water for long periods (or absorbing
humidity from the environment in the case of hydrophilic materials). Consequently, it can maintain
the essential characteristics of a suitable breeding site for tiger mosquitoes, such as water and
humidity, for an extended period. Furthermore, it is possible to regulate gel's mechanical properties
and degradation times [67], surface morphology, viscosity, pH, salinity, substrate texture etc.,
simulating physical and chemical conditions of natural oviposition substrates of tiger mosquitoes
and/or other container-breeding species [68]. 2-Hydroxyethylcellulose (HEC) and Sodium alginate
(SA) are two typical biocompatible biopolymers physical and crosslinked hydrogels forming without
high temperature or toxic crosslinking agents.
Consequently, they allow the inclusion of a biopesticide. Entomopathogenic fungi such as Beauveria
bassiana (Bb) or Metarhizium anisopliae are examples of potentially embeddable biopesticides
proven as active against tiger mosquitoes [69]. The possibility to embed these insect pathogens have
already been proven [70, 71], in particular at the conidial stage, by suspending conidia into
biopolymeric solutions able to form micro shells or spheres (e.g. alginate) containing the pathogens
and to preserve their vitality and persistence when applied (usually by spraying after rehydration)
[72, 73].
Considering the link between conidial growth and the presence of water, nutrients, and specific
ranges of pH and temperature [74, 75], HEC and SA hydrogels could be potentially able to promote
oviposition and fungal survival and growth at the same time. Consequently, by coupling the
biomimetic design of the hydrogel and the inclusion of the bioinsecticide, is possible to obtain a
biomimetic lure and kill substrates.
The biomimetic lure and kill substrate can be used as oviposition substrate instead of standard paper
or wood made substrates with the final aim to induce the largest number of female mosquitoes to
select our substrate as an oviposition site rather than the other present in the area, improving control
efficacy. In addition, the trap could be made of biodegradable polymers such as or cardboard
10
polylactic acid (similar to glue-based sticky traps [76-79]), avoiding trap disposal at the end of its
working period and ensuring a possible industrial scale-up and a very low cost and easily storable final
product. Finally, another possible advantage is the presence of the so-called auto-dissemination
mechanism of the insecticide spread by insects after oviposition (Figure 2.1). This mechanism can
power the effect of the bioinsecticide, especially for insects that do not lay all eggs in a single site
(skip oviposition), such as Aedes aegypti or Aedes albopictus [80, 81]. A representation of lure and kill
steps in a trap employing the biomimetic oviposition substrate are reported in Figure 2.1.
Figure 2.1 Example of a biomimetic lure and kill trap. A: lure phase; B: oviposition phase; C: lethal infection of
eggs and adult and auto-dissemination phase.
11
Conclusions
The increasing problems associated with the reduced efficacy and high ecological impact of
traditional pest management methods highlight the need for adopting "precision methods", focusing
on new effective and sustainable tools. The proposed approach, modulated by tissue engineering
and based on biomimetic principles, identifies biomaterials as a helpful tool as multifunctional
materials capable of satisfying biomimetic lure, biocompatibility and bioinsecticide growing.
As here described for the tiger mosquito, the approach can be efficient for several pest species.
This approach might be cost-competitive, increasing the effectiveness of substrates and devices as
much as possible. Competitivity can be obtained by identifying promising specific behavioural habits
of target species and designing an ideal substrate able to mimic the natural conditions necessary to
trigger a specific response in the target species, precisely as it is done in the scaffold-cell interaction
of TE approaches.
Developing a new pest control approach is a multidisciplinary process, then a strong interaction
among different research areas is needed to find specific behavioural patterns in insects and quantify
their driver parameters. Consequently, entomology, mycology and materials engineering are crucial.
The following steps necessary to development are to verify i) which hydrogel characteristics (e.g.
humidity, pH, salinity, composition) mostly influence the oviposition behaviour and in what range of
values; ii) the oviposition preference of Aedes albopictus for a hydrogel appropriately tuned for major
microhabitat parameters as compared to natural larval habitats. The role of material engineering is
instrumental in finding the best materials and fitting processing, technically and economically, in a
continuous transfer of knowledge from materials engineering to entomology. This with the final aim
to discover, underline and optimize for each target pest species the best lethality results with lower
environmental impact, thus obtaining pest control tools with the highest safety and large areas of
applicability.
12
References
1. Grayson WL, Martens TP, Eng GM, Radisic M, Vunjak-Novakovic G. Biomimetic approach to
tissue engineering. Seminars in Cell & Developmental Biology. 2009;20 6:665-73; doi:
https://doi.org/10.1016/j.semcdb.2008.12.008.
https://www.sciencedirect.com/science/article/pii/S1084952108001547.
2. Langer R, Vacanti JP. Tissue engineering. Science. 1993;260 5110:920; doi:
10.1126/science.8493529. http://science.sciencemag.org/content/260/5110/920.abstract.
3. Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, et al. Biodegradable
Polymer Scaffolds for Tissue Engineering. Bio/Technology. 1994;12 7:689-93; doi:
10.1038/nbt0794-689. https://doi.org/10.1038/nbt0794-689.
4. Diffenbaugh NS, Krupke CH, White MA, Alexander CE. Global warming presents new
challenges for maize pest management. Environmental Research Letters. 2008;3 4:044007;
doi: 10.1088/1748-9326/3/4/044007. http://dx.doi.org/10.1088/1748-9326/3/4/044007.
5. Powell JR. Mosquitoes on the move. Science. 2016;354 6315:971; doi:
10.1126/science.aal1717. http://science.sciencemag.org/content/354/6315/971.abstract.
6. Hemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria
disaster: will insecticide resistance derail malaria control? The Lancet. 2016;387 10029:1785-
8; doi: 10.1016/S0140-6736(15)00417-1. https://doi.org/10.1016/S0140-6736(15)00417-1.
7. Sparks TC, Storer N, Porter A, Slater R, Nauen R. Insecticide resistance management and
industry: the origins and evolution of the Insecticide Resistance Action Committee (IRAC) and
the mode of action classification scheme. Pest Management Science. 2021;77 6:2609-19; doi:
https://doi.org/10.1002/ps.6254. https://doi.org/10.1002/ps.6254.
8. Mitra A, Chatterjee C, Mandal F. Synthetic Chemical Pesticides and Their Effects on Birds.
Research Journal of Environmental Toxicology. 2011;5; doi: 10.3923/rjet.2011.
9. Ndakidemi B, Mtei K, Ndakidemi P. Impacts of Synthetic and Botanical Pesticides on Beneficial
Insects. Agricultural Sciences. 2016;07:364-72; doi: 10.4236/as.2016.76038.
10. Ahmad L, Mahdi SS. Precision Pest Management. In: Ahmad L, Mahdi SS, editors. Satellite
Farming: An Information and Technology Based Agriculture. Cham: Springer International
Publishing; 2018. p. 119-27.
11. Bonds JA. Ultra-low-volume space sprays in mosquito control: a critical review. Med Vet
Entomol. 2012;26 2:121-30; doi: 10.1111/j.1365-2915.2011.00992.x.
12. Esu E, Lenhart A, Smith L, Horstick O. Effectiveness of peridomestic space spraying with
insecticide on dengue transmission; systematic review. Trop Med Int Health. 2010;15 5:619-
31; doi: 10.1111/j.1365-3156.2010.02489.x.
13. Taranta C, Levy, T., & Benlahmar, O.: Method for Controlling Arthropods Comprising the Spot-
Wise Application of a Gel. In:
https://patentsgooglecom/patent/US20110184040?oq=Patent+US+2011+%2f+184040A1.
Edited by USPTO, vol. 184040A1. US2011.
14. Wooding M, Naudé Y, Rohwer E, Bouwer M. Controlling mosquitoes with semiochemicals: a
review. Parasit Vectors. 2020;13 1:80; doi: 10.1186/s13071-020-3960-3.
15. Fraser KA-O, Mwandigha L, Traore SF, Traore MM, Doumbia S, Junnila A, et al. Estimating the
potential impact of Attractive Targeted Sugar Baits (ATSBs) as a new vector control tool for
Plasmodium falciparum malaria. 1475-2875 (Electronic).
16. Traore MM, Junnila A, Traore SF, Doumbia S, Revay EE, Kravchenko VD, et al. Large-scale field
trial of attractive toxic sugar baits (ATSB) for the control of malaria vector mosquitoes in Mali,
13
West Africa. Malaria Journal. 2020;19 1:72; doi: 10.1186/s12936-020-3132-0.

https://doi.org/10.1186/s12936-020-3132-0.
17. Canyon D, Muller R. Oviposition and olfaction responses of Aedes aegypti mosquitoes to
insecticides. Tropical biomedicine. 2013;30:691-8.
18. Kraemer MUG, Reiner RC, Brady OJ, Messina JP, Gilbert M, Pigott DM, et al. Past and future
spread of the arbovirus vectors Aedes aegypti and Aedes albopictus. Nature Microbiology.
2019;4 5:854-63; doi: 10.1038/s41564-019-0376-y. https://doi.org/10.1038/s41564-019-
0376-y.
19. Organization WH: Draft global vector control response 2017-2030. Edited by Organization
WH2016: 53
20. Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, et al. A Review of
the Invasive Mosquitoes in Europe: Ecology, Public Health Risks, and Control Options. Vector-
Borne and Zoonotic Diseases. 2012;12 6:435-47; doi: 10.1089/vbz.2011.0814.
https://doi.org/10.1089/vbz.2011.0814.
21. Bellini R, Michaelakis A, Petrić D, Schaffner F, Alten B, Angelini P, et al. Practical management
plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus). Travel
Medicine and Infectious Disease. 2020;35:101691; doi:
https://doi.org/10.1016/j.tmaid.2020.101691.
22. Braks M, Medlock J, Hubálek Z, Hjertqvist M, Perrin Y, Lancelot R, et al. Vector-Borne Disease
Intelligence: Strategies to Deal with Disease Burden and Threats. Frontiers in Public Health.
2014;2; doi: 10.3389/fpubh.2014.00280.
23. Kampen H, Medlock JM, Vaux AGC, Koenraadt CJM, van Vliet AJH, Bartumeus F, et al.
Approaches to passive mosquito surveillance in the EU. Parasites & Vectors. 2015;8 1:9; doi:
10.1186/s13071-014-0604-5. https://doi.org/10.1186/s13071-014-0604-5.
24. Amraoui F, Failloux A-B. Chikungunya: an unexpected emergence in Europe. Current Opinion
in Virology. 2016;21:146-50; doi: https://doi.org/10.1016/j.coviro.2016.09.014.
25. Gratz NG. Critical review of the vector status of Aedes albopictus. Medical and Veterinary
Entomology. 2004;18 3:215-27; doi: https://doi.org/10.1111/j.0269-283X.2004.00513.x.
https://doi.org/10.1111/j.0269-283X.2004.00513.x.
26. Weaver SC, Reisen WK. Present and future arboviral threats. Antiviral Research. 2010;85
2:328-45; doi: https://doi.org/10.1016/j.antiviral.2009.10.008.
27. Benedict MQ, Levine RS, Hawley WA, Lounibos LP. Spread of The Tiger: Global Risk of Invasion
by The Mosquito Aedes albopictus. Vector-Borne and Zoonotic Diseases. 2007;7 1:76-85; doi:
10.1089/vbz.2006.0562. https://doi.org/10.1089/vbz.2006.0562.
28. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution
and burden of dengue. Nature. 2013;496 7446:504-7; doi: 10.1038/nature12060.
https://doi.org/10.1038/nature12060.
29. Smith LB, Kasai S, Scott JG. Pyrethroid resistance in Aedes aegypti and Aedes albopictus:
Important mosquito vectors of human diseases. Pesticide Biochemistry and Physiology.
2016;133:1-12; doi: https://doi.org/10.1016/j.pestbp.2016.03.005.
30. Dusfour I, Vontas J, David J-P, Weetman D, Fonseca DM, Corbel V, et al. Management of
insecticide resistance in the major Aedes vectors of arboviruses: Advances and challenges.
14
PLOS Neglected Tropical Diseases. 2019;13 10:e0007615; doi:

10.1371/journal.pntd.0007615. https://doi.org/10.1371/journal.pntd.0007615.
31. Moyes CL, Vontas J, Martins AJ, Ng LC, Koou SY, Dusfour I, et al. Contemporary status of
insecticide resistance in the major Aedes vectors of arboviruses infecting humans. PLOS
Neglected Tropical Diseases. 2017;11 7:e0005625; doi: 10.1371/journal.pntd.0005625.
https://doi.org/10.1371/journal.pntd.0005625.
32. Baldacchino F, Caputo B, Chandre F, Drago A, della Torre A, Montarsi F, et al. Control methods
against invasive Aedes mosquitoes in Europe: a review. Pest Management Science. 2015;71
11:1471-85; doi: https://doi.org/10.1002/ps.4044. https://doi.org/10.1002/ps.4044.
33. Johnson BJ, Ritchie SA, Fonseca DM. The State of the Art of Lethal Oviposition Trap-Based
Mass Interventions for Arboviral Control. Insects. 2017;8 1; doi: 10.3390/insects8010005.
34. Fouet C, Kamdem C. Integrated Mosquito Management: Is Precision Control a Luxury or
Necessity? Trends in Parasitology. 2019;35 1:85-95; doi: 10.1016/j.pt.2018.10.004.
https://doi.org/10.1016/j.pt.2018.10.004.
35. Hans von H, Norbert B. Cost-benefit analysis of mosquito control operations based on
microbial control agents in the upper Rhine valley (Germany). European Mosquito Bulletin.
2009;27.
36. Blanford S, Shi W, Christian R, Marden JH, Koekemoer LL, Brooke BD, et al. Lethal and Pre-
Lethal Effects of a Fungal Biopesticide Contribute to Substantial and Rapid Control of Malaria
Vectors. PLOS ONE. 2011;6 8:e23591; doi: 10.1371/journal.pone.0023591.
https://doi.org/10.1371/journal.pone.0023591.
37. Lee SJ, Kim S, Yu JS, Kim JC, Nai Y-S, Kim JS. Biological control of Asian tiger mosquito, Aedes
albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain. Journal of
Asia-Pacific Entomology. 2015;18 2:217-21; doi:
https://doi.org/10.1016/j.aspen.2015.02.003.
38. Clark TB, Kellen WR, Fukuda T, Lindegren JE. Field and laboratory studies on the pathogenicity
of the fungus Beauveria bassiana to three genera of mosquitoes. Journal of Invertebrate
Pathology. 1968;11 1:1-7; doi: https://doi.org/10.1016/0022-2011(68)90047-5.
https://www.sciencedirect.com/science/article/pii/0022201168900475.
39. Velo E, Kadriaj P, Mersini K, Shukullari A, Manxhari B, Simaku A, et al. Enhancement of Aedes
albopictus collections by ovitrap and sticky adult trap. Parasites & Vectors. 2016;9 1:223; doi:
10.1186/s13071-016-1501-x. https://doi.org/10.1186/s13071-016-1501-x.
40. Mackay AJ, Amador M, Barrera R. An improved autocidal gravid ovitrap for the control and
surveillance of Aedes aegypti. Parasites & Vectors. 2013;6 1:225; doi: 10.1186/1756-3305-6-
225. https://doi.org/10.1186/1756-3305-6-225.
41. Pereira-dos-Santos T, Roiz D, Lourenço-de-Oliveira R, Paupy C. A Systematic Review: Is Aedes
albopictus an Efficient Bridge Vector for Zoonotic Arboviruses? Pathogens. 2020;9 4; doi:
10.3390/pathogens9040266.
42. Meena A, Choudhary N. Container breeding preference of Aedes albopictus in urban
environment. 2019.
43. Dormont L, Mulatier M, Carrasco D, Cohuet A. Mosquito Attractants. J Chem Ecol. 2021;47 4-
5:351-93; doi: 10.1007/s10886-021-01261-2.
44. Dibo M, Chiaravalloti-Neto F, Battigaglia M, Mondini A, Favaro E, Barbosa A, et al.
Identification of the best ovitrap installation sites for gravid Aedes (Stegomyia) aegypti in
residences in Mirassol, State of Sao Paulo, Brazil. Memórias do Instituto Oswaldo Cruz.
2005;100:339-43; doi: 10.1590/S0074-02762005000400001.
15
45. Faraji A, Unlu I. The Eye of the Tiger, the Thrill of the Fight: Effective Larval and Adult Control
Measures Against the Asian Tiger Mosquito, Aedes albopictus (Diptera: Culicidae), in North
America. 1938-2928 (Electronic).
46. Ritchie S, Long S, McCaffrey N, Key C, Lonergan G, Williams C. A Biodegradable Lethal Ovitrap
for Control of Container-Breeding Aedes. Journal of the American Mosquito Control
Association. 2008;24:47-53; doi: 10.2987/5658.1.
47. Eiras AE, Costa LH, Batista-Pereira LG, Paixão KS, Batista EPA. Semi-field assessment of the
Gravid Aedes Trap (GAT) with the aim of controlling Aedes (Stegomyia) aegypti populations.
PLOS ONE. 2021;16 4:e0250893; doi: 10.1371/journal.pone.0250893.
48. Blanford S, Jenkins NE, Christian R, Chan BHK, Nardini L, Osae M, et al. Storage and persistence
of a candidate fungal biopesticide for use against adult malaria vectors. Malaria Journal.
2012;11 1:354; doi: 10.1186/1475-2875-11-354. https://doi.org/10.1186/1475-2875-11-354.
49. Acharya N, Seliga RA, Rajotte EG, Jenkins NE, Thomas MB. Persistence and efficacy of a
Beauveria bassiana biopesticide against the house fly, Musca domestica, on typical structural
substrates of poultry houses. Biocontrol Science and Technology. 2015;25 6:697-715; doi:
10.1080/09583157.2015.1009872. https://doi.org/10.1080/09583157.2015.1009872.
50. Norris EJ, Mullis AS, Phanse Y, Narasimhan B, Coats JR, Bartholomay LC. Biodistribution of
degradable polyanhydride particles in Aedes aegypti tissues. PLOS Neglected Tropical
Diseases. 2020;14 9:e0008365; doi: 10.1371/journal.pntd.0008365.
51. Paquette CCH, Phanse Y, Perry JL, Sanchez-Vargas I, Airs PM, Dunphy BM, et al. Biodistribution
and Trafficking of Hydrogel Nanoparticles in Adult Mosquitoes. PLOS Neglected Tropical
52. Barrera R, Mackay, A. J., & Amador, M. : Methods and apparatus for surveillance and control
of insect vectors . In: USPTO, vol. 923,774,1. US2012
53. Gaugler R, Suman, D., & Wang, Y. : Autodissemination of an Insect-Growth Regulator for
Insect Management. In:
https://patentsgooglecom/patent/US20130303574A1/en?oq=US+patent+20130303574
Edited by USPTO, vol. 20130303574 US2011
54. Buczkowski G, Roper E, Chin D. Polyacrylamide Hydrogels: An Effective Tool for Delivering
Liquid Baits to Pest Ants (Hymenoptera: Formicidae). Journal of Economic Entomology.
2014;107 2:748-57; doi: 10.1603/EC13508. https://doi.org/10.1603/EC13508.
55. Tay J-W, Choe D-H, Mulchandani A, Rust MK. Hydrogels: From Controlled Release to a New
Bait Delivery for Insect Pest Management. Journal of Economic Entomology. 2020;113
5:2061-8; doi: 10.1093/jee/toaa183. https://doi.org/10.1093/jee/toaa183.
56. Sannino A, Madaghiele M, Demitri C, Scalera F, Esposito A, Esposito V, et al. Development and
characterization of cellulose-based hydrogels for use as dietary bulking agents. Journal of
Applied Polymer Science. 2010;115 3:1438-44; doi: https://doi.org/10.1002/app.30956.
https://doi.org/10.1002/app.30956.
57. Nicodemus GD, Bryant SJ. Cell Encapsulation in Biodegradable Hydrogels for Tissue
Engineering Applications. Tissue Engineering Part B: Reviews. 2008;14 2:149-65; doi:
10.1089/ten.teb.2007.0332. https://doi.org/10.1089/ten.teb.2007.0332.
58. Au - Khetan S, Au - Burdick J. Cellular Encapsulation in 3D Hydrogels for Tissue Engineering.
JoVE. 2009; 32:e1590; doi: doi:10.3791/1590. https://www.jove.com/t/1590.
16
59. Chi C, Li X, Zhang Y, Miao S, Chen L, Li L, et al. Understanding the effect of freeze-drying on
microstructures of starch hydrogels. Food Hydrocolloids. 2020;101:105509; doi:
https://doi.org/10.1016/j.foodhyd.2019.105509.
https://www.sciencedirect.com/science/article/pii/S0268005X19312469.
60. Mwingira V, Mboera LEG, Dicke M, Takken W. Exploiting the chemical ecology of mosquito
oviposition behavior in mosquito surveillance and control: a review. J Vector Ecol. 2020;45
2:155-79; doi: 10.1111/jvec.12387.
61. Benelli G, Wilke ABB, Beier JC. Aedes albopictus (Asian Tiger Mosquito). Trends in
Parasitology. 2020;36 11:942-3; doi: 10.1016/j.pt.2020.01.001.
https://doi.org/10.1016/j.pt.2020.01.001.
62. Che Dom N, Azman M, Mokhtar M, Australia T. Development and oviposition preferences of
field collected Aedes albopictus based on different water characteristics. Malaysian Journal
of Fundamental and Applied Sciences. 2019;15; doi: 10.11113/mjfas.v15n2019.1127.
63. Bentley MD, Day JF. Chemical Ecology and Behavioral Aspects of Mosquito Oviposition.
Annual Review of Entomology. 1989;34 1:401-21; doi:
10.1146/annurev.en.34.010189.002153.
https://doi.org/10.1146/annurev.en.34.010189.002153.
64. Carrasco D, Lefèvre T, Moiroux N, Pennetier C, Chandre F, Cohuet A. Behavioural adaptations
of mosquito vectors to insecticide control. Current Opinion in Insect Science. 2019;34:48-54;
doi: https://doi.org/10.1016/j.cois.2019.03.005.
65. Gaburro J, Paradkar PN, Klein M, Bhatti A, Nahavandi S, Duchemin J-B. Dengue virus infection
changes Aedes aegypti oviposition olfactory preferences. Scientific Reports. 2018;8 1:13179;
doi: 10.1038/s41598-018-31608-x. https://doi.org/10.1038/s41598-018-31608-x.
66. Reiskind MH, Zarrabi AA. Water Surface Area and Depth Determine Oviposition Choice in
Aedes albopictus (Diptera: Culicidae). Journal of Medical Entomology. 2012;49 1:71-6; doi:
10.1603/ME10270. https://doi.org/10.1603/ME10270.
67. Shen X, Shamshina JL, Berton P, Gurau G, Rogers RD. Hydrogels based on cellulose and chitin:
fabrication, properties, and applications. Green Chemistry. 2016;18 1:53-75; doi:
10.1039/C5GC02396C. http://dx.doi.org/10.1039/C5GC02396C.
68. Madeira N, Macharelli CA, Carvalho L. Variation of the Oviposition Preferences of Aedes
aegypti in Function of Substratum and Humidity. Memórias do Instituto Oswaldo Cruz.
2002;97:415-20; doi: 10.1590/S0074-02762002000300025.
69. Shigemitsu H: Immobilization of microorganisms antagonistic to plant pathogenic
microorganisms. In:
https://patentsgooglecom/patent/US4647537A/en?oq=US+Pat+No+4%2c647%2c537.
Edited by USPTO, vol. 4,647,5371984.
70. Marois JJ, Fravel, D. R., Connick, W. J., Walker, H. L., & Quimby, P. C.: reparation of pellets
containing fungi for control of soilborne diseases In:
71. Lewis JA, Papavizas, G. C., & Connick, W. J. : Preparation of pellets containing fungi and
nutrient for control of soilborne plant pathogens. In:
72. Batista DPC, de Oliveira IN, Ribeiro ARB, Fonseca EJS, Santos-Magalhães NS, de Sena-Filho JG,
et al. Encapsulation and release of Beauveria bassiana from alginate–bentonite
17
nanocomposite. RSC Advances. 2017;7 42:26468-77; doi: 10.1039/C7RA02185B.

http://dx.doi.org/10.1039/C7RA02185B.
73. Damir M. Effect of Growing Media and Water Volume on Conidial Production of Beauveria
bassiana and Metarhizium anisopliae. Journal of Biological Sciences. 2006;6; doi:
10.3923/jbs.2006.269.274.
74. Ritz K, Young IM. Interactions between soil structure and fungi. Mycologist. 2004;18 2:52-9;
doi: 10.1017/S0269915X04002010. https://www.cambridge.org/core/article/interactions-
between-soil-structure-and-fungi/A7D00C76F8FE0F3AA1F763BAE5C85C87.
75. Jaronski ST. Chapter 11 - Mass Production of Entomopathogenic Fungi: State of the Art. In:
Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI, editors. Mass Production of Beneficial
Organisms. San Diego: Academic Press; 2014. p. 357-413.
76. Degefa T, Yewhalaw D, Zhou G, Lee M-C, Atieli H, Githeko AK, et al. Evaluation of the
performance of new sticky pots for outdoor resting malaria vector surveillance in western
Kenya. Parasites & Vectors. 2019;12 1:278; doi: 10.1186/s13071-019-3535-3.
https://doi.org/10.1186/s13071-019-3535-3.
77. Facchinelli L, Valerio L, Pombi M, Reiter P, Costantini C, Della Torre A. Development of a novel
sticky trap for container-breeding mosquitoes and evaluation of its sampling properties to
monitor urban populations of Aedes albopictus. Med Vet Entomol. 2007;21 2:183-95; doi:
10.1111/j.1365-2915.2007.00680.x.
78. Lau SM, Chua TH, Sulaiman WY, Joanne S, Lim YA, Sekaran SD, et al. A new paradigm for Aedes
spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit. Parasit Vectors.
2017;10 1:151; doi: 10.1186/s13071-017-2091-y.
79. Pombi M, Guelbeogo WM, Kreppel K, Calzetta M, Traoré A, Sanou A, et al. The Sticky Resting
Box, a new tool for studying resting behaviour of Afrotropical malaria vectors. Parasites &
Vectors. 2014;7 1:247; doi: 10.1186/1756-3305-7-247. https://doi.org/10.1186/1756-3305-
7-247.
80. Devine GJ, Perea EZ, Killeen GF, Stancil JD, Clark SJ, Morrison AC. Using adult mosquitoes to
transfer insecticides to Aedes aegypti larval habitats. Proceedings of
the National Academy of Sciences. 2009;106 28:11530; doi: 10.1073/pnas.0901369106.
http://www.pnas.org/content/106/28/11530.abstract.
81. Caputo B, Ienco A, Cianci D, Pombi M, Petrarca V, Baseggio A, et al. The “Auto-Dissemination”
Approach: A Novel Concept to Fight Aedes albopictus in Urban Areas. PLOS Neglected Tropical
18
Chapter 3
Preparation and evaluation of biopolymer-based hydrogels as biomimetic oviposition

substrate for trapping devices against Aedes albopictus.
 Biomimetic approach principles are specialized and verified for tiger mosquitoes.
 The rationale for parameters and polymers selection is set, and several cellulose and sodium
alginate-based hydrogels are prepared and characterized.
 Hydrogels are lab/semi-field tested to verify the lure ability as an oviposition substrate
 2-Hydroxyethylcellulose (HEC) based preparations, designed following some key oviposition
parameters, are up to 5 times more attractive than the standard wooden substrate.
 A low-cost cardboard trap using the most attractive hydrogel preparation is up to 7 times
more attractive than a standard device on a semi-field 30 days long oviposition assay.
Abbreviations: Lethal ovitrap, LOT; 2-Hydroxyethylcellulose, HEC; Sodium alginate, SA; Sorbitol, S;
Turbidity, Tb, Relative Humidity, RH; Precision pest management, PPM.
19
3.2 Introduction
As already described in chapter 2, the biomimetic lure and kill approach points to attract insects by
imitating some specific natural conditions related to their behaviour and, at the same time, to host a
natural bioinsecticide (such as fungi, bacteria) to obtain a lure and kill oviposition substrate bypassing
both pesticide and semiochemical use. Then, the hydrogel can be applied into a lethal ovitrap to
obtain a lure and kill device better performing in eggs collected. Ovitraps are one of the possible
precision control methods for Aedes albopictus [1, 2]. Using a biomimetic approach could make them
evolve from a standard setup (water and insecticide filled container and a moist wooden oviposition
substrate) to a more performing and low-cost version. Hydrogels were considered the most
promising to specialize biomimetic approach to tiger mosquito due to their easiness in properties
variations, for its water uptake, biocompatibility and biodegradability [3-7]. Furthermore, for this
specific application, hydrogel features (such as water retention, biodegradation, biocompatibility) are
helpful to fix ovitraps limits related to costs, duration, maintenance/disposal, and synthetic
insecticides use that still limit trap’s mass employment as mass control method [8-14].
3.2.1 Rationale for oviposition parameters selection

Oviposition is a complex and multifactorial phenomenon during which the gravid female, guided by
several driving parameters, identifies the most suitable sites for offspring survival [15]. Given the
extreme adaptability of Aedes albopictus at larval level [16], both in natural and in anthropized
contexts of different geographical areas [17], many factors/parameters can play a role in identifying
micro-environments for site selection [18-22], and their selection for substrate design is crucial.
The essential condition for container-breeding mosquitos oviposition (a group including tiger
mosquitoes) is the presence of a water container (natural or artificial) to create a suitable
environment for larval development after hatching. However, differently from other genera, the tiger
mosquito does not lay down its eggs directly on the water surface but on a wet substrate inside the
container (or on the container's inner wall), waiting for the water level to rise to submerge them to
hatch (condition reproduced by standard ovitraps) [23]. In natural environments, and in general, in
infested areas where the insect has several places potentially suitable for oviposition, it is possible to
assume that the site's choice takes place after the search, identification, and selection of a specific
oviposition container/substrate, among many others. Numerous factors could influence each phase,
but the main drivers are not identified and quantified univocally through a model [24]. Focusing on
the container selection phase, the most significant part of studies identify container's water features
as crucial (pH, salinity, turbidity, presence of chemical deterrent such as pesticides etc.) [25-30]. In
addition, other properties strictly related to oviposition substrate, such as surface texture,
morphology and slope, moisture content, water vapour emission, are reported as crucial in the final
choice of a site [31, 32]. Even though the lack of defined ranges, these features, in addition to the
knowledge of the primary natural environments exploited as oviposition sites (e.g. hollow trunks,
plant leaves, muddy embankments), can provide a reference for the design of a biomimetic substrate
(biopolymer selection and hydrogel and device design). In particular, considering that water
properties will be transferred to oviposition substrate (by absorption), the ideal hydrogel should
20
match water and substrate features. Following the rationale, a panel of possible parameters have
been selected and reported in Table 3.1.
Table 3.1 A panel of possible oviposition influencing parameters and possible suitable conditions.
Parameters
Water
Substrate Substrate
pH Salinity content Morphology Turbidity
Composition texture/orientation
wt%
Suitable
Mild mud÷wood- Cloudy
conditions Mild Organic >0% Rough
acidic÷basic like/Sloped water
[24-30]
3.2.2 Rationale in biopolymer and substrate composition selection

It has been shown how biopolymers and in particular hydrogels, adapt well to approaches based on
biocompatibility and biomimetic principles. Therefore, they have been chosen as the preferred
materials to develop the biomimetic approach [33-35]. Celluloses and alginates, in particular 2-
Hydroxyethylcellulose (HEC) and Sodium alginate (SA), were chosen due to their hydrophilic nature
for their chemicals nature similar to natural sites. HEC and SA were processed to form physical (non-
crosslinked) and chemically crosslinked (CL) hydrogels replicating the suitable condition for the
oviposition parameter selected within specific ranges (due to the lack of defined indications) to check
a single parameter range of effect.
3.2.3 Aim of the work

The aim of the work was i) to verify oviposition on preliminarily selected hydrogels (to exclude a
repellent effect); ii) evaluate the effect of the parameters on oviposition to optimize their
combination; ii) characterize the substrate to evaluate possible material-oviposition relations and its
possibility to be integrated into an ovitrap. A preliminary set of hydrogels was thus prepared and
tested compared to standard deposition substrates such as absorbing paper or Masonite. Further
parameters were identified, and their effect on attractiveness (in the laboratory and later in the field)
was evaluated. Tested substrates were also characterized to explore possible relationships between
substrate properties and oviposition and evaluate their suitability for a convenient application inside
a trap.
3.3 Materials and methods
3.3.1 Materials
2- Hydroxyethylcellulose in powder (HEC, Aldrich Chemistry, average Mw 720000) and Sodium
alginate (SA, SAFC, average Mw 120000) were the polymers used to produce substrates.
Chemically crosslinked hydrogels were produced through chemical crosslinking processes carried out
using water-soluble liquid Poly(ethylene glycol) diglycidyl ether (PEGDE, Aldrich average Mw 500) as
biocompatible crosslinking agent and Sodium Hydroxide pellets (NaOH, Aldrich) as catalyst of the
21
reaction. Calcium chloride (CaCl2, Anhydro Beads, ≥99.9%, Sigma-Aldrich) was used as the
crosslinking agent in SA chemically crosslinked hydrogels. Standard high weight and rough white
absorbing paper (filtering paper-like) and Masonite were used in oviposition test. D-Sorbitol (D-
sorbitol 99%, Sigma-Aldrich), NaCl (Sodium chloride BioXtra, ≥99.5%, Sigma Aldrich), NaOH, (Sodium
hydroxide BioXtra, ≥98%, pellets (anhydrous), Sigma-Aldrich), Lactic acid (L-(+)-Lactic acid ≥98%,
Sigma-Aldrich), lab-made Masonite maceration were added to preparations in order to produce
oviposition parameters variations respectively in water evaporation rate, pH and turbidity. Masonite
maceration was lab produced by boiling 3 new Masonite sticks for 10 minutes in 0.5 l of distilled
water. Sticks were then soaked in water for 10 days to obtain a dark brown liquid to be diluted with
water.
3.3.2 Hydrogels preparation

Physical hydrogels
Physical hydrogels (non cross-linked) were produced by adding HEC and SA powders to distilled water
and stirring at 1500-2000 rpm for about 30 minutes at room temperature. Polymer concentration
ranged within 2-30%wt, and sample produced were respectively HEC2-30 and SA2-30.
Chemical hydrogels
Crosslinked Hydroxyethylcellulose hydrogels, CL-HEC, were produced through the following protocol
already reported in other works [36-38]. 90 ml of distilled water containing 4ml of PEGDE were
prepared, then HEC within polymer concentrations range 2-30%wt (CL-HEC 2-30) was added and
stirred at 1500-2000 rpm at room temperature for about 30 min (solution A). A separate 10 ml
solution (solution B) of distilled water and 4g of NaOH was prepared. After complete mixing, solution
B was added to A and incubated at room temperature overnight to obtain a water-insoluble HEC
hydrogel. A third solution (neutralization solution, solution C) was prepared by adding 4.10 ml of 37%
HCl (Aldrich chemistry) to 96 g of distilled water to neutralize NaOH. The hydrogel was i) cut in pieces
and immersed in solution C until neutral pH was restored ii) repeatedly washed in distilled water,
iii)submerged until completely swollen (equilibrium state).
SA crosslinked hydrogel samples with polymer concentration 2-30 wt % (CL-SA 2-30) were prepared
by adding SA powder to distilled water and stirring at 1500-2000 rpm at room temperature for about
30 min. In another container was prepared a 1 molar solution of calcium chloride (CaCl2) used as
crosslinking agents for SA [39]. Due to the difficulties to shape CL-SA to perform an oviposition test,
a Masonite stick 2x10 cm (surely not repellent oviposition substrate) was dip-coated (2-3 mm layer X
5 cm) into SA solution. Then, the coated stick was immersed into CaCl2 solution at 35°C for 1 h to
promote e deeper penetration of the crosslinker.
Parameters variation
Water-soluble sorbitol powder (S) was added as a humectant within 0-10%wt to produce S0-10
samples to change water evaporation. A salinity range was obtained by adding NaCl to distilled water
and monitoring salinity variation through a portable conductivity meter (Bormac model 5 COND 70+).
The same method was used to produce hydrogels with different pH (4.5<pH<10) by adding NaOH, or
22
Lactic acid. Here, pH was adjusted during preparation by using a Mettler Toledo model pH8 benchtop
pH-meter. Parameters were changed at constant polymer concentration, and their ranges are
reported in Table 3.2.
Table 3.2 Parameter variation ranges.

Parameter Range
pH 4.5-10
Salinity % 0-5
Turbidity
0-8
(McFarland)
Sorbitol wt% 0-10
Finally, to evaluate the effect of turbidity on oviposition, Lab-prepared Masonite maceration was
added to distilled water at different ratios to obtain a colour range (Figure 3.1). Turbidity values were
monitored by a densitometer (Biosan DEN 1). McFarland was preferred to NTU units due to the high
presence of particles in the suspension.
Figure 3.1 Example of different turbidity on gel. From left to right Turbidity (Tb) 0,4,8.
3.3.3 Aedes albopictus oviposition assay

Aedes albopictus colony
An Aedes albopictus colony was raised in an insectary placed at the Parasitology Section of the
Department of Public Health and Infectious Diseases of the Sapienza University of Rome. Colony
breeding conditions were: T 25±1°C, RH 70%, light hours /darkness hours ratio was 14:10. Mosquitoes
made a blood meal 48h before the oviposition test. This time was enough to complete the
gonotrophic cycle (eggs maturation) of the species.
Lab-Oviposition assay
At the end of the feeding, 30 gravid females were maintained in every cage giving them the tested
substrates (T) and the control (absorbing paper, C) placed inside a white plastic container containing
30 ml of distilled water. All the tests were performed comparing the same cage hydrogels with the
same features (e.g. HEC16 vs SA16 or CL-HEC16 vs CL-SA16) against the same size absorbing paper.
10 grams of physical hydrogels were spread on 2x5 cardboard then placed into the plastic container
whereas 2x5 cm CL samples were simply placed on the cardboard. A scouting test (Oviposition 1) was
conducted to find the best performing substrate composition within a 24h oviposition period. A
second test (Oviposition 2) was performed as previously described to evaluate, using the best
23
performing preparation in oviposition 1, the effect of pH, salinity and turbidity within a 24h
oviposition period. A third prolonged (two weeks, same setup and control previously described) lab-
oviposition assay (Oviposition 3) aimed to compare in a single cage the hydrogels composition best
performing during oviposition 1 and 2 and against Masonite stick using. Every test was performed in
triplicate. The results are reported for each sample as T/C, which is the ratio between the total
number of eggs laid on the sample (T) and control (C) ± Standard deviation (SD).
3.3.4 Oviposition substrates characterization

Calorimetric analysis of free water content
Free water percentage (FW%) is defined as the water not chemically bonded to the material and that
therefore it is still free to change its state (e.g. solid to liquid). Free water is also the first to be released
through evaporation in hydrogels. Then its amount could influence oviposition. The free water
amount can be evaluated through calorimetric analysis by comparing the melting enthalpy of known
weight samples (equal to the area of endothermal peak) to water melting enthalpy per grams. Free
water percentage is (free water measured/sample weight)x100. Calorimetric analysis was performed
through Differential scanning calorimetry (DSC) using a Q2000 Series DSC from TA Instruments DSC.
Samples with the same weight (5g) were subjected to a thermal cycle in a nitrogen atmosphere
composed of a cooling ramp of 5 ° C/minute to -20 ° C and then a heating ramp of 5°C/minute up to
50°C [40]. Then, melting peak areas were measured with the specific functionality of the software
Q2000 Series software analysis. Analyses were performed on HEC, SA and standard controls e.g.
absorbing paper and Masonite (used as the lower amount of free water necessary for oviposition).
Controls were tested after 4 days of water immersion (time to reach the equilibrium condition i.e. no
more absorption). CL-SA was removed from Masonite stick before testing.
Water release
The tests aimed to evaluate humectant (sorbitol) and polymer concentration (viscosity) effects on
water evaporation in HEC and SA samples. The Sorbitol effect was tested by varying the Sorbitol
concentration in a range 2-10 wt% (S2-10) intoHEC16 (resukted as best performing in oviposition) at
different. In order to test the effect of viscosity on water release, sorbitol concentration in samples
was constant (6wt%, S6) for different HEC concentrations. The test was performed in a BINDER
climatic chamber at 25°C, RH=80% on sample (about with the same starting weight and shape) placed
singularly in a plastic container. Samples were weighed every 24h using a Sartorius microbalance (10-
5 sensitivity).
Water release in plastic trap set-up at controlled conditions

The weight loss in trap setup test aimed to measure water evaporation from HEC16 and HEC16/S
substrates in field-like conditions in a possible application setup. It was performed in a Binder model
KBF 115 climatic chamber at controlled conditions (25°C and 80% RH) for 24 days. About 200 g of
HEC16 and HEC16/S were spread on silicone paper stripe then bonded to the wall of a plastic
commercial trap (Polytrap©, Figure 3.2) Water evaporation was monitored by evaluating daily the
weight of trap+gel system using a Sartorius microbalance (10-5 sensitivity). Samples were compared
24
to a 2x15 Masonite stick placed in the same trap at the same conditions immerged in 200 ml of water.
Results are reported as normalized weights of the gel with time.
Figure 3.2 Traps setup in controlled conditions and on-field testing. Left: Setup of poly trap employed for
water release in controlled condition test. Right: Easy trap used for oviposition and duration test on
field. Commercial traps provided by Gea Srl-Mialn, Italy.
At the end of the testing period, few grams of HEC16 and HEC16/S6 and Masonite were tested
through DSC analysis performed as described above to evaluate the residual free water.
Yield stress
Considering that Aedes albopictus oviposition needs a sloped surface (almost vertical), to replicathìe
the condition, the gel must not flow down after application (e.g. the wall of a trap or a cardboard
stick). Physical hydrogels (the only flowing) have to present a thixotropic behaviour (toothpaste-like),
i.e. flowing only when critical shear stress (or yield stress, τ0) is exceeded. Considering that the only
applied force on the gel spread on a wall will be its own weight (Fw), the condition required for
application on a vertical wall is τ0gel> Fw.. Here, the average shear stress value due to Fw action (τlimit)
was calculated as Fw/At (where At is the cross-sectional area of material with area parallel to the
applied force vector). In particular, was calculated for a 5 mm layer of hydrogel spread on the inner
surface of a cylinder 10x15 cm (trap-like configuration) assuming a)hydrogel density as the same as
water; b) gel spread uniformly on the surface; c) At as the whole application surface. In these
conditions τlimit=50Pa. The yield test is then used to measure τ0 value. The yield stress test was
performed with a Malvern Kinexus Pro rotational rheometer. The yield point was evaluated through
a standard test (based on Bingham model) by the use of Malvern Kinexus Pro rotational rheometer
software (rSpace) in a shear rate range 0-100 s-1 in the plate-plate configuration at 25°C. It was
performed on HEC and SA samples to evaluate the lower polymer concentration in tested physical
gels able to stand on a vertical surface.
Gel viscosity
Shear Viscosity of a fluid is a measure of its resistance to deformation at a given rate (e.g. syrup has
a higher viscosity than water [41, 42]) and its measurement is a way to characterize a hydrogel. CL
samples, due to chemical stabilization, are fragile and unable to flow, furthermore, their viscosity is
about 2 magnitude orders higher than physical HEC and SA samples, then can be considered as
infinite. Viscosity was measured on HEC and SA samples with a single shear rate test with a Malvern
25
Kinexus Pro rotational rheometer in a plate-plate configuration (50s-1 of shear rate for 5 minutes,
25°C and atmospheric pressure). The test was performed on physical hydrogels to show the variation
of viscosity with the polymer concentration.
Morphological analysis
Scanning an electronic microscope through SEM EVO® 40, Carl Zeiss AG on CL-HEC and CL-SA samples
was performed to evaluate the surface morphology and possible effect in oviposition.
On-field Oviposition assay and water release
The best performing samples were subjected to a single preliminary test on field conditions in a
restricted area (5x5m backyard) in Settimo Milanese (Milan, Italy) in the period September 2019 using
a commercial low-cost cardboard trap (cockroaches-like trap) with an active surface of 12x15 cm
(Easy trap, Figure 3.2). A black standard oviposition trap with Masonite stick in distilled water was
used as control. Traps were placed in a shaded, humid and mosquito populated place of the backyard
for 30 days. Oviposition was monitored every 15 days and traps’ weight daily. Average environmental
temperature and humidity were 23±1°C, 75% RH during the test period (data from Fondazione
Osservatorio Metereologico Milano Duomo).
3.4 Results and discussion
3.4.1 Oviposition 1
The assay was performed to evaluate hydrogels suitability as oviposition substrates and, eventually,
which composition is the most promising for Aedes albopictus oviposition. Oviposition was expressed
through the T/C ratio (sample performed better than control when T/C>1).
1.6
A B
1.6
1.4 1.4
1.2 1.2
1.0 1.0
T/C
0.8
T/C
0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
2 6 8 10 16 20 30 2 6 8 10 16 20 30
Polymer concentration wt% Polymer concentration wt%
HEC SA CL-HEC CL-SA
Figure 3.3 Oviposition on HEC and SA physical hydrogel (A) and chemically crosslinked HEC and SA based
hydrogels (B). Oviposition tests refer to absorbing paper as control and T/C is the ratio between eggs
laid on sample (T) and control (C). All the tests were carried at 25°C and 80%, relative humidity (RH),
pH=6.5, Salinity<1%.
From the assay emerged that hydrogels can act as oviposition substrates. The assay (Figures 3.3 A
and B) showed that physical hydrogels were more attractive than CL-samples than a standard control
26
(absorbing paper). In particular, HEC samples were more attractive than SA samples and control
(starting from HEC8). Generally, a polymer concentration-related trend appeared, and a peak of T/C
was registered for HEC16 (T/C=1.41) and SA20. The presence of a peak could indicate that the luring
effect could not depend only on the polymer type (in terms of a polymer luring potential) but on
some concentration-dependent factors, such as free water amount, water release, substrate viscosity
etc.. Differently, the absence of oviposition on CL hydrogels (Figure 3.3B) could be related to a
repelling effect of PEGDE as crosslink agents or neutralization agent (respectively NaOH, CaCl2 and
HCl) [26] or to surface morphology [43] (e.g. smooth surface, usually not luring for oviposition as
previously explained).
3.4.2 Oviposition 2
The second oviposition test (Oviposition 2) was performed to evaluate the most attractive hydrogel
preparation in oviposition 1 (HEC16), the effect of pH, salinity and turbidity variations. pH, salinity
and turbidity seem to have a heavy impact on T/C. In particular, are clearly identified ranges of pH
and salinity useful for oviposition (respectively 4.5<pH<7.5 and salinity<3%, Figure 3.4-A and B). pH,
salinity probably, outside some ranges, negatively impacts Aedes albopictus sense (tactile and
olfactory) [44]. Humectant (sorbitol) concentration seems to not have a high impact on oviposition
(T/C range between 1.2 (HEC16) and 1.46 (HEC16/S6)), but it seems to reduce T/C when increased
(T/C close 1 for S10) (Figure 3.4-C). This could be related to an excessively low water release (free
water evaporation) that can make substrate less attractive [45]. This lead to suppose a role of
evaporation rate in lure activity. In particular, this hypothesis could explain oviposition reduction at
high polymer and sorbitol % as water release (or free water evaporation) is ruled by humectant and
polymer viscosity [45, 46].
HEC16/S6 showed the higher T/C (=1.46), and it was identified as the best sorbitol concentration and
used in all other tests. Turbidity level (Figure 3.4-D) seems to impact positively on oviposition. In
particular, for in particular, Tb=8 T/C is doubled compared to HEC/Tb0. Oviposition dependence from
turbidity could be related to a higher organic presence in water used to produce hydrogels (as already
reported for water in breeding sites) or to a wood-like colour better simulating a natural oviposition
surface (e.g. tree holes, tree bark etc.).
27
A B
pH=6.5 Salinity<1%
4 1.6
3.5 1.4
3 1.2
2.5 1
T/C
T/C
2 0.8
1.5 0.6
1 0.4
0.5 0.2
0 0
10 8-9 6.5-7.5 5.5-6.5 4.5-5.5 <4.5
Salinity % pH
C D
pH=6.5, Salinity<1% pH=6.5, Salinity<1%
4 4
3.5 3.5
3 3
2.5 2.5
T/C
T/C
2 2
1.5 1.5
1 1
0.5 0.5
0 0
S10 S8 S6 S4 S2 S0 Tb10 Tb8 Tb6 Tb4 Tb2 Tb0
(HEC16) (HEC16)
Sorbitol concentration (S) %wt Turbidity (Tb) McFarland
Figure 3.4 Effects of salinity (A), pH (B), Sorbitol concentration, S (C) and turbidity Tb (D) on HEC16 oviposition.
All test refers to absorbing paper as control and T/C is the ratio between eggs laid on sample (T) and
control (C). All the tests were carried at 25°C and 80% of relative humidity (RH).
3.4.3 Oviposition 3
This test was carried out to compare all the compositions best performing in the previous oviposition
test and against Masonite (standard oviposition substrate employed in ovitraps). Furthermore, the
test wanted to assess oviposition in two weeks to evaluate a possible role of humectant as the
promoter of a long-lasting lure effect. HEC16/Tb8 showed the highest oviposition level (T/C=5.1,
Figure 3.5) in the first and second weeks. Generally, all gel samples presented a higher oviposition
level compared both to control and Masonite. Although HEC16/Tb8 was the best performing aver
two weeks, HEC16/S6 was the only to register an increase in T/C value between weeks 1 and 2 (from
1.2 to 2.1). This result could be related to the sorbitol presence, and its effect as humectant can retain
a higher amount of water than the other samples. Furthermore, the higher performance of all
hydrogels without Masonite over Masonite stick excludes an olfactory role of Masonite in lure
performance of Masonite maceration containing samples both in oviposition 3 and 2.
28
4
T/C
3
Week 1
2 Week 2
0
Masonite HEC 16 HEC16/S6 HEC16/Tb8
stick
Oviposition Substrate
Figure 3.5 Lab oviposition test. Masonite, absorbing paper (control) vs best-performing gels (two weeks test).
Oviposition test covered a two weeks period in order to evaluate lure with time. All tested samples
refer to absorbing paper as control and T/C= eggs on sample (T) /eggs on control (C). Test
parameters: 25°C, (RH=80%), gel pH=6.5, salinity<1%.
3.4.4 Water content and water releaseat controlled conditions

Free water percentage (FW%) was measured through DSC analysis and its release was assessed
through weight loss measurement performed on gel samples (with and without sorbitol) directly
exposed to controlled conditions (25°C, 80% RH) and in ovitraps setup condition in HEC/S6, HEC16
and Masonite.
Free water percentage was inversely proportional to polymer concentration. From weight
measurement emerged an inversely proportional dependence of water release rate from Sorbitol
and polymer concentration (slope of the segment between day 1 and 2 in Figure 3.6B and C) due to
the humectant effect of sorbitol to the lower permeability in higher polymer concentrations. Water
release in trap setup in a climatic chamber ((Figure 3.6D) was slower than gel samples due to the
presence of a semi-closed container (Figure 3.2). HEC16/S6 presented a slower water release
compared to HEC16 (final residual weights were 45% and 31% of the initial weight) and both
hydrogels release water slower than Masonite stick.
29
A B
100% 1.60
90% 1.0
1.40
80%
Normalized weight
1.20 0.8
70%
60% 1.00
FW%
0.6
T/C
50% 0.80
40% 0.60 0.4
30%
0.40
20%
0.20 0.2
10%
0% 0.00 0.0
2 6 8 10 16 20 30
Polymer concentration wt% 1 2 3 4 5
Day
HEC SA
HEC16/S6 HEC8/S6 HEC2/S6
Masonite Absorbing paper
HEC T/C Oviposition 1 SA T/C Oviposition 1 HEC16/S0 HEC8/S0 HEC2/S0
C D
100%
1.0 90%
80%
Normalized weight
Normalized weight
0.8
70%
60%
0.6
50%
0.4 40%
30%
0.2 20%
10%
0.0 0%
1 2 3 4 5 0 5 10 15 20
Day
Day
HEC 16/S10 HEC16/S8 HEC16/S6
HEC16/S4 HEC16/S2 HEC16/S0 HEC16% Masonite HEC16/S6
Figure 3.6 (A) Free water % in substrates compared to oviposition values. Water release weight variations with
time: (B) effect of sorbitol concentration S on HEC16 (C) effect of polymer concentration for S6 (D)
Water release at 25°C, RH=80%. All results are expressed as value± SD.
Finally, DSC free water analysis revealed 25% and 18% of residual free water respectively in HEC16/S6
and HEC16 and no residual free water in Masonite stick after 24 days. Free water analysis merged
with oviposition results (Figure 3.6A) and showed no clear correlation with mosquito substrate
preference: the highest oviposition was registered at the same FW%=80% (HEC16) as absorbing
paper (control in oviposition assay). Consequently, it is possible to suppose that water contents are
not the only driver in oviposition site selection. By analysing water release behaviour and reduction
in oviposition for higher polymer and sorbitol concentration reported in oviposition 2, it is possible
to suppose a role of evaporation in substrate selection. On the other hand, considering the difference
in oviposition between HEC and SA for the same FW% is possible to suppose a role of substrate
texture (described through viscosity).
Consequently, the right combination of viscosity, FW% and water release is needed. Evaporation
trend was confirmed, even though slower when applied into semi-closed traps setup. The presence
30
of free water after 24 days indicates potential residual lure activity of hydrogels, suggesting the
possibility to improve the trap’s duration.
3.4.5 Yield stress and Shear viscosity

Viscosity can be related to water release rate and substrate texture. On the other hand, the yield
stress is a parameter that determines the possibility of the substrate to be spread on an inclined wall
without flow, as needed for physical hydrogels employment inside ovitraps.
A B
40 140
35 Butter-like 120
40 Pas [29]
Shear viscosity Pas
30
100
Honey-like
25
10 Pas [27] 80
τ0 Pa
20
Oil-like 60
15
0.2 Pas [26]
10 40
5 20
0 0
0 4 8 12 16 20 24 28 32 0 4 8 12 16 20 24 28 32
Polymer concentration wt% Polymer concentration wt%
HEC SA SA τlimit HEC
Figure 3.7 (A) Shear viscosity values ± SD for different HEC and SA concentrations (wt%) evaluated in a single
shear rate test (50s-1) at 25°C in plate-plate configuration. (B) Yield stress values ± SD with polymer
concentration evaluated in a shear rate range 0-100 s-1 at 25°C with plate-plate configuration.
Values are reported in Figure 3.7 and Table 3.3. As expected, viscosity and yield stress depend on
polymer concentration
Table 3.3 Average Shear Viscosity ± SD and Yield stress ± SD at 25°C registered in single shear viscosity and
Yield stress test.
Polymer
Shear viscosity (Pas) Yield stress (Pa)

Concentration
(wt%)
HEC SA HEC SA
2 0.4±0.03 0.2±0.01 9.6±2.8 8±2.7

4 1.5±0.11 0.8±0.06 25.9±3.2 12±3.1
6 2±0.14 0.7±0.05 28.3±3.1 20.1±4.3
8 4.5±0.32 2.55±0.18 39±4.1 28±2.1
10 7.5±0.53 5.2±0.36 53±3.8 43±4.3
16 14.1±0.99 11±0.77 102±2.4 55±5.2
20 18.3±1.28 14.9±1.04 110±4.2 61±5.1
30 34.7±2.43 33.1±2.32 120±5.3 73±5.3
31
From viscosity values analysis and oviposition 1 results, it emerged as oil-like textures (e.g. HEC2-4,
SA2-4, [41]) obtained low T/C levels (<1), resulting as less attractive than others. HEC16-20, SA16-20
concentrations (honey-like, 11-18 Pas [42], e.g. HEC16) resulted as the most attractive. A further
increase of concentration (butter-like texture, [47], e.g. HEC30) leads to a decrease in T/C. For HEC,
the viscosity range that seems to guarantee the right compromise between evaporation rate and
substrate texture is 5-20 Pas (HEC8-20). However, it is possible to exclude that the lower deposition
on HEC30 was due to an excessive viscosity of the substrate as it is certainly less viscous than standard
substrates such as Masonite. Consequently, it has to be related to other features such as water
behaviour (content or evaporation rate). Yield stress becomes higher than the threshold value (50
Pa, for vertical wall, evaluated as previously reported) at HEC10; consequently, the lower spreadable
concentration is HEC10. Yield stress does not seem to influence oviposition (T/C>1 at HEC8), but it
guarantees that physical hydrogels can be spread without flowing. Sorbitol samples and Masonite
maceration prepared gel samples were also tested in the Viscosity and Yield stress test (performed
as described), and no influence was found on rheological behaviour.
3.4.6 Morphological analysis

SEM analysis was conducted on CL samples as previously described. SEM micrographs revealed a
smooth surface with no pores (Figure 3.8 A and B) and salt clusters (Fig. A).
The absence of a pattern or a rough surface could be a deterrent for Aedes albopictus oviposition,
which usually prefers rough surfaces. Nevertheless, it is not possible to exclude a possible repellent
effect due to the presence of NaOH or HCl residuals from the neutralizing solution that can change
salinity or pH out from oviposition ranges previously reported.
Figure 3.8 SEM micrographs (scale bar 100 µm): (A) CL-HEC8 and (B) CL-SA8 samples as an example of
crosslinked hydrogel surface.
32
3.4.7 On-field water release and oviposition

HEC16/S6/Tb8 (best performing in oviposition), HEC16 a were spread onto a cardboard trap (a
commercial cockroaches-like trap, Figure 3.2) and tested to evaluate duration in terms of water
contents and oviposition against a standard trap with Masonite paddle as oviposition substrate.
Although with different conditions, HEC16/S6/Tb8 preparation confirmed on-field test in cardboard
traps setup that: a) water release was slower than HEC16 and Masonite; b) hydrogels can be used as
oviposition substrates for a more extended period than Masonite on field test in cardboard traps
setup. Weight loss analysis showed similar behaviour to climatic but as expected due to different
trap’s shape and environmental conditions reported lower final residual water values chamber water
evaporation test (36% and 21% respectively for HEC16/S6/Tb8 and HEC16). Oscillation in weight
values can be related to a hygroscopic behaviour of the material (hydrogel and cardboard), able to
rescue water from environmental humidity (especially during the rainy days). Oviposition showed no
large difference in short term period (e.g. first 15 days) between HEC16 and HEC16/S6, but the
difference in water release (slower evaporation) due to sorbitol presence led to an evident
preference on hec16/s6/Tb8 preparation in the second 15 days period (T/C=7.18 vs T/C=3.8). In the
late part of the test, oviposition presence also confirmed an extended duration of the device
containing the gel and its possible use into a low-cost cardboard trap. Comparison between these
two different trap setups is confirmed as gel-based traps perform better than standard Masonite
traps.
A B
100% 8
90% 7
80%
Normalized weight
6
70%
60% 5
T/C
50% 4
40%
3
30%
2
20%
10% 1
0% 0
0 10 20 30 0-15 15-30
Day Day
HEC16/S6/Tb8 HEC16 Masonite in water HEC16 HEC16/S6/Tb8
Figure 3.9 On-field Water evaporation analysis through weight evaluation (A) and oviposition (B) (Period=30
days, gels in cardboard trap vs standard Masonite ovitrap). T/C= eggs on sample (T) /eggs on control
(C). Environmental conditions were 23±1°C, 75% RH (data from Fondazione Osservatorio
Meterologico Milano Duomo). All the samples have pH=6.5 and salinity<1%.
Conclusions
The main output reported in Chapter 3 was the identification of the hydrogel preparation
HEC16/S6/Tb8 that was 2 up to 5 times more capturing than controls and standard substrate
33
(Masonite) in lab oviposition assay. HEC16/S6/Tb8 was 4-7 times more capturing in semi-field
oviposition assay than a standard ovitrap when applied in a low-cost cardboard trap. Furthermore,
the composition showed an activity extended to 30 days rather than 1 week (average active period
of standard ovitraps. A set of parameter ranges and the specific values for the best preparation have
been indicated and reported in Table 3.4. Substrate preference behaviour in tiger mosquitoes
generally confirmed the choice of those oviposition parameters selected for this preliminary
biomimetic substrate design and the possibility of controlling some of them by changing the
hydrogel’s features.
Table 3.4 A panel of range for oviposition parameters and substrate composition identified through tests.
Oviposition parameters
Water Yield
Turbidity Sorbitol Composition Viscosity
pH Salinity content stress Morphology
(McF) wt% wt% (Pas)
wt% (Pa)
Attractive 4.5÷ HEC8÷20/ 4.5÷18.3
<3% 0-10 0-10 >0%
ranges 7.5 SA16÷30 /11÷33.1
>50 Rough
Best 6.5
<1% 8 6 HEC16/S6/Tb8 80% 14.1
preparation ÷7.5
In fact, Sorbitol (S) and Masonite maceration addition to HEC16 to control water release rate and
obtain a specific turbidity (Tb) level, enhanced substrate lure. Sorbitol humectant action contributed
to extending substrate activity. Generally, this experimental section demonstrated that sodium
Alginate and Hydroxyethylcellulose physical hydrogels formulation with neutral pH and low salinity
showed no repellent effect on tiger mosquito (Aedes albopictus). Starting from SA12 and HEC1O, this
composition presented compatible yield stress values for the spread on sloped surfaces as oviposition
substrates as requested for ovitraps use.
Tiger mosquitoes showed a preference dependent on HEC and SA-based physical hydrogels
concentration, probably related to substrate texture and/or water release rate. Differently, insects
rejected chemically cross-linked hydrogels probably for the presence of chemical cross-linker and/or
NaOH, HCl or CaCl2 residuals, or an inadequate surface morphology could cut down oviposition.
Through this preliminary study, it was possible only to underline for some parameter (e.g. water
release) an active role in promoting or depress oviposition. Other and more specific studies and
experimental designs are needed to define a specific range of values.
In order to broaden the study, it is necessary to progress in the knowledge of breeding sites eventually
through the use of sensors dedicated to live-monitoring the effect of specific parameters changes
and oviposition behaviour.
The next step within this research for biomimetic lure and kill substrate preparation is to verify the
possibility of host living bioinsecticides. Alternatively, future studies can be carried out to obtain a
mechanical insecticide action (i.e. due only to the material without using active substances).
34
References
1. Barrera R, Mackay, A. J., & Amador, M. : Methods and apparatus for surveillance and control of
insect vectors . In: USPTO, vol. 923,774,1. US2012
2. Dibo M, Chiaravalloti-Neto F, Battigaglia M, Mondini A, Favaro E, Barbosa A, et al. Identification
of the best ovitrap installation sites for gravid Aedes (Stegomyia) aegypti in residences in
Mirassol, State of Sao Paulo, Brazil. Memórias do Instituto Oswaldo Cruz. 2005;100:339-43; doi:
10.1590/S0074-02762005000400001.
3. Sannino A, Demitri C, Madaghiele M. Biodegradable Cellulose-based Hydrogels: Design and
Applications. Materials. 2009;2 2; doi: 10.3390/ma2020353.
4. Chang C, Zhang L. Cellulose-based hydrogels: Present status and application prospects.
Carbohydrate Polymers. 2011;84 1:40-53; doi: https://doi.org/10.1016/j.carbpol.2010.12.023.
5. Demitri C, Del Sole R, Scalera F, Sannino A, Vasapollo G, Maffezzoli A, et al. Novel superabsorbent
cellulose-based hydrogels crosslinked with citric acid. Journal of Applied Polymer Science.
2008;110 4:2453-60; doi: https://doi.org/10.1002/app.28660.
6. Kulikowski R. Potential of Hydrogel Application for Plant Protection. Ecological Chemistry and
engineering. 2009;16 9:1191-8.
7. Sannino A, Madaghiele M, Demitri C, Scalera F, Esposito A, Esposito V, et al. Development and
characterization of cellulose-based hydrogels for use as dietary bulking agents. Journal of Applied
Polymer Science. 2010;115 3:1438-44; doi: https://doi.org/10.1002/app.30956.
9. Degefa T, Yewhalaw D, Zhou G, Lee M-C, Atieli H, Githeko AK, et al. Evaluation of the performance
of new sticky pots for outdoor resting malaria vector surveillance in western Kenya. Parasites &
Vectors. 2019;12 1:278; doi: 10.1186/s13071-019-3535-3. https://doi.org/10.1186/s13071-019-
3535-3.
10. Eiras AE, Costa LH, Batista-Pereira LG, Paixão KS, Batista EPA. Semi-field assessment of the Gravid
Aedes Trap (GAT) with the aim of controlling Aedes (Stegomyia) aegypti populations. PLOS ONE.
2021;16 4:e0250893; doi: 10.1371/journal.pone.0250893.
11. Facchinelli L, Valerio L, Pombi M, Reiter P, Costantini C, Della Torre A. Development of a novel
sticky trap for container-breeding mosquitoes and evaluation of its sampling properties to
monitor urban populations of Aedes albopictus. Med Vet Entomol. 2007;21 2:183-95; doi:
10.1111/j.1365-2915.2007.00680.x.
12. Johnson BJ, Ritchie SA, Fonseca DM. The State of the Art of Lethal Oviposition Trap-Based Mass
Interventions for Arboviral Control. Insects. 2017;8 1; doi: 10.3390/insects8010005.
13. Mackay AJ, Amador M, Barrera R. An improved autocidal gravid ovitrap for the control and
surveillance of Aedes aegypti. Parasites & Vectors. 2013;6 1:225; doi: 10.1186/1756-3305-6-225.
https://doi.org/10.1186/1756-3305-6-225.
35
14. Ritchie S, Long S, McCaffrey N, Key C, Lonergan G, Williams C. A Biodegradable Lethal Ovitrap for
Control of Container-Breeding Aedes. Journal of the American Mosquito Control Association.
2008;24:47-53; doi: 10.2987/5658.1.
15. Mwingira V, Mboera LEG, Dicke M, Takken W. Exploiting the chemical ecology of mosquito
oviposition behavior in mosquito surveillance and control: a review. J Vector Ecol. 2020;45 2:155-
79; doi: 10.1111/jvec.12387.
https://doi.org/10.1016/j.pt.2020.01.001.
albopictus an Efficient Bridge Vector for Zoonotic Arboviruses? Pathogens. 2020;9 4; doi:
10.3390/pathogens9040266.
18. Che Dom N, Azman M, Mokhtar M, Australia T. Development and oviposition preferences of field
collected Aedes albopictus based on different water characteristics. Malaysian Journal of
Fundamental and Applied Sciences. 2019;15; doi: 10.11113/mjfas.v15n2019.1127.
19. Bentley MD, Day JF. Chemical Ecology and Behavioral Aspects of Mosquito Oviposition. Annual
Review of Entomology. 1989;34 1:401-21; doi: 10.1146/annurev.en.34.010189.002153.
https://doi.org/10.1146/annurev.en.34.010189.002153.
20. Carrasco D, Lefèvre T, Moiroux N, Pennetier C, Chandre F, Cohuet A. Behavioural adaptations of
mosquito vectors to insecticide control. Current Opinion in Insect Science. 2019;34:48-54; doi:
https://doi.org/10.1016/j.cois.2019.03.005.
21. Gaburro J, Paradkar PN, Klein M, Bhatti A, Nahavandi S, Duchemin J-B. Dengue virus infection
changes Aedes aegypti oviposition olfactory preferences. Scientific Reports. 2018;8 1:13179; doi:
10.1038/s41598-018-31608-x. https://doi.org/10.1038/s41598-018-31608-x.
22. Reiskind MH, Zarrabi AA. Water Surface Area and Depth Determine Oviposition Choice in Aedes
albopictus (Diptera: Culicidae). Journal of Medical Entomology. 2012;49 1:71-6; doi:
10.1603/ME10270. https://doi.org/10.1603/ME10270.
23. Hawley WA. The biology of Aedes albopictus. J Am Mosq Control Assoc Suppl. 1988;1:1-39.
24. Alcalay Y, Tsurim I, Ovadia O. Female mosquitoes disperse further when they develop under
predation risk. Behavioral Ecology. 2018;29 6:1402-8; doi: 10.1093/beheco/ary113.
https://doi.org/10.1093/beheco/ary113.
25. Day JF. Mosquito Oviposition Behavior and Vector Control. Insects. 2016;7 4:65; doi:
10.3390/insects7040065. https://pubmed.ncbi.nlm.nih.gov/27869724.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198213/.
26. Jinguji H, Fujiwara Y, Ohtsu K, Morimoto M. Effects of Sodium Chloride on Oviposition Behavior
of Aedes albopictus. Journal of the American Mosquito Control Association. 2021;36 4:253-6;
doi: 10.2987/20-6962.1. https://doi.org/10.2987/20-6962.1.
27. Madzlan F, Dom NC, Tiong CS, Zakaria N. Breeding Characteristics of Aedes Mosquitoes in Dengue
Risk Area. Procedia - Social and Behavioral Sciences. 2016;234:164-72; doi:
https://doi.org/10.1016/j.sbspro.2016.10.231.
environment. 2019.
36
29. Reiter P, Amador MA, Colon N. Enhancement of the CDC ovitrap with hay infusions for daily
monitoring of Aedes aegypti populations. J Am Mosq Control Assoc. 1991;7 1:52-5.
30. Thavara U, Tawatsin A, Chompoosri J. Evaluation of attractants and egg-laying substrate
preference for oviposition by Aedes albopictus (Diptera: Culicidae). Journal of vector ecology :
journal of the Society for Vector Ecology. 2004;29:66-72.
31. Madeira N, Macharelli CA, Carvalho L. Variation of the Oviposition Preferences of Aedes aegypti
in Function of Substratum and Humidity. Memórias do Instituto Oswaldo Cruz. 2002;97:415-20;
doi: 10.1590/S0074-02762002000300025.
32. Okal MN, Francis B, Herrera-Varela M, Fillinger U, Lindsay SW. Water vapour is a pre-oviposition
attractant for the malaria vector Anopheles gambiae sensu stricto. Malaria Journal. 2013;12
1:365; doi: 10.1186/1475-2875-12-365. https://doi.org/10.1186/1475-2875-12-365.
33. Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, et al. Biodegradable
Polymer Scaffolds for Tissue Engineering. Bio/Technology. 1994;12 7:689-93; doi:
10.1038/nbt0794-689. https://doi.org/10.1038/nbt0794-689.
34. Grayson WL, Martens TP, Eng GM, Radisic M, Vunjak-Novakovic G. Biomimetic approach to tissue
engineering. Seminars in Cell & Developmental Biology. 2009;20 6:665-73; doi:
https://doi.org/10.1016/j.semcdb.2008.12.008.
35. Langer R, Vacanti JP. Tissue engineering. Science. 1993;260 5110:920; doi:
10.1126/science.8493529. http://science.sciencemag.org/content/260/5110/920.abstract.
36. Kono H. Characterization and properties of carboxymethyl cellulose hydrogels crosslinked by
polyethylene glycol. Carbohydrate Polymers. 2014;106:84–93; doi:
10.1016/j.carbpol.2014.02.020.
37. Kono H. Carboxymethyl Cellulose-Based Hydrogels. 2015. p. 243-58.
38. Ghorpade VS. Chapter 4 - Preparation of hydrogels based on natural polymers via chemical
reaction and cross-Linking. In: Chen Y, editor. Hydrogels Based on Natural Polymers: Elsevier;
2020. p. 91-118.
39. Shalapy A, Zhao S, Zhang C, Li Y, Geng H, Ullah S, et al. Adsorption of Deoxynivalenol (DON) from
Corn Steep Liquor (CSL) by the Microsphere Adsorbent SA/CMC Loaded with Calcium. Toxins.
2020;12 4; doi: 10.3390/toxins12040208.
40. Yoshida H, Hatakeyama T, Hatakeyama H. Characterization of water in polysaccharide hydrogels
by DSC. Journal of Thermal Analysis. 1993;40 2:483-9; doi: 10.1007/BF02546617.
https://doi.org/10.1007/BF02546617.
41. Fellows P. Food processing technology: Principles and practice: Third edition. Food Processing
Technology: Principles and Practice: Third Edition. 2009:1-913.
42. Yanniotis S, Skaltsi S, Karaburnioti S. Effect of moisture content on the viscosity of honey at
different temperatures. Journal of Food Engineering - J FOOD ENG. 2006;72:372-7; doi:
10.1016/j.jfoodeng.2004.12.017.
43. Falsone L, Brianti E, Severini F, Giannetto S, Romi R. Oviposition substrate in Asian tiger mosquito
surveillance: Do the sizes matter? Journal of Vector Ecology. 2015;40 2:256-61; doi:
https://doi.org/10.1111/jvec.12162. https://doi.org/10.1111/jvec.12162.
44. Gopalakrishnan R, Das M, Baruah I, Veer V, Dutta P. Physicochemical characteristics of habitats
in relation to the density of container-breeding mosquitoes in Asom, India. Journal of vector
borne diseases. 2013;50:215-9.
37
45. Gaudin S, Lourdin D, Le Botlan D, Ilari JL, Colonna P. Plasticisation and Mobility in Starch-Sorbitol
Films. Journal of Cereal Science. 1999;29 3:273-84; doi: https://doi.org/10.1006/jcrs.1999.0236.
46. Bakry N, Mohamad Isa MIN, Sarbon N. Effect of sorbitol at different concentrations on the
functional properties of gelatin/carboxymethyl cellulose (CMC)/chitosan composite films.
International Food Research Journal. 2017;24:1753-62.
47. Citerne G, Carreau P, Moan M. Rheological properties of peanut butter. Rheologica Acta.
2001;40:86-96; doi: 10.1007/s003970000120.
38
Chapter 4
Growth and effectiveness on Aedes albopictus eggs of Beauveria bassiana added to

biomimetic hydrogel oviposition substrate for ovitrap use.
 Beauveria bassiana (Bb)was embedded into some oviposition-attractive hydrogel

compositions to form the lure and kill Bb/Gel matrix.
 The inclusion of Beauveria bassiana (Bb) is the last step for preparing the biomimetic lure and
kill substrate.
 It was tested Bb survival and growth when embedded inside different hydrogel attractive
preparations and Bb/Gel matrix efficacy on tiger mosquito eggs.
 All Gel-like media tested sustained for 24 days Bb growth and eggs mortality compared to
liquid controls, probably due to matrix texture.
 About 90% of mortality was reached after 10 days of egg-hydrogel contact by using 2-
Hydroxyethylcellulose (HEC) Bb/Gel matrix composition.
 Hydrogel controls showed a lethal effect comparable to Bb/Gel matrix also without
mechanical trapping.
Abbreviations: Beauveria bassiana, Bb; lethal ovitrap, LOT; Potato Dextrose Agar PDA; Conidial
Solution, CS; Chitosan, Cs; 2-Hydroxyethylcellulose, HEC; Sodium alginate, SA; Vegetative growth
assay, VG; concentration in weight, wt; Standard error of the mean, Sem; European Paralament, EP.
39
4.2 Introduction
Besides mechanical trapping (ovitraps), biological control through bioinsecticides is one of the most
explored control strategies evaluated as an alternative to pesticides [1]. There are different examples
of bioinsecticides such as microbial larvicides (e.g. Bacillus thuringiensis var. Israelensis or Wolbachia
[2]), natural essential oils (e.g. Mentha pulegium or Ruta chalepensis [3-5]) and entomopathogenic
fungi (Metarhizium anisopliae or Beauveria bassiana, Bb) [6-8].
In particular, the use of entomopathogenic fungi spores (conidia) to manage insect pests
(mycoinsecticides) has been steadily gaining popularity. They are employed as bioinsecticide against
many pests in dry or liquid (aqueous or oily) formulations or after encapsulation in granules, pellets
etc destined mainly for spraying applications or the release through baits or other precision delivery
devices [9]. They are active against Coleoptera Hymenoptera Lepidoptera Homoptera Diptera [10].
In particular, Beauveria bassiana is lethal to many mosquito species such as C. tarsalis, C. pipiens, A.
aegypti, A. sierrensis, A. nigromaculis, and A. Albimanus [11] but also Aedes albopictus and Anopheles.
[12, 13].
Fungi are safe for human and animal health and act as a parasite on different arthropods and
mosquitoes species [11-15], infecting and then killing them in a period long enough to allow the
autodissemination process [16].
Consequently, over the last 50 years, advances in mass production have occurred in response to the
increasing popularity, and more and more fungi are commercialized worldwide [9]. Currently, Bb
based productive steps involve conidia mass-produced through conidia solid substrate fermentation
(e.g. on rice), drying and separation from the substrate, reprocessing to produce commercial forms
and final dehydration for storage [9-17].
Conidial formulations might be a valid alternative to chemical pesticides because of their comparable
lethality with lower toxicity (to humans and the environment) and lower risk of resistance
phenomena in insects [18, 19]. However, they present some weakness that limits their employment
as a replacement of pesticides having superior effectiveness, low production cost and easiness of
application [20-23]. For example, Bb survival after field application is strongly affected by
environmental factors such as low survival to heat, UV radiation, substrate composition and drying
[24] (See Section A.3). Furthermore, Bb use is still limited due to the short storage period that does
not reach the minimum storage period (two years in WHO/FAO regulations) without loss in efficacy
unless drug-like costly storage conditions are used (<5°C and controlled humidity).
Finally, focusing on its use against tiger mosquitoes, Bb presents the same delivery of synthetic
pesticides when sprayed due to Aedes albopictus behaviour [25] (e.g. difficulty to reach mosquitoes
shaded resting places etc.).
Consequently, new Bb application methods such as low viscosity water or oil-polymer solutions,
granules, pellets, floating formulations, or feeding systems [26] have been developed to improve field
performance. Bb encapsulation was also studied in sprayable biopolymers matrices such as alginate-
chitin-kaolin- wheat bran microcapsules [27]. However, Bb has never been used as an active
ingredient inside a biomaterial-based bulk substrate such as a biomimetic substrate for lethal ovitrap.
Biomaterial-based hydrogels, known as biocompatible materials from Tissue engineering (TE), are the
perfect candidate to prevent any possible toxicity for the fungus [28] and to provide a
40
microenvironment suitable for its growth. Beauveria will be embedded into the biocompatible
hydrogels substrate already optimized for oviposition, particularly to the most promising
formulations, to obtain a Beauveria bassiana/Gel matrix (Bb/Gel). The production of a Bb/Gel is
possible because the values of the oviposition-related parameter effective ranges (e.g. pH, Salinity,
Composition, water content wt%, substrate texture, Surface morphology etc) presents an
overlapping to the conditions for Bb productions [29, 30] as reported in Table 4.1.
Table 4.1 Overlapping of key parameters and substrate composition for the growth of Beauveria bassiana and
oviposition activity.
Parameters
Water content Substrate Surface
pH Salinity % Composition
wt% texture morphology
Not
Oviposition Natural
4-7 <3% >0% Soft to solid smooth/mm
lure [17] susbtrates
pores
Natural
Solid
Bb growth susbtrates
5-7 <3% 20-80 wt% substrate -
[9] rich in sugars
(fermentation)
(e.g. cereals)
The biomimetic approach can improve the employment of bioinsecticides in biological control. The
system could improve at the same time, conidial field survival and improve delivery against tiger
mosquitoes exploiting the attractivity of the oviposition substrate.
The aim of this research section in the development of the biomimetic lure and kill substrate is to
evaluate the biocompatibility and the effectiveness of Beauveria bassiana.
Consequently, a biodegradable and biocompatible hydrogel with proven oviposition attraction
capability for tiger mosquitoes was used to encapsulate Bb conidia and hyphae. This section of the
study aimed to i) prepare a Bb/Gel system with different Bb suspension composition and natural
polymer concentrations; ii) evaluate the survival to the embedding process and the most suitable
hydrogel composition to sustain the growth of Bb for 24 days (as long as the duration of a long-lasting
cardboard trap employing hydrogel substrate already tested in chapter 3), evaluating; iii) assess the
efficacy of the Bb/Gel systems against Aedes albopictus eggs and larvae and iv) evaluate the most
effective Bb/gel system and contact time, studying the relationship between Bb viability and its
efficacy against Aedes albopictus.
4.3 Materials and methods
4.3.1 Beauveria bassiana origin and infection suspension preparation

An autochthonous strain of Beauveria bassiana, Bb (CD1123) was obtained from naturally infected
ticks locally collected in a private dog shelter in Putignano, (Bari, Italy). The strain was morphologically
and molecularly identified as described in Cafarchia et al. [14], then maintained on Potato Dextrose
Agar (PDA, Liofilchem) at 4°C. Two Bb conidial and hyphae infection suspensions (CS1 and CS2) were
41
obtained by culturing 15 strains on PDA for 3 weeks at 25±1°C. Plates were washed with 10 ml of
sterile distilled water containing 0.1% v/v Tween 80 (Fluka) (CS1) or with 10 ml of sterile 1%wt
peptone water (Pep, Sigma-Aldrich) containing 0.1% v/v Tween 80, and 2%wt chitosan powder (CS2).
A total of 400ml of CS1 and CS2 were prepared, vortexed and spectrophotometrically adjusted for
turbidity (Biosan DEN 1) to reach an optical density of 7 McFarland. The number of conidia and
hyphae for both suspensions was evaluated by quantitative plate counts of colony-forming unit/ml
(CFU/ml) on PDA plates (i.e., 1–7 × 106 conidia/ml).
4.3.2 Preparation of Beauveria bassiana/Gel systems

Two different natural polymer powders were used for gel preparation: 2-Hydroxyethylcellulose (HEC,
Aldrich Chemistry, average Mw 720000) and Sodium alginate (SA, SAFC, average Mw 120000). HEC
powder was added to CS1 and CS2 solutions and stirred at 1500-2000 rpm at room temperature for
about 30 min. Two different concentrations were arranged: 16%wt (HEC16) and 30%wt (HEC30). SA
was prepared at 16%wt (SA16) using the same solutions (CS1 and CS2) and protocol as above. The
following Bb/Gel systems were obtained: CS1-HEC16, CS1-SA16, CS1-HEC30 and CS2-HEC16, CS2-
SA16, CS2-HEC30. Control gels without Bb were also prepared using the same polymer powders (HEC
and SA) solubilized in sterile distilled water to obtain the concentration of 16%wt and 30%wt as above
(i.e., HEC16, SA16, HEC30). All Gel systems were used for the survival and growth test of Bb (bioassay
1), and their efficacy was assessed against Aedes albopictus eggs through a hatching test. The tests
were performed using the most simple hydrogel formulation resulted attractive in oviposition and
not HEC16/S6/Tb8 (the best one). However, all the components in HEC16/S6/Tb8 formulation are
natural, non-toxic and already employed in solid substrate fermentation of Bb.
4.3.3 Ades albopictus eggs

For hatching assay, Aedes albopictus eggs were obtained from an insectary colony of the Department
of Public Health and Infectious Diseases of Sapienza University (Rome). Colony breeding conditions
were: T 25±1°C, RH 70%, light/dark hours ratio 14:10. The eggs were superficially sterilized by putting
them under laminar flow hood using a UV lamp at 500 W for 10 min. The absence of fungi on the
eggs surface was verified by culturing 10 eggs on PDA for 6 days at 25°C. Then, the vitality of eggs
was assessed by performing a hatching test on a random batch of 100 eggs placed in 200 ml of sterile
distilled water. The above protocol was repeated for every batch of tested eggs.
4.3.4 Bioassay 1: Evaluation of Beauveria bassiana viability

The viability of Bb in gel systems was assessed by evaluating the growth, the conidia viability and
vegetative growth.
Beauveria bassiana growth evaluation assay
Beauveria bassiana survival and growth rate in Bb/Gel systems were assessed by evaluating the
number of conidia through quantitative plate counts of CFU/ml on PDA plates. Briefly, the bottles
containing the gel system with and without Bb were placed for 24 days at controlled conditions (i.e.,
42
25±1°C and 90% RH) inside a dark Binder Climatic Chamber and monitored every 4 days. CS1 and CS2
were used as positive controls and No Bb gel (i.e., HEC16, SA16, HEC30 and SA30) as negative control
to exclude contaminations. The experiment was repeated three times, and the number of conidia
was expressed as mean values of Log10 of CFU/ml. Conidia concentration values normalized respect
to day 0 with standard error of the mean (Sem), were expressed as growth curves.
Beauveria bassiana conidial viability assay
Conidia viability assay was performed only on 24 days old CS-2 samples (CS2-HEC16, CS2-SA16 and
CS2-HEC30, that were the best performing compositions in growth assay) to quantify the number of
conidia acting on eggs in the hatching test. On the 24th day of incubation, 0.1 g of gel systems were
harvested from each tested sample then diluted into 9.9ml of sterile distilled water. Solutions were
vortexed, left to settle for 15 minutes, and then supernatant removed.
The remaining suspension was filtered with sterile 8µm Watman filters and then centrifuged
(3000rpm × 5 min), washed twice in 1 ml of phosphate-buffered saline solution (PBS) and re-
suspended in 1 ml of PBS. Finally, conidia number was determined by quantitative PDA plate count
of colony-forming units (CFU)/ml after 4 days incubation at 25±1◦C and 90% RH. The conidia vitality
was expressed as a percentage ratio between active conidia and conidial number at day 24 (known
from Bb growth evaluation assay). All the experiments were performed in duplicate and repeated
three times on different days.
Beauveria bassiana vegetative growth assay
The vegetative growth of Bb was evaluated in the gel composition showing the best results in the
conidial growth. CS2-HEC (16%wt and 30%wt) and CS2-SA (16%wt). CS2-HEC16, CS2-SA16 and CS2-
HEC30 were arranged through the same mixing protocol already described and placed in the 80mm
Petri dish.
A Bb mycelial plug (i.e., 10 mm in diameter) was placed upside down cultured onto the centre of each
80 mm Petri dish containing the gel preparations. Bb colonies diameters after 10 days incubation at
controlled conditions (25±1◦C, RH 80%) were measured. All the experiments were performed in
triplicate and vegetative growth was expressed as mycelia colony diameter percentage compared to
initial diameter ± Sem.
4.3.5 Bioassay 2: Effect of Beauveria bassiana/Gel systems on Aedes albopictus eggs

Bb/Gel systems efficacy on Aedes albopictus eggs and larvae after 24 days growth was evaluated
through a hatching test. Systems action mechanisms on eggs and larvae were also examined through
morphological analysis and gel viscosity measurement.
Eggs hatching test
2g (or 2ml for liquids) of each gel containing Bb or liquid and gel controls (Table 4.1) previously
incubated for 24 days at 25±1°C and 90% RH were harvested and uniformly spread on sterile paper
(20x20x1 mm, Whatman N. 1, Labor 67 g/m2, Tecnochimica Moderna). Papers were placed inside a
40mm Petri dish, and 30 Aedes albopictus eggs (5 days old, previously described) were laid on each
paper with the help of a sterile feather. These samples were incubated in a Binder Climatic Chamber
43
at 25±1°C and 90% RH for different contact periods (5, 10 and 15 days). Sterile distilled water was
used as positive control. After the incubation period, papers were removed from Petri dishes and
placed into sterile plastic containers (hatching containers) filled with 80 ml of sterile distilled water.
Hatching containers were stored in an air-conditioned room at 25°C and 80% RH (monitored and
recorded by a data logger) for 5days, and the hatching was evaluated by counting the number of
larvae hatched. The experiment was repeated three times and the number of hatching eggs were
averaged. The corrected mortality rates were calculated using Schneider-Orelli’s formula using
distilled water as reference [i.e. Corrected mortality% (CM%)= (Mortality% in Bb/Gel−Mortality% in
Distilled water control)/(100−Mortality% in Distilled water control) × 100] where Mortality%= (1-
Living larvae/Tested eggs)x100) [38]± Standard Error of the Mean (Sem).
Table 4.2 Samples tested in hatching test for 5, 10, 15 days of contact. Chitosan (Cs) 2%wt, Peptone water,
were further controls. Distilled water was the reference control for CM% evaluation.
CS1 and CS2 Bb Control Gel Control No Bb Liquid control
1%
Distilled Chitosan2%wt
HEC16 SA16 HEC30 CS1 CS2 SA16 HEC30 SA30 Peptone
water (Cs)
water
4.3.6 Substrate characterization

Morphological analysis
Bb/Gel samples and eggs were observed with an optical microscope (Greenough Leica S) and
scanning electronic microscope (SEM EVO® 40, Carl Zeiss AG). Samples were observed hydrated at
optical microscope and SEM after dehydrated for 5 hours at 50°C. The presence of fungal structures
and the Bb/Gel systems effect on eggs and larvae were evaluated.
Gel viscosity measurement
The viscosity of a fluid is a measure of its resistance to deformation at a given rate (e.g. syrup has a
higher viscosity than water), and its measurement is a way to characterize a hydrogel. Analysis was
performed through the viscosity comparison between 24 days old CS2 samples (CS2-HEC16 and CS2-
SA16) and same age no Bb gel control (HEC16 and SA16, which represent the viscosity reference).
After 24 days at 25±1 °C and 90% RH, both CS2 gel samples and gel controls were harvested from
bottles and viscosity was measured with a single shear rate test with a Malvern Kinexus Pro rotational
rheometer in a plate-plate configuration (50s-1 of shear rate, 30°C and atmospheric pressure). The
test was performed to evaluate the relevance of the mechanical trapping effect of the gel on Aedes
albopictus eggs and larvae.
44
4.4 Results
4.4.1 Bioassay 1: Beauveria bassiana growth evaluation in Bb/gel system

Growth of Bb in all gel samples and controls was reported in Figure 4.1 as normalised conidial
concentration (NCC). No growth of Bb was found in negative Gel control (i.e. HEC16, HEC 30, SA16
and SA30), confirming no contaminations. All Bb/Gel systems presented higher growth values than
those registered in CS1 and CS2 Bb controls (Figure 4.1 and 4.2). Conidial concentration decreased in
all samples and controls within 4 days. On day 8, an increasing trend was observed in Gel samples
(+41% in CS1-HEC16, +112% in CS2-HEC30). Differently, liquid controls CS2-Bb and CS1-Bb decreased
to 0 within day 16.
The highest Bb concentration at 24 days were always registered in gels prepared with CS2. In
particular, CS2-HEC30 at day 24 was 1 order of magnitude higher than starting initial concentration
and about 2 than liquid controls evaluated on normalised values at day 24 [NCC =(day 24normalized
Bb/Gel systems CFU/ml values at) / (day 24 Normalized CFU/ml values at in Bb control)] ± Sem (Figure
4.2).
CS2-HEC16 CS2-SA16 CS2-HEC30 CS1-HEC16

CS1-SA16 CS1-HEC30 CS2-Bb Control CS1-Bb Control
3.50
2.50
NCC at day 0 CFU/ml
1.50
.50
0 4 8 12 16 20 24
-.50
Incubation Time (Days)
Figure 4.1 Bb growth for CS1 and CS2 preparations with and without polymer. Results expressed as normalized
(respect to day 0 concentration values, starting conidia concentration 1–7 × 106 conidia/ml)
expressed as conidia number values ± Sem.
45
1.E+03
Normalized concentartion at day 24 CFU/ml
1.E+02
1.E+01
1.E+00
Figure 4.2 Comparison between conidial concentration in Bb/Gel vs liquid control CS1-Bb and CS2-Bb.
Results expressed as [(day 24normalized Bb/Gel systems CFU/ml values at) / (day 24 Normalized
CFU/ml values at in Bb control)].
4.4.2 Conidia viability in Beauveria bassiana/Gel systems and vegetative growth assay
Conidia viability assay and vegetative growth were performed on Bb/Gel systems CS2-HEC16, CS2-
SA16, and CS2-HEC30 (best performing in growth analysis). Conidia viability (Figure4.3 A) was
measured by evaluating the ratio between active conidia (able to sporulate) and total conidia
measured through plate count.
100% 2.5
A B
Final Mycelium Diameter (cm)
%Active conidia/Total conidia
80% 2.0
60% 1.5
40% 1.0
20% 0.5
0% 0.0
CS2-HEC16 CS2-HEC30 CS2-SA16
Active conidia/Total conidia Initial diameter
Figure 4.3 Conidia viability in Bb/Gel systems and vegetative growth assay. (A) Active conidia/Total Conidia %
± SEM, (B) Final mycelium colony diameters, Diameter (cm) ± SEM (Initial diameter=1cm) in CS2-
HEC16, CS2-SA16 and CS2-HEC30 (24 days old) after 4 days at 25°C, 90%RH.
46
Vegetative growth (Figure4.3 B) was evaluated through the measurement of the mycelium diameter.
CS2-HEC16 and CS2-HEC30 presented higher active conidia values than CS2-SA16. A higher mycelium
development percentage was observed in CS2-HEC16 (100%) than CS2-SA16 (30%) and CS2-HEC30
(13.3%).
4.4.3 Bioassay 2: Beauveria bassiana/Gel effect on Aedes albopictus eggs hatching

Hatching assay was performed by placing eggs for 5, 10 or 15 days on 24 days old Bb/Gel substrates
and controls then placing them in distilled water for 5 days. Corrected mortality percentages CM% in
Bb/gel systems and CS1 and CS2 liquid controls was measured by evaluating hatching (Figure 4.4).
Corrected mortality in all CS1 and CS2 made Bb/gel systems was higher than respective Bb liquid
controls (CS1 and CS2). HEC based Bb/Gel systems (both CS1 and CS2 made) at any concentration
showed higher CM% than SA based Bb/gel systems. The highest CM% values was registered after 10
contact days in CS2-HEC16 (88%). Generally, 10 days is the most effective contact time.
100% 1.0E+07
90% 9.0E+06
80% 8.0E+06
70% 7.0E+06
60% 6.0E+06
CFU/ml
CM%
50% 5.0E+06
40% 4.0E+06
30% 3.0E+06
20% 2.0E+06
10% 1.0E+06
0% 0.0E+00
5 Days 10 Days 15 Days CFU/ml at 24 Days
Figure 4.4 Hatching test after 5, 10 and 15 contact days (primary axis) and (secondary axis) CFU/ml of conidia
at contact (24th day of growth). Results expressed as corrected mortality % ± Sem.
Surprisingly CM% at 15 days was lower than those registered after 10 days regardless the gel
composition. Very high mortality levels were registered in No Bb gel controls (ranging from 77.5% in
No Bb el control SA16 to 94% in SA30).
4.4.4 Morphological assays on Beauveria bassiana/Gel systems

SEM micrograph (Figure 4.5 A) showed the emergence of fungal structures (blue arrows) from an egg
(green bracket) after 10 days of contact in CS2-HEC16. Mycelium and gel material trapping action on
47
eggs was also observed by using optical microscope (respectively in Figure 4.5 B and Figure 4.5 C-D,
trapped larvae in gel just after the hatching from an egg).
Figure 4.5 Morphological analysis on of sample CS2-HEC16 (10 days contact time): (A) SEM micrographs (scale
bar 100 µm) egg (green bracket) with fungal system (blue arrows). (B) Optical microscope
observations: mycelium on egg (green arrow, scale bar: 1 mm). (C, D) Optical microscope analysis
(scale bar 500 µm): dead larvae trapped (red arrows) inside the gel and eggs (green arrows).
4.4.5 Gel viscosity measurement

Active conidia analysis and vegetative growth results do not explain higher mortality in No Bb Control
Gels than Bb/Gel system. Therefore, due to the absence of Bb, results must be associated to amaterial
mechanical trapping action of the. Consequently, a viscosity test was performed to evaluate its
possible role in trapping action. The viscosity of 24 days of growth CS2 Gel samples (CS2-HEC 16 and
CS2-SA16) (was compared to same-age No Bb Gel controls HEC16 and SA16) to evaluate viscosity
values. From the test (Figure 4.6) emerged a drop in viscosity values in Bb/Gel systems compared to
No Bb controls. CS2-SA16 samples revealed a higher reduction (-72%) in viscosity than No Bb gel
SA16, whereas reduction in CS2-HEC16 was -25% compared to HEC16-No Bb Gel control.
48
20
18
16
14
Shear viscosity (Pas)
12
10
0
SA16 HEC16
NoBb Gel Control CS2 Gel samples
Figure 4.6 Shear viscosity ± SEM of Bb/Gel CS2-HEC16 and CS2-SA16 and respective No Bb Gel control after
24 days (25±1 °C at 90% RH).
4.5 Discussion
Fungal survived the hydrogel preparation process and grew inside the gel, suggesting the
biocompatibility between gel preparation and Bb. In particular, hydrogel microenvironment
conditions, components effects, processing conditions and material properties, pH, salinity, water
presence (already tested in oviposition) do not affect Bb survival negatively.
On the contrary, all hydrogel substrates promoted Bb growth compared to liquid media CS1 and CS2
(about 1 order of magnitude higher, i.e. 107CFU/ml concentration). Nutrients presence (CS1 or CS2)
and type of polymer (2-Hydroxyethylcellulose or Sodium Alginate) and their concentration (16%wt or
30%wt) affected Bb conidial production and vegetative growth. Differences in results are probably
due to the difference in texture between gels and liquid media and different gel's concentrations.
Soil's intrinsic bulk density can affect the fungal growth (and living tissue) and activity [31], as well as
reported in cell-tissue interaction in the medical field [32]. Consequently, Bb/Gel system
concentrations and polymer type related properties (such as bulk density, viscosity, stiffness or water
or oxygen content etc.) could have influenced and fostered the behaviour of Bb.
CS2-HEC16 is the best Bb/Gel system emerged as the most suitable composition in fostering both the
vegetative growth and conidia production
All 24 days old Bb/Gel systems showed a higher lethality compared to the reference controls. Polymer
type and concentration, together with the contact time, affected mortality. In particular, it emerged
that CS2-HEC16 /10 days are the most effective combination to prevent hatching (CM=88%). A
positive aspect is that 10 days is compatible with the life cycle of Aedes albopictus, then eggs infection
can occur before hatching.
49
CS2-HEC16 and No Bb Gels showed comparable mortality percentages at 10 days, respectively 88%
and 90% in No Bb-HEC16. In addition, no relation between active conidia and mortality levels was
found (see Figure4.3 A and 4.4). Consequently, it is possible to hypothesize that effectiveness could
not be linked only to the Bb conidia viability but also to other action mechanisms.
SEM and optical microscope observations seem to support the hypothesis of a combination of Bb
pathogenicity (fungal structures on eggs) and gel's mechanical trapping (e.g. trapped larvae inside
the matrix) related to polymer viscosity. Trapping action might be related to material viscosity (how
much the egg can sink inside the matrix). In fact, HEC16 has higher mortality than, although having
comparable active conidia. Shear viscosity comparison between 24 days old systems (CS2-HEC16 and
CS2-SA16) and respective No Bb Gel control showed how Bb/Gel systems (lower lethality) were less
viscous than No Bb Gel during the hatching test. In addition, the higher difference in viscosity was
registered between CS2-SA16 and SA16- No Bb that also presented a significant difference in
mortality (respectively CM=94% vs 30% at 10 days of contact). In a frame of cooperating active
systems, it is possible to suppose that the fungal action could have non balanced the reduction in
viscosity in CS2-SA16 gel, leading to a drastic reduction in eggs mortality differently from CS2-HEC16,
which presented a higher conidial level and a lower viscosity reduction during at hatching test.
Although further analysis is needed to confirm this, it is also possible that fungal growth might have
affected the viscosity by degrading polymer structure as already reported for natural rubber [31, 33].
In fact, viscosity drop could not be related to polymer ageing because the comparison was among
same age gels.
Conclusions
The research section aimed to evaluate the survival, growth, and effectiveness of Beauveria bassiana
into a natural hydrogel-based substrate as a fundamental step for producing a biomimetic lure and
killing oviposition substrate. Assays proved that Bb survived hydrogel encapsulation and indicated an
influence of hydrogel composition and polymer concentration on fungal growth and viability. All
hydrogel substrates promoted Bb growth compared to liquid media CS1 and CS2 (about one order of
magnitude higher CFU/ml concentration). CS2-HEC16 (Hydroxyethylcellulose-based gel enriched
with peptone as nutrients) performed better than liquid controls and other gels to sustain and
promote growth for 24 days.
Surprisingly, the most lethal substrate was without Bb (SA16-No Bb, 94%), and CS2-HEC16 /10 days
are the most effective combination to prevent hatching (CM=88%). Consequently, different lethal
action mechanisms emerged, and in possible to suppose an essential role for mechanical trapping
action that might be viscosity related and managed through material concentration variation.
In conclusion, by adding bioinsecticides inside a hydrogel in a trapping device, the efficiency and
possibilities of using the trapping approach can be increased, but it would also benefit from using
lower starting fungal concentrations. The effectiveness of the substrate without an active substance
and the role of viscosity is a highly positive unexpected result. The absence of a biocide could lead to
economic advantage in reducing production costs and biocide registration. Furthermore, the absence
of toxic substances enlarges the application scenario giving a commercial advantage. On the other
50
hand, the presence of the biopesticide allows the autodissemination approach, but its concentration
has to be adequately evaluated to avoid an excessive reduction in mechanical trapping action.
Further studies will focus on verify oviposition on Bb/Gel systems and their effect on adult
mosquitoes and to verify the biocompatibility between the most capturing composition
HEC16/S6/Tb8 and the bioinsecticide. However, all the components contained in this formulation are
natural, non-toxic and already employed in solid substrate fermentation of Bb.
51
References
11:1471-85; doi: https://doi.org/10.1002/ps.4044. https://doi.org/10.1002/ps.4044.
2. Jiggins FM. The spread of Wolbachia through mosquito populations. PLOS Biology. 2017;15
6:e2002780. https://doi.org/10.1371/journal.pbio.2002780.
https://doi.org/10.1016/j.pt.2020.01.001.
4. M. Domingues P, Santos L. Essential oil of pennyroyal (Mentha pulegium): Composition and
applications as alternatives to pesticides—New tendencies. 2019;139:111534.
5. Immediato D, Figueredo LA, Iatta R, Camarda A, de Luna RLN, Giangaspero A, et al. Essential oils
and Beauveria bassiana against Dermanyssus gallinae (Acari: Dermanyssidae): Towards new
natural acaricides. Veterinary Parasitology. 2016;229:159-65; doi:
https://doi.org/10.1016/j.vetpar.2016.10.018.
6. Gopalakrishnan R, Das M, Baruah I, Veer V, Dutta P. Physicochemical characteristics of habitats
in relation to the density of container-breeding mosquitoes in Asom, India. Journal of vector
borne diseases. 2013;50:215-9.
7. Hans von H, Norbert B. Cost-benefit analysis of mosquito control operations based on microbial
control agents in the upper Rhine valley (Germany). European Mosquito Bulletin. 2009;27.
8. Blanford S, Shi W, Christian R, Marden JH, Koekemoer LL, Brooke BD, et al. Lethal and Pre-Lethal
Effects of a Fungal Biopesticide Contribute to Substantial and Rapid Control of Malaria Vectors.
PLOS ONE. 2011;6 8:e23591; doi: 10.1371/journal.pone.0023591.
Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI, editors. Mass Production of Beneficial Organisms.
San Diego: Academic Press; 2014. p. 357-413.
10. Keswani C, Singh S, Singh H. Beauveria bassiana: Status, Mode of action, Applications and Safety
issues. 2013;3:16.
11. Clark TB, Kellen WR, Fukuda T, Lindegren JE. Field and laboratory studies on the pathogenicity of
the fungus Beauveria bassiana to three genera of mosquitoes. Journal of Invertebrate Pathology.
1968;11 1:1-7; doi: https://doi.org/10.1016/0022-2011(68)90047-5.
12. Lee J, Woo R, Choi C, Shin T, Gwak Ws, Woo S. Beauveria bassiana for the simultaneous control
of Aedes albopictus and Culex pipiens mosquito adults shows high conidia persistence and
productivity. 2019;9.
13. Lina BF-W, Alejandro PR, Robert WB, Ephantus JM. Characterization of Tolypocladium
cylindrosporum (Hypocreales: Ophiocordycipitaceae) and Its Impact Against Aedes
aegypti and Aedes albopictus Eggs at Low Temperature. Journal of the American
Mosquito Control Association. 2017;33 3:184-92. https://doi.org/10.2987/16-6596R.1.
52
14. Cafarchia C, Immediato D, Iatta R, Ramos RAN, Lia RP, Porretta D, et al. Native strains of Beauveria
bassiana for the control of Rhipicephalus sanguineus sensu lato. 2015;8 1:80.
https://doi.org/10.1186/s13071-015-0693-9.
albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain. Journal of Asia-
Pacific Entomology. 2015;18 2:217-21; doi: https://doi.org/10.1016/j.aspen.2015.02.003.
16. Immediato D, Camarda A, Iatta R, Puttilli MR, Ramos RAN, Di Paola G, et al. Laboratory evaluation
of a native strain of Beauveria bassiana for controlling Dermanyssus gallinae (De Geer, 1778)
(Acari: Dermanyssidae). 2015;212 3:478-82.
17. Ying S-h, Feng M-G. Medium components and culture conditions affect the thermotolerance of
aerial conidia of fungal biocontrol agent Beauveria bassiana. 2006;43:331-5.
of a candidate fungal biopesticide for use against adult malaria vectors. Malaria Journal. 2012;11
1:354; doi: 10.1186/1475-2875-11-354. https://doi.org/10.1186/1475-2875-11-354.
19. Ajibade FO, Akosile SI, Oluwatuyi OE, Ajibade TF, Lasisi KH, Adewumi JR, et al. Bacteria removal
efficiency data and properties of Nigerian clay used as a household ceramic water filter. Results
in Engineering. 2019;2:100011; doi: https://doi.org/10.1016/j.rineng.2019.100011.
Diseases. 2012;6 8:e1793. https://doi.org/10.1371/journal.pntd.0001793.
Necessity? Trends in Parasitology. 2019;35 1:85-95; doi: 10.1016/j.pt.2018.10.004.
https://doi.org/10.1016/j.pt.2018.10.004.
23. Thavara U, Tawatsin A, Chompoosri J. Evaluation of attractants and egg-laying substrate
preference for oviposition by Aedes albopictus (Diptera: Culicidae). 2004;29:66-72.
24. Sandhu SS, Sharma AK, Beniwal V, Goel G, Batra P, Kumar A, et al. Myco-Biocontrol of Insect
Pests: Factors Involved, Mechanism, and Regulation. Journal of Pathogens. 2012;2012:126819.
https://doi.org/10.1155/2012/126819.
of a candidate fungal biopesticide for use against adult malaria vectors. 2012;11 1:354.
https://doi.org/10.1186/1475-2875-11-354.
26. Chandel K, Suman DS, Wang Y, Unlu I, Williges E, Williams GM, et al. Targeting a Hidden Enemy:
Pyriproxyfen Autodissemination Strategy for the Control of the Container Mosquito Aedes
albopictus in Cryptic Habitats. PLOS Neglected Tropical Diseases. 2016;10 12:e0005235.
27. Nicodemus GD, Bryant SJ. Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering
Applications. Tissue Engineering Part B: Reviews. 2008;14 2:149-65.
https://doi.org/10.1089/ten.teb.2007.0332.
in Function of Substratum and Humidity. 2002;97:415-20.
53
29. Gerding-González M, France A, Sepulveda ME, Campos J. Use of chitin to improve a Beauveria
bassiana alginate-pellet formulation. Biocontrol Science and Technology. 2007;17 1:105-10.
https://doi.org/10.1080/09583150600937717.
30. Hong T, Gunn J, Ellis R, Jenkins N, Moore D. The effect of storage environment on the longevity
of conidia of Beauveria bassiana. 2001;105:597-602.
31. Guimarães CF, Gasperini L, Marques AP, Reis RL. The stiffness of living tissues and its implications
for tissue engineering. 2020;5 5:351-70. https://doi.org/10.1038/s41578-019-0169-1.
32. Ritz K, Young IM. Interactions between soil structure and fungi. Mycologist. 2004;18 2:52-9.
https://www.cambridge.org/core/article/interactions-between-soil-structure-and-
fungi/A7D00C76F8FE0F3AA1F763BAE5C85C87.
33. Borel M, Kergomard A, Renard MF. Degradation of Natural Rubber by Fungi imperfecti.
Agricultural and Biological Chemistry. 1982;46 4:877-81.
https://doi.org/10.1080/00021369.1982.10865189.
54
Chapter 5
Conclusions and future perspectives
The research activities have focused on the development of an innovative, environmentally

compatible pest management method. It has been conceptualized and tested an innovative approach
called “biomimetic lure and kill” based on biomimetics and biocompatible biopolymers to replicate
some specific environmental conditions to attract insects and simplify the target-selective delivery of
biopesticides. The approach has been specialized for the tiger mosquito (Aedes albopictus) by
employing a biomaterial-based hydrogel designed to reproduce the key natural parameters involved
in oviposition site selection and host the fungus Beauveria bassiana as biopesticides and then using
it as an ovitrap oviposition substrate.
The guidelines in conceptualization, design and production of the biomimetic substrate were mainly
environmental and economic sustainability. The final aim was to obtain an insecticidal device that is
theoretically valid to provide a lure and kill precision pest management approach for insects not
responsive to semiochemical stimuli but with a competitive cost/ratio compared to chemical
pesticides based approaches. The biomimetic approach generally points to improving effectiveness
in insect lure and kill strategy lacking effective attractive systems different from semiochemicals.
However, when specialized for tiger mosquitoes, the biomimetic approach points not only to improve
the efficacy (more capturing traps) but also to reduce costs by enhancing devices durability and
reducing management (e.g. less water refill and biodegradation) and integrating two approaches
(mechanical trapping and biopesticides) that separately are technically and economically
unsustainable.
The main research activities focused on substrate design and preparation, evaluating lure effect
through oviposition, and assessing Bb/Gel biocompatibility (conidial growth) and system lethality
against Aedes albopictus eggs (hatching assay).
The main output of the first experimental section (Chapter 3) was to indicate a preferred biomimetic
hydrogel preparation HEC16/S6/Tb8. Hydrogel preferred preparation was 2-5 times more capturing
than controls and standard substrate (Masonite) in lab oviposition assay, and 4-7 times more
capturing in semi-field oviposition assay than a standard ovitrap when spread into a low-cost
cardboard trap. Furthermore, the composition showed an activity extended up to 30 days rather than
one week (average active period of standard ovitraps).
The second experimental section focused on Beauveria bassiana-hydrogel biocompatibility
evaluating the hydrogel as a growth matrix for the biopesticides and then the efficacy of Bb/Gel
matrix against tiger mosquito eggs.
The tests showed that hydrogel substrates sustain Bb growth for 24 days, increasing the initial
conidial concentration by about 1 order of magnitude, reaching 107CFU/ml concentration.
Surprisingly, the most lethal substrate was without Bb (SA16-No Bb, 94%), and CS2-HEC16 /10 days
are the most effective combination to prevent hatching (CM=88%). Consequently, different lethal
action mechanisms emerged. Among the, it was possible to suppose an essential role for mechanical
55
trapping action. The possibility of having lethality without using a biocide but only exploiting the
material's action opens a new scenario in which lethality can be managed through material design
and not through an active substance. The absence of a biocide reduces biocide registration costs
pending on devices, making the method extremely competitive on the market. However, these
results do not reduce the role of Bb in the composition that could be essential in autodissemination
strategy.
Consequently, the two main goals, growth and efficacy, of Chapter 4 have been confirmed, and the
Bb/Gel matrix can potentially be employed as the biomimetic lure and kill oviposition substrate as
expected in the research project. This possibility was also confirmed by analysing the overlapping of
attractive ranges for oviposition and growth reported in Table 4.1 in chapter 4.
The natural follow-up of this study is to carry out tests on adults using substrates containing Bb to
verify both lure (even though Bb have been proven as not repellent on insects), lethality on adults
and autodissemination effect. Subsequently, it will be necessary to carry out adequately structured
large-scale trials, using lethal ovitraps containing the engineered substrate, which evaluate the
effectiveness in population reduction and the durability of the device in the field under more
challenging conditions. In addition, it may be appropriate to carry out the field test over a more
extended period to assess the effect of environmental conditions on devices and catches.
The present study can be expanded by carrying out entomological trials focused on identifying
"universal" characteristics of oviposition sites and expanding the knowledge of breeding side
selection main drivers. Another focus for future studies might be to explore the use of different
biopesticides or characteristics that can increase the virulence of Bb or other bioactive species, such
as new growth substrates or material related properties (e.g. mechanical properties or morphology
dependent fungi structures). In addition, another aspect of improvement could be the optimization
of hydrogel substrates to promote conidia storage for a more extended period, for example, through
freeze-drying procedures.
Finally, a possible follow-up of the study is to evaluate an industrial scale-up of the substrate. For
example, the presence of a physical hydrogel having honey-like viscosity would allow the use of
coating lines to be used to position the gel inside cardboard traps using the same processing
employed for adhesive traps based on pheromones. The possible absence of biocides would favour
the process, allowing processing at higher temperatures and with lower control levels.
56
Appendix
A.1 Aedes albopictus
A.1.1 Aedes albopictus: description and behaviour1

Mosquitoes are present worldwide, occurring throughout the tropical and temperate regions and
northwards into the Arctic Circle. Among the most relevant medically relevant mosquitoes, there are
Culicinae, which are accounted Aedes species and consequently Aedes albopictus, commonly called
tiger mosquito.Aedes are a vector of yellow fever, dengue, West Nile virus, Chikungunya and other
arboviruses2. The adult Asian tiger mosquito is less than 10 mm long from end to end with a striking
white and black pattern. The body size variation in adult mosquitoes depends on the density of the
larval population and food supply within the breeding water. Apart from some characteristics
between genera, mosquitoes are all structurally similar (Figure A1).
Diagrammatic representation of a female adult mosquito [1].
1 Main reference for Entomological information within all the chapter: Jaronski ST. Chapter 11 - Mass Production of Entomopathogenic
Fungi: State of the Art. In: Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI, editors. Mass Production of Beneficial Organisms. San Diego:
Academic Press; 2014. p. 357-413.
2 Arbovirus is a term used to refer to a group of viruses that are transmitted by arthropods.
57
The distinctive mark is the arrangement of black and white on the dorsal surface of the thorax. It is
the Aedes mosquitoes distinctive patterns (e.g. the reason to call Aedes albopictus tiger mosquito).
Palps and antennae have a role in breeding site research and selection as through them, mosquitoes
can identify water and water vapour, taste surfaces, water, and detect chemicals presence [1].
With a few exceptions, a female mosquito must bite a host and take a blood meal to obtain the
necessary nutrients for the development of her eggs after hatching. Then the gravid female searches
for the suitable breeding site in which eggs can hatch and larvae can survive and complete their
development cycle (eggs, larvae, pupae, adult)shown in Figure A.2. Other shared features are [2]:
 All mosquito larvae require water to develop; no mosquito has larvae that can withstand
desiccation, although they may be able to survive short periods, for example, in wet mud.
 Mosquito larvae must come to the water surface to breathe. Atmospheric air is taken in
through a pair of spiracles situated dorsally.
 Mosquito larvae feed on yeasts, bacteria, protozoans and numerous other microorganisms,
as well as on decaying plant and animal material found in the water.
Oviposition behaviour and consequently breeding site choice is probably the most significant
difference between Anophelinae (e.g. Anopheles) and Culicinae (e.g. Aedes albopictus). In particular,
Anopheles lay down their eggs directly on the water surface. Eggs can hatch within 2–3 days up to 7–
14 days, depending on temperature, probably because they cannot survive when dried and at too
low temperature. Almost any permanent or temporary water collection can be a mosquito larval
habitat, but larvae are usually absent from large expanses of uninterrupted water such as lakes,
especially if they have large numbers of fish and other predators. Generally, water presents specific
parameters such as pH, salinity, organic content, turbidity etc.
Aedes, and in particular Aedes albopictus, do not lay eggs on the water surface. Oviposition occurs in
shaded places/containers of temporary water, and eggs are laid singly on wet surfaces (usually
container walls) just above the waterline. Many natural containers provide breeding places (Figure
A.3), such as water-filled tree-holes, rock-pools, bamboo stumps, bromeliads, pitcher plants, leaf axils
in bananas, pineapples, and other plants water-filled split coconut husks and even snail shells [3].
Differently, in anthropized environments of temperate countries, the tiger mosquito adapted to
oviposit mostly on artificial habitats (cartons, trashed containers, used tires etc.). Larval emergence
occurs after rainfall raises the water level in the containers. The eggs may require several
submersions before hatching.
Additionally, oxygen (O2) tension significantly affects egg hatch. Several studies have shown that low
O2 tension stimulates the hatching of Aedes albopictus eggs and is a more critical factor than flooding
or temperature on inducing egg hatch . Development is temperature dependent, but the larvae
usually pupate after five to ten days, and the pupal stage lasts two days [4]. From this oviposition
behaviour derived the name of container breeding mosquitos. Aedes albopictus eggs can withstand
desiccation, can remain dry for months on the container walls but remain viable, ready to hatch when
covered with water.
58
Chart of the principal characters of the stages in mosquitoes life cycle. The main difference is the
position of eggs compared to the waterline [1].
In addition, environmental conditions can influence eggs hatching. For example, in temperate
regions, Aedes eggs may also enter a state of diapause, that is not hatching until some specific
environmental stimulus such as a change in day length and/or temperature breaks diapause and the
eggs hatch. In particular, tiger mosquitoes produce cold (0-5°C) and dry-resistant eggs and females
can survive to cold temperatures (> 9°C [5]), one of the reasons for their great spread in Europe.
Another difference between the two species is the activity period. In particular, Aedes are a diurnal
insect differently from Anophelinae (nocturnal). Biting behaviour is crucial for their control strategy
management. In fact, due to diurnal activity, tiger mosquito control in the urban area cannot be done
quickly through insecticide spraying due to the human presence leading to the need for more
targeted and safe methods [4].
59
Meta-analysis of natural larval breeding sites exploited by Aedes albopictus [3].
A.1.2 Aedes albopictus as vector: spread and mosquito-borne diseases

Aedes albopictus originated in Southeast Asia but has spread during the last 30–40 years [2, 4]. Its
success in colonizing new geographic locations is due to its ability to adapt to different climates [6]
and to oviposition behaviour adaptation (e.g. laying drought-resistant eggs in casual containers that
can withstand the long journeys between continents) [7]. In 2009, the Invasive Species Specialist
Group was listed as one of the top 100 invasive species and was considered the most invasive
mosquito species globally [8]. Since its first appearance in Albania in 1979 and Italy in 1990, Aedes
albopictus has been reported in 20 European countries, considered a severe threat to public health.
It landed in Europe internationally, especially trade in used tires and lucky bamboo [9]. The species is
now widely established and reportedly as nuisance species, particularly in Italy [10], France [11, 12]
and Spain [13], and generally in Mediterranean counties [14], and its expansion will be enhanced by
trades improvement and climate changes [15]. Mosquito population growth will promote biting
nuisance and overall mosquito-related medical risk of mosquito-borne diseases (MBDs) outbreaks.
60
Aedes albopictus distribution on Europe in May 2020. Italy is the most heavily infested country in
Europe [4].
Mosquitoes can cause millions of deaths every year (Figure A5). From the medical point of view,
Aedes albopictus can transmit to humans many dangerous arboviruses such as Eastern equine
encephalitis virus (EEEV), La Crosse virus (LACV), but overall tiger mosquito is known as vector of
dengue, chikungunya, yellow fever and west Nile fever [15]. Symptoms are usually mild and can
include mild fever, skin rash, inflammation of the eyes (conjunctivitis), muscle and joint pain, etc.
However, all of them are potential disabling (e.g. Zika infection during pregnancy causes
microcephaly, fetal brain malformations [16]) or fatal diseases, mainly when contracted in
underdeveloped countries, leading to a pandemic with both health and economic issues [17, 18]. The
fast and uncontrolled spread of a disease lead presents economic loss, especially in tourism-based
economies [17]. It has been estimated for some Latin American and the Caribbean region countries
a loss as high as 1.6% of GDP [19].
Vector-borne disease annual number of cases and death [8].

61
Anexample of the dangerousness of Aedes albopictus spreading across the Europe (promoted by
climatic changes) are the Chikungunya outbreaks in Italy in 2007 and 2017[20] and other cases were
reported in France [21].
A.2 Mosquito control methods
A.2.1 Importance of vector control

Not all mosquitoes are virus vectors, but intuitively, the risk is proportional to the victor diffusion
once the virus is present. Consequently, vector control is crucial in MBD control.
A probably overtaken but still representative of the effect of control on MBD is the McDonald model
expressed through the vectorial capacity V reported in Equation 1 [22].
𝒎𝒂𝟐 𝒑𝒏
𝑽= Equation1
− ln 𝒑
Where the parasite's extrinsic incubation period (EIP, n days); the ratio of mosquitoes to humans (m);
mosquito survival through one day (p); and human biting rates (a)
An intuitive restatement of Equation1 is that an infectious person will be subject to the attention
of m mosquitoes (assuming everyone is equally attractive) and will receive ma bites each day. For
those mosquitoes to become infectious, they must survive the extrinsic incubation period (with
probability p n). The adult mosquitoes (on average) live for 1/(−ln(p))1/(−ln(p)) days biting and
potentially infecting humans at a rate of per day [22].
The average vector/human ratio influences vector capacity sice the other parameters are fixed.
Consequently, it is possible to conclude that the only control parameter is the vector number Thus,
vector control is the only way to reduce the risk and halt a disease.
Generally, it is possible to conclude that moving from Aedes albopictus to adapt to new
environments, its predicted spread and establishment in Europe, and its confirmed involvement in
pathogen transmission cycles makes the surveillance and control of this species hugely important.
Malaria control strategy gives an example of the effectiveness of the control action on the vector to
contain the spreading of MBDs [23]. Even in the absence of disease transmission, Aedes albopictus is
a severe nuisance biting species, particularly in urban areas, where control can become an economic
burden to local municipalities due to numerous larval development sites [24].
A.2.2 Chemical-based control methods: advantages and drawbacks

Control measures can be directed to immature aquatic stages, adults, or both stages simultaneously.
However, larval control is prevalent to prevent adult emergence. Both approaches are mainly
chemical-based but differ in the habitat. Larval stages live in water differently from adults
consequently are easier to be targeted.
Insect growth regulators (IGRs) and pyrethroids are the prevalent chemicals used worldwide for
mosquito control [25]. Following Directive 98/8/EC (Biocidal Products Directive) and EU Regulation
528/2012 (Biocidal Products Regulation), certain biocides are banned from use in Europe, such as
62
temephos3 , due to health concerns [26], effects on non targeted insects and biodiversity [27] and to
promote less persistent and biodegradable insecticides [28]. Other organophosphates, such as
Fenthion (C10H15O3PS2) or Chlorpyrifos (C9H11Cl3NO3PS), can be used only as emergence and in
polluted water.
Larval control usually involves insect growth regulators (IGRs), differently pyrethroids prevails in adult
control [25].
Insect growth regulators (IGRs) are hormone-like compounds able to stop insect growth, usually
formulated as liquids granules or briquettes. For example, pyriproxyfen (C20H19NO3,) is a juvenile
hormone analogue that arrests larval development of insects. Differently, other compounds such as
diflubenzuron (C14H9ClF2N2O2, one of the more employed against Aedes albopictus), can inhibit chitin
formation in the immature stages [29].
Larvicides are usually applied as emulsions or oil solutions [30]. Theoretically, larvicides could benefit
from being more environmentally friendly because they have long persistence (up to 100 days,
reducing application numbers), are more specific in killing mosquitoes, and there are still few cases
of resistance [31]. Furthermore, mosquitoes have not been reported as developing, so larval control
by IGR can be a preferential way to control mosquitoes. However, some studies do not agree on this
point [32].
IGRs are usually directly delivered on larval sites from knapsack-type sprayers carried on operators'
backs, but they are sometimes dispersed from vehicle-mounted spraying machines. Large or
inaccessible areas may require aerial spraying from helicopters or small fixed-wing aircraft.
IGRs could be suitable to the autodissemination approach (adult mosquitoes as carriers from one site
to another of insecticide compounds previously spread on breeding sites). Nevertheless, this
approach gave no results using pyriproxyfen formulation to treat tyres or vegetation due to climatic
conditions such as high rainfall, but it could be successful if employed in ovitraps [33].
On the other hand, pyrethroids are synthetic compounds structurally derived from pyrethrum
(naturally contained in Chrysanthemum cineraria folium, which is allowed in biological agriculture due
to low toxicity but low persistence). Pyrethroids such as permethrin and deltamethrin (respectively
C21H20Cl203 and C22H19Br2NO3) are generally used against adult stages of mosquitoes. Larvicidal use
can be effective, but it is not allowed due to their destructive action on numbers of other aquatic
insects and animals (even crustaceans and fish) [34]. Water-based aerosols deliver pyrethroids, mists
and fogs (respectively < 50; 51–100; 5–15 μm as droplets size) using motorized knapsack mist-
blowers, shoulder-carried thermal foggers or boat- or vehicle-mounted machines in order to kill
outdoor-resting (exophilic) adult mosquitoes. They are a wide spectrum of approaches, then not
environmental and human safe [35]. Granules, pellets or gelatine capsules, often containing
pyrethroids, can be used to penetrate dense growths of aquatic vegetation. Slow-release granules
and pyrethroids resistance to environmental conditions allow scattering formulations before the
areal flooding, killing drought-resistant residual larvae as the granules release their toxicants into the
water.
3Dimetoxy-sulfaylidene-phosphorane used in developing world during dengue outbreak. There are suggestions that temephos could
be toxigenic and mutagenic.
63
In emergencies, aerial spraying gives fast and effective vector control, e.g., used to stop dengue
transmission. However, it is an untargeted method involving numerous non-infesting species rather
than having a high impact on biodiversity and public health [36]. The effectiveness of sprays is
affected by droplet size distribution, meteorological conditions (temperature, wind speed and
direction), habitat type (vegetation cover, open or secluded locations) and the time of application
(flight activity of target species). These parameters are crucial for Aedes mosquitoes to be controlled
efficiently through spraying. In fact, (in particular albopictus) as diurnal insects are expected to be
targeted more efficiently during their diurnal and/or crepuscular flight activity. However, the
presence of people represents a constraint for the implementation of diurnal and/or crepuscular
sprayings, particularly in urban areas [37]. Furthermore, shaded rest places (e.g. in gardens) reduce
the possibility of correctly targeting them. Although spray applications have been successfully used
in some control campaigns against Aedes mosquitoes, this method is debatable due to low efficacy
and high residual effects with potential impact on non-target species.
Unlike other pests, the so-called precision methods directed to specific mosquito genus do not exist
for mosquitoes because they are not pheromone responsive insects then, lure and kill devices (attract
insects through poisoned baits or pheromones) cannot be used. Consequently, pesticide spraying
based control can only run after mosquitoes. For example, Aedes albopictus breeding sites are usually
occasional artificial (often small man-made water containers) hard to individuate and where
insecticides can be easily removed or inactivated by water action [38, 39]. Adult rest sites in urban
areas are in private houses, gardens and consequently difficult to be reached through extensive
public methods. Furthermore, prevention is complex because some species developed poisoned
places avoiding behaviour, e.g. changes in biting period activity and insecticide avoidance [40]. In
addition, the development of insecticide resistance to some classes of molecules in the adult and
larval stages remains a real threat for vector control [41]. In particular, the effect of pyrethroids on
Aedes albopictus resistance was found throughout the world [42, 43].
Finally, another side effect of pesticide-based approach is their indirect environmental and health
action [44]. In fact, targeting difficulties have resulted in only a small amount of chemicals hitting the
target, the other significant part hitting water and soil, and the food chain affecting [45].
A.2.3 Environmental friendly mosquito control methods

As previously shown in the previous paragraph, the insecticide-based approach presents some
environmental and health issues and methodological limitations. Therefore, alternative management
strategies have been explored. Among the most studied approach, it is possible to find environmental
(oviposition sites reduction), mechanical (trapping), biological (use of natural active substances, or
introduction of predators), genetic (introduction of genetically modified mosquitoes) [46-48].
Source reduction consists of preventing Aedes mosquitoes from using potential breeding sites.
Effective source reduction, especially for Aedes albopictus, requires scrupulous and repeated
cleaning or treatment of containers for everyday use, so it relies on extensive homeowner
collaboration [49].
Traps are widely used for the survey and monitoring of mosquito populations. Ovitraps exploit the
propensity of Aedes mosquitoes to lay their eggs in small containers. In particular, in the most
64
straightforward setup are a black plastic pot (e.g. small flowerpot) filled with water and oviposition
substrate soaked in the water (usually Masonite or cardboard) [46]. Aedes albopictus, if present, will
use the substrate as oviposition site. The addition of a larvicide or an autocidal mechanism (e.g larval
drought, or emerging adult trapping) allows to use them as control method. Organic infusions such
as grass, hay or oak, or CO2 source can be added to ovitraps to improve their attractivity [50, 51].
However, traps suffer for higher costs and for natural breeding sites, reducing their lure capability
[52]. The biological approach can be based on natural competition (e.g. predator introduction such
as fish in water sources, clearly not suitable for Aedes albopictus). Higher efficacy can have the use
of natural bioactive substances such as entomopathogenic fungi (e.g. Beauveria bassiana and
Metarhizium anisopliae), bacteria (e.g. Bacillus thuringiensis var. israelensis (Bti), Lysinibacillus
sphaericus, Wolbachia), essential oils or fermentation products (e.g. Pennyroyal oil and
Saccharopolyspora spinose fermentation) [53, 54]. Finally, genetically modified mosquitoes are the
last to appear and are mainly based on the production of sterile male and female mosquitoes to be
released in the environment.
Generally, these methods' advantage relies on low environmental impact and high safety profile foe
users and animals. At the same time, in particular, natural active substances have low persistence to
environmental stresses such as sunlight, drought, and high temperature. Furthermore, their survival
and efficacy is strictly dependent on substrate of application -. In particular, classic aspersion methods
already used in pesticide application on the field can be employed to spread these substances [55].
Higher costs than chemical pesticides, needs of a higher number of applications, and new application
methods limited the diffusion of biopesticides. For example, the use of Bacillus based larvicides is
restricted by the economic constraints of application on widespread ephemeral water bodies, their
rapid inactivation through UV radiation [56] and sedimentation in the soil and leaf litter. This poor
residual activity requires frequent reapplication, hampering intervention and efficacy. Consequently,
developing new delivery strategies and methods to extend active natural substances lifespan are
among the biggest challenges in bio-based control.
A.2.4 Lethal Ovitraps as possible alternative to pesticide spraying

Ovitraps (OTs) are one of the possible alternative methods for tiger mosquito control [57].
Ovitraps action mechanism is based on the oviposition habit of the tiger mosquito females previously
described, providing a poisoned artificial breeding spot and substrate to tiger mosquitoes. Ovitraps
were used in the past almost exclusively for the collection of eggs for scientific monitoring and
arbovirus surveillance and dengue surveillance. Monitoring ovitraps were small, black plastic pots
filled with tap water were provided with a masonite paddle (20 × 2 cm) as an oviposition substrate.
The masonite paddle was periodically replaced, and the ovitrap refilled and put back in place by an
operator [58].
Recently, the emerging evidence of pesticide-related drawbacks pushed to use traps as a control
method and consequently to dvelope new and more efficient types of trap [46] able to lure and then
kill insects (then named lethal ovitraps, LOTs). The transition from a monitoring tool to a control
device led first to add synthetic larvicide to water active on emerging larvae (passive lure and kill
traps). Then new types of traps emerged, considerably more complex, which avoid the use of
65
insecticides but provide elements for the active capture of adults (e.g. fans to suck them) or carbon
dioxide cylinders and/or light signals to attract (active lure and kill traps)[59, 60]. Nevertheless, this
type of trap is intended for domestic use given the need for electrical power and the high costs.Other
lethal ovitraps can be classified depending on lethal mechanism. For example, autocidal ovitraps
allow oviposition but prevent the emergence of females by trapping them. Sticky ovitraps, act on
mosquito when it lands on the surface using an adhesive strip [58].
Left: Common ovitraps used in recent mass trapping campaigns [41]. Right: sticky surface from a
sticky trap [58].
The problems relating to this last class of traps (defined as passive "lure and kill" traps as they do not
use elements that do work to attract or capture, such as fans) are: a) the need to constantly top up
the evaporated water and insecticide to function, b) keep them continuously monitored and
therefore costs related to personnel, c) use of chemical insecticides with consequent environmental
and interaction risk for animals and humans and non-target insects, d) need to recover traps when
not active to prevent them from becoming new deposition foci if filled by rain (and therefore further
costs for recovery and eventual disposal), e) low effectiveness due to competition with natural
deposition sites in their vicinity; f) not targeted for a single species of mosquito (e.g. also Anopheles,
Culex, Aedes use water for oviposition).
Consequently, extending their use to mass control campaigns (i.e. numerous traps to place and
survey on a large area [61]). Thus insecticide based ovitraps (monitoring-like) are still the most used
in large-scale control [62].
A small number of trials of trap-based control campaigns have been performed mainly in Dengue
endemic countries such e.g. Brazil, Thailand and Australia. Generally, these trials emerged that
campaigns can positively affect vector number reduction [63] when coupled with site reduction and
preventive insecticide spraying, particularly if traps are placed on at least 80% of the area.
Furthermore, citizen's collaboration is crucial due to mosquito presence in private spaces (e.g.
gardens and houses) [64]. Thus, traps optimization, cost reduction and devices safety (to allow
indoor/garden safety use) are crucial for trap cost-effectiveness and then for mass trapping
campaigns [65, 66]. The solutions proposed are biodegradable traps oriented with the aim to reduce
66
maintenance and disposal costs (e.g. starch made ovitraps [67]). However, presented biodegradable
ovitrap are insecticide based and still have short active periods [64]. In addition, the presence of
larvicidal products, can acts as repellent on adult female able to detect (through antennae and palps)
the chemical presence in water and consequently on oviposition substrate, reducing the number of
laid eggs [68, 69]. A possible solution could be using natural active substances.
A.3 Entomopathogenic fungi as bioinsecticides
A.4.1 Biology of entomopathogenic fungi 4

An entomopathogenic fungus is a fungus that can act as a parasite of insects and kills or seriously
disables them. These fungi usually attach to the external body surface of insects in the form of
microscopic spores (usually asexual, mitosporic spores, also called conidia). Under the right
conditions, particularly temperature and humidity, these spores germinate as hyphae and colonize
the insect's cuticle, which they bore through enzymatic hydrolysis, reaching the insects' body cavity
(hemocoel) [70]. Then, after 4-14 days, the insect is usually killed (sometimes by fungal toxins), and
new spores (sporulation, if humidity is usually required) can be formed in/on the insect. An example
of the life cycle and infection process is reported in Figure A.7.
Schematic life cycle of the entomopathogenic fungi, exemplified by Beauveria bassiana [71].
Describing more in detail, the life cycle is based on the ascomycetes. It begins when the spore
contacts the arthropod cuticle, attaching initially by van der Waals forces and then adheres more
firmly through an anchoring structure. The hypha penetrates the arthropod cuticle by means of
several enzymes and mechanical pressure. Once in the hemocoel, the fungus proliferates by means
of yeast-like bodies (e.g. mycelium). As the host dies, the fungus rapidly transforms into mycelium
and, under ideal conditions, emerges to conidiate on the exterior of the insect (Figure A.8).
4
Main reference for Entomological information within all the the chapter: Jaronski ST. Chapter 11 - Mass Production of
Entomopathogenic Fungi: State of the Art. In: Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI, editors. Mass Production of Beneficial
Organisms. San Diego: Academic Press; 2014. p. 357-413.
67
A.4.2 Fungi as biopesticide

As previously presented, the use of entomopathogenic fungi can be an alternative control method
[48]. Fungal infection has been shown to cause a substantial reduction in transmission potential of a
range of different vector species, including mosquitoes [72-74]. Among the first explored fungal
pathogens able to infect some mosquito populations (Anopheline), were Lagenidium, Coelomomyces,
and Culicinomyces. Nevertheless, many other fungi were found as active against mosquitoes at the
larval and/or adult stage, such as Metarhizium anisopliae, Beauveria tenella, etc. [75].
Nevertheless, their slower action than chemical insecticides [73], the interest in fungal application
against pests derives from their low toxicity, the ability to spread easily through insect population,
and the low presence of resistance phenomena in target insects, probably due to their infective
mechanism. For example, adult malaria vectors emerged as insecticide-resistant, remain fully
susceptible to fungal infection [73]. In addition, fungi are generally known as safe to people and with
low toxic residues.
Fungi as insecticides are characterized by contact, differently from other types of biopesticide
(bacteria and viruses) active only when ingested. This aspect is helpfu for insects not respondent to
feeding stimuli as mosquitoes.
Numerous studies showed that fungi can infect adult mosquitoes (included Aedes albopictus) [76]
when applied to a range of substrates, suggesting potential for use as Indoor Residual Sprays (IRS),
or via novel delivery strategies included ovitraps substrate [77].
Consequently, numerous commercial products are already available on the market. Currently,
commercial formulations of fungal conidia, including water or oil-based suspensions, granules, dusts,
and floating formulations, have been used against mosquitoes (mainly used for larval control due to
water presence in action habitat that can extend fungal life and activity) [78].
One of the principal difficulties in fungal application against adult female mosquitoes is their low
persistence on-field and storage for an extended period. Two fundamental parameters used to define
the quality of an insecticide. In particular, World Health Organization indicated as two years is the
minimum storage period without significant efficacy loss of the active ingredient for mosquito
dedicated insecticides.
Currently, chemical insecticides dedicated to indoor applications have 2-6 months of residual action
after application. Studies investigating the persistence of fungal spores have shown that the infective
period of biopesticides was days or weeks when applied in agriculture [79] due to solar UV radiations
and delivery methods [80]. Differently, when spores are applied in more hidden environments, such
as soil inoculation, using less aggressive systems than spraying, persistence can be extended to
months or even years [81]. The increase in the infective period in a protected environment could
allow the use of fungi for indoor application. However, they are still more expensive than chemicals
and registration costs are too high in relation to the current market size, deterring companies from
pursuing registration.
Storage depends on the fungal strain, production conditions, temperature, humidity, and spore
moisture content [82, 83]. Currently, the spores of many species of entomopathogenic fungi can be
mass-produced on a variety of culture media and so are potentially suitable for production on an
industrial scale. Under ideal conditions of low temperature (e.g. 5°C) and low spore moisture content
68
(<5%), entomopathogenic fungi (in particular Beauveria bassiana) have been shown to store with
minimal loss of viability for over 2 years, as long as initial viability is high, but only usingcostly drug
storage-like conditions [84].
A.4.3 Beauveria bassiana

Beauveria bassiana (Bb) was the first entomopathogenic fungus to be used against pests. (Augostino
Bassi's 1835), and its efficacy was later proved against mosquitoes. As reported for other fungal
bioinsecticides, infection is by contact through destruction of the cuticle by some enzymes and
release of the toxin beauvericin [85].
This entomopathogenic fungus acts as a parasite of different insects (Coleoptera Hymenoptera
Lepidoptera Homoptera Diptera [86] and it is lethal to many mosquito species such as C. tarsalis, C.
pipiens, A. aegypti, A. sierrensis, A. nigromaculis, and A. Albimanus but also Aedes albopictus and
Anopheles. [87-89].
Mycelium and conidiophores of Beauveria bassiana on an engorged larva of Rhipicephalus

sanguineus at 20 days post infection [90].
Beauveria effect was proved also in other hematophagous parasites such as Rhipicephalus
sanguineus (ticks) [90], as showed in Figure A.8. Consequently, Bb has assumed a key role in the
management of numerous agricultural, veterinary and forest pests but also for the control of
mosquito populations and, currently, Bb is mass-produced and more than 40 commercial products
are available in the market.
Beauveria bassiana is typically administered in one or more applications of conidia that are released
in dry or liquid formulations and in aqueous or oily solutions, granules, pellets or floating formulations
that exploit the possibility of the spores multiplying in an aquatic environment. The use of Bb in
69
biological control does not present any risk to human or animal health. Given its safety, suitability for
shaded place and lack of repellent effect when applied on surfaces, Bb use could be suitable for
indoor applications [86].
However, Bb suffers from low persistence in the outdoor environment, and also indoor activity is
related to the substrate of application. A strong dependence of persistence on environmental
conditions and the substrate of application has emerged. For example, when Bb is applied on soil, it
has a persistence comparable to a common pesticide (up to 4-5 months), while if applied on wood or
concrete, its persistence is reduced (from 1 week to 2-3 months). Differently, if used in operating
conditions (exposed to heat, UV radiation and drying of the substrate), the persistence drops to days
[84, 91]. Not only persistence is affected by substrate, but also efficacy. For example, the spores of
Bb have been applied by spraying on earth, concrete and wood tiger mosquitoes were exposed for 1
hour to the treated substrate and then removed. After removal, survival was observed over the next
14 days, and it showed a strong dependence on the Bb application substrate (confirming the link
between application substrate and Bb efficacy). Effectiveness and residual effect were also verified
on plywood, bricks, plastic and glass surfaces [91].
The characteristics of resistance and tolerance to thermal stresses are also strongly influenced by the
composition of the growth medium of Bb. For example, if carbohydrates are added to the preparation
used for growth, Bb can withstand up to about 50°C [92]. Conidia encapsulation within polymeric
matrices has been proven helpful to preserve vitality and persistence. Generally, many commercial
patented products are formulated as Bb conidia encapsulation in pellets or spheres made of polymers
able to provide water and create a physical barrier to UV (e.g. silica or silico-aluminate-based gel or
formulations based on water-soluble alginate pellets or microspheres [93].
For example, the US 5141744 patent describes the preparation of an insecticidal composition as a
hydrated synthetic macrogel (acrylic polymers, polyacrylamides and polyurethanes) capable of
retaining hydration with the function of acting as a reserve of water for the entomopathogen. The
purpose of the macrogel is to maintain the vitality of the pathogenic organism and act as a poisoned
bait for insects. Nevertheless, this kind of application neutralizes one of the essential advantages of
fungal biopesticides, i.e. their ability to act without ingestions. In fact, for some insects, food baits are
a valuable tool, but not for vectors of diseases such as mosquitoes. Therefore, the use of Bb-based
baits could reduce the persistence problem, but it is not a proper tool against mosquitoes. Similarly,
US patent 5273749 describes a process for the coating of microbial pesticides but such as to be
applied on leaves against plant weeds and subsequently ground and processed to be applied by
spraying on plants or seeds.
Although encapsulation can improve Bb persistence, encapsulated formulation acts mainly as a
protection to the pathogen. There is also the disadvantage of dispersing a non-biodegradable
synthetic polymer into the environment that could also be ingested by non-target animals, children,
insects, incurring limitations of use and therefore contradicting the use of a natural agent.
Furthermore, considering the application methods of the commercial formulations (e.g. consumable
matrix, pellets, water or oil-based solution for spraying), none of them solves the core problem of
mosquito control of new and more efficient and targeted delivery methods or to improve the existing
ones such as traps.
70
A.4.4 Beauveria bassiana mass production

The analysis of production on an industrial scale also plays a fundamental role in this work. It provides
an essential indication of the conditions and substrates most suitable for the proliferation of
Beauveria bassiana (or other entomopathogens), facilitating the choice of biomaterials to use.
Many commercial products based on entomopathogenic fungi are now available on the market;
about 40% are based on Bb. For the production in the laboratory of limited quantities of
entomopathogenic fungi, a substrate based on Potato Dextrose Agar (PDA) is used as a well-
established practice, but it is entirely different from mass production. Currently, the most mass
production used method is solid substrate fermentation (particularly for Bb production). It is based
on the natural conidiation processes, in which fungi proliferate on natural organic substrates
(fermentation). The process aims to yields aerial conidia as the final product (infectious part of Bb).
Solid substrate fermentation is biphasic, with an initial step being a liquid fermentation to produce
inoculum to obtain the maximum yield of the process (in terms of production of active conidia). It
must take place under specific conditions. Wide ranges of media have been used for the liquid
fermentation phase. The simplest contains dextrose/sucrose as the carbon source and yeast extract
as a source of nitrogenous compounds and vitamins. The ideal liquid fermentation should produce
mostly, or entirely, blastospores and short hyphae for optimal dispersion of inoculum through the
substrate starting from the suitable inoculum media. Then, the liquid fermentation requires vigorous
aeration.The solid substrate, whether organic or inert, must be hydrated and sterilized before the
inoculum procedure. In some cases, published protocols using rice substrate have used nutritional
additives in hydrating the substrate to increase conidial production (e.g. dextrose, cane molasses
etc.). The typical duration of the solid substrate phase is 7–14 days, but depending on the nature of
the inoculum (conidia or blastospores/mycelia), the substrate is colonized by fungus within the first
24–48 h, after which there is active mycelial proliferation through the substrate.
Many organic materials have been evaluated as substrates for the ascomycetes, but rice and barley
seem to be the most suitable (although expensive). Mineral carriers such as granules of calcined
diatomaceous earth are another low-cost option for production (e.g. diatomite has a high surface to
volume ratio and absorb aqueous liquids up to 110–140% of its weight). They have the advantage of
allowing flexible control of nutrients and can be recycled after washing and sterilization.
Most academic and industrial systems have used traditional solid substrate fermentation. In
situations where labour costs are low, allowing labour-intensive approaches, polypropylene
autoclavable bags are used. The solid substrate is inserted inside the bags, and then the initial liquid
solution containing the fungus is inoculated for initial mycelial colonization and growth, and then
transfer the cultures to open, nonsterile, plastic laundry hampers or tubs for the sporulation phase
within a controlled environment. On the other hand, in technically advanced environments, another
direction of research pertains to the use of trays within controlled environment chambers.
A critical moisture level is needed for optimal fungal growth and sporulation, and it is generally
evaluated through water chemical activity (aw). Generally, the ideal value is 0.998 for B. bassiana.
Because the solid substrate phase is an active fermentation, oxygen needs to be readily available to
all parts of the substrate, but carbon dioxide and heat must be drawn off. Another aspect to take in
account is light exposure. Fermentation seems to be more productive in dark environments.
71
Finally, for all purposes except immediate use, the conidia produced on the solid substrate must be
dried down to a moisture content <9%w/w (for compliance to commercial regulations but also to
optimal shelf life). Even trace of moisture in the conidia results in foreshortened shelf life. Generally,
drying is obtained by opening plastic fermentation bags or transferring the sporulated substrate to
open table tops or using air-lift devices. However, extreme desiccation of conidia (aw <0.1) can lead
to a significant problem in conidia vitality at rehydration. Consequently, the drying process is usually
stopped when the conidia have reached a water activity of 0.3. Then, conidia are separated from
growth media mainly through mechanical methods stored in a closed package.
The most significant problem in solid substrate fermentation is the scale-up to large capacity at
commercial levels. For a mycoinsecticide to succeed commercially, a huge number of conidia must
be produced as cheaply and efficiently as possible to compete with chemical insecticides. Currently,
the most expensive part of the process is related to substrates and their sterilization process.
Consequently, the research of recyclable (or waste-derived) solid substrate is highly desirable.
A.4 Hydrogel
A.4.1 Principal concepts

Hydrogel products constitute a group of polymeric (synthetic and natural including biodegradable
[94]) materials. The hydrogels' structure is usually an elastic and hydrophilic porous network. The
structure can be designed by acting on processing, compositions and bonds involved in 3D structure,
to consents both to absorb and gradually release a large volume of water or solutions containing, for
example, drugs, nutrients or cells [95] generally remaining insoluble in aqueous solutions [96, 97].
These networks are composed of homopolymers or copolymers based on the method of preparation.
Due to the extensive range of properties, hydrogels can be applied from agriculture to soft electronics
for water release in soil, diagnostic, drug delivery, tissue engineering, and pharmaceutical
applications [98, 99]. Hydrogels are generally characterized by several parameters such as the final
capability to absorb liquids (swelling thermodynamics), (swelling kinetics), free and chemically
bonded water, degradation time, as well as their mechanical and rheological properties in swollen or
dry conditions [100].
A.4.2 Hydrogels classification, use and properties

Hydrogels can be divided into many groups (Figure A.9 [101] based on composition, physical
properties, method of preparation, ionic charges, etc. The more relevant division for the thesis is
between physical and chemical. Physical hydrogels are based on weak physical bonds. Physical
hydrpgels can be defined as polymeric networks bound together via polymer chain entanglement
and/or weak non-covalent interactions between polymer chains [99]. The attractive forces holding
these networks together are typically based on hydrogen bonding, electrostatic or hydrophobic
interactions and thus, the gels can be reversibly dissolved under certain conditions that would
weaken these attractive forces, i.e. a change in pH, shear rate, solvent presence etc. [99] They do not
possess a defined and stable morphological structure and usually are a continuous, viscous matrix,
72
sometimes liquid-like. However, also physical gel can be made of water even though it remains stable.
On the other hand, chemical hydrogels [101] are stable network (e.g. not soluble in water) made
ofcovalent bonds between polymer chains. These gels are usually formed through monomer
polymerization in the presence of a crosslinking agent, which is typically a monomer with at least two
polymerizable functional moieties [102]. In crosslinked hydrogels, the network density (crosslinking
density) is mainly due to the crosslinker action for the same polymer and concentration. In physical
hydrogels, polymer chain network depends on polymer molecular weight and concentration, then
generally more viscous physical hydrogels possess also a more structured network [103]. . Both
physical and chemical hydrogel can be obtained from natural macromolecules (such as
carbohydrates, proteins etc. [104]) and synthetic polymers.
Especially in the last ten years, natural hydrogels had significant diffusion, particularly in medical
applications.
On the other hand, there are chemical crosslinked gels.
Classification of hydrogels following different criteria [101].
Physical gel formation both for HEC and SA can be briefly described as a hydration reaction of
polysaccharides. Chemical hydrogel can be synthesized in a several "classical" chemical ways. These
include one-step procedures like polymerization and parallel crosslinking of multifunctional
monomers and multiple step procedures involving synthesis of polymer molecules with reactive
groups and their subsequent crosslinking [102, 103]. Two examples of physical hydrogel based on
natural polymers employed are Sodium Alginate (SA) and Hydroxyethylcellulose (HEC). The same two
polymers have been chemically crosslinked respectively using Poly(ethylene glycol diglycidyl ether)
and CaCl2.
A.4.3 Hydrogel for precision pest management

Hydrogel versatility due to properties regulation possibility make them suitable also for pest
management applications. According to Sharma et al. (2019), about 3.5 million tons of pesticides are
73
sprayed yearly worldwide (50–60% herbicides, 20–30% insecticides, and 10–20% fungicides), but less
than 1% of these pesticides come in direct contact with or are consumed by target pests [25, 105].
Consequently, pest management needs to move from a predominantly broad use of chemical
insecticides to a more sophisticated path of "precision" approaches reducing (and eliminating, when
possible) quantities and toxicity of insecticides involved in applications or improving the delivery of
natural substances (e.g. biopesticide encapsulation) [106]. The new generation of precision control
tools points to increase pest targeting and pesticide delivery mainly combining attractant and active
substance in the same matrix [40] (the "lure and kill" approach). Among the most common strategies
based on semiochemicals are the use of sexual or food pheromones or natural/synthetic baits to
convey the insecticidal agent more directly and effectively by attracting a specific insect on traps toxic
surfaces or to favour ingestion of the toxic bait. Although based on the use of chemicals, the
"precision" approach represents a step forward that reduces the volumes of insecticides used and
facilitate indoor employment in the presence of people and/or animals or sensible products (e.g. food
industry). Due to their properties, hydrogels can be employed to produce precision pest management
tools and devices. For example, controlled release properties can achieve a controlled and very
targeted delivery of various contact insecticides or absorb and deliver small volumes of toxicants
within liquid baits [60, 106]. Therefore, hydrogels have great potential pest management tools to
develop new low impact devices or to use dangerous or banned substances (e.g. broad-spectrum
insecticides, such as organophosphates (e.g. chlorpyrifos) and carbamate (e.g. carbaryl)) through
controlled release [44, 107]. Usually, controlled release is made through beads or capsules. In the
first, the pesticide is contained in a polymeric core coated by a shell (natural or synthetic)able to
contain also pheromones to obtain a better lure and kill action [108]. Differently, capsules are
produced through the microencapsulation process. Oil, soft polymeric or water-based droplets
containing an active ingredient are sprayed into a crosslinking agent solution that can produce a
reaction at droplet-crosslinker interface (e.g. sodium alginate sprayed in CaCl2) [109]. The procedure
leads to a microcapsule containing the pesticide, which can be dispersed in a medium and sprayed
(generally is possible to control the capsule dimension acting on droplets size). Common polymeric
encapsulated wall materials are natural (e.g. gelatin, gum arabic, starch, sugar, ethylcellulose,
carboxymethylcellulose, sodium alginate etc.) or synthetic (e.g. polypropylene, polystyrene,
polyacrylamide, polyethers, polyesters, polyamides etc.)[110].
Other methods are based on swelling, mainly in bait-based methods (e.g. against fire ants). For
example, superabsorbent spheres can be immersed in a pesticide liquid and a sucrose solution to
create a bait [111]. Generally, polyacrylamide hydrogels have been used in pest management.
However, this hydrogel class is not biodegradable, and its release on the field can be as dangerous as
the free spraying of pesticides. Consequently, precision pest management is shifting to biopolymers
such as alginates or cellulose. Nevertheless, it is evident that using hydrogels baits cannot be a
solution for insects not responsive to feeding stimuli like mosquitoes [112, 113].
74
A.4.4 Crosslinking mechanism in HEC - PEGDE hydrogels5

A possible example of a reaction scheme between HEC and Poly ethyleneglycol diglycidyl ether
(PEGDE, a bi-functional molecule containing an epoxide group) with NaOH as catalyst will be
reported. It is a reaction between epoxy groups and hydroxyl groups.
Hydroxyethylcellulose (HEC) is a biocompatible, biodegradable, nontoxic, hydrophilic, non- ionic
water soluble derivative of cellulose. It is broadly used in biomedical field, paint industry, as a soil
amendment in agriculture, coal dewatering, cosmetics, absorbent pads, wastewater treatment and
gel electrolyte membranes [114].
Numerous papers have been published on preparing cellulose‐based hydrogels using crosslinking by
epoxides, alkyl halides, and compounds with both epoxy and halide groups (e.g., epichlorohydrin).
The crosslinking reactions are generally carried out in strongly basic pH at high temperatures.
Poly(ethyleneglycol diglycidyl ether) (PEGDE) has also been used as a macromolecular crosslinker to
design cellulose‐based gels. HPC‐PEGDE hydrogels were synthesized through the reaction between
epoxy groups and hydroxyl groups as reported in Figure A10.
Typical chemical reactions for cellulose‐based gels synthesis: (A) epoxide crosslinking; (B) alkyl
halide crosslinking. (C) The specific reaction between HEC and PEGDE with basic catalyst NaOH.
The reaction between halide functional groups and the hydroxyl groups requires strong alkaline pH,
which has limited its application in preparing cellulose‐based hydrogels. A combination of the halide
and epoxide functional groups, such as epichlorohydrin, is frequently used in crosslinking for
cellulose-based hydrogels synthesis. Cellulose hydrogels can be "one‐step" synthesized from
cellulose in NaOH/urea aqueous solution. The gelation can be controlled by a synergy of chemical
and physical crosslinking processes: the etherification reaction between cellulose and PEGDE and the
self‐association and entanglement of cellulose chains via hydrogen bonding reconstruction from
cellulose/NaOH aqueous solutions.
5
Main reference for chemical crosslinking in cellulose reaction are taken from: Kang, H., Liu, R., & Huang, Y. (2016). Cellulose-Based
Gels. Macromolecular Chemistry and Physics,217(12), 1322–1334. https://doi.org/10.1002/macp.201500493
75
A.4.5 Crosslinking mechanism in Sodium Alginate - CaCl2 hydrogels6

Sodium alginate (NaC6H7O6) is a water-soluble polysaccharide that can be isolated from brown
seaweed and kelps. It is built up of two uronic acids residues, L-guluronic and D-mannuronic acid.
Alginates are biodegradable, biocompatible, and non-toxic polymers. Their most important property
is related to their stabilizing and gelling properties as well as their ability to retain water. Owing to
these properties, they have a broad range of applications, mainly in the food industry as thickeners,
stabilizing agents and emulsifiers, and they are also used in the cosmetic and drug industry [115].
When sodium alginate is put into a solution of calcium ions, the calcium ions replace the sodium ions
in the polymer to form calcium alginate. In particular, sodium alginate reacts with calcium chloride
(CaCl2) to generate calcium alginate (C12H14CaO12), which is a gelatinous material due to a rearranged,
in which they bond in a way called egg-box model.
It is reported the crosslinking mechanism in Sodium alginate using CaCl2 as crosslinker.
Crosslinking in SA is a substitution reaction in which Ca2+ substitute Na and act as coordinator atom,
creating a bridge between polymer chains. The reaction starts immediately at the gel-solution
interface, then it advances by diffusion mechanism, as CaCl2 penetrates in Sodium alginate.
Egg-box shell model after SA crosslinking reaction [116].
6Main reference for chemical crosslinking in cellulose reaction are taken from: Shalapy, A., et al. (2020). Adsorption of Deoxynivalenol
(DON) from Corn Steep Liquor (CSL) by the Microsphere Adsorbent SA/CMC Loaded with Calcium. Toxins, 12(4), 208.
https://doi.org/10.3390/toxins12040208
76
Crosslinking process leads to a gelatinous material having mechanical and rheological properties
depending from the crosslinking time and concentration. At molecular level, crosslinked SA can be
described through the eggbox shell mode reported in Figure A12.
77
References
1. Service M. Medical entomology for students, fourth edition. 2008.

2. Hawley WA. The biology of Aedes albopictus. J Am Mosq Control Assoc Suppl. 1988;1:1-39.
albopictus an Efficient Bridge Vector for Zoonotic Arboviruses? Pathogens. 2020;9 4.
4. Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, et al. A Review of the
Invasive Mosquitoes in Europe: Ecology, Public Health Risks, and Control Options. Vector-Borne
and Zoonotic Diseases. 2012;12 6:435-47. https://doi.org/10.1089/vbz.2011.0814.
5. Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, et al. Integrated Aedes
management for the control of Aedes-borne diseases. PLOS Neglected Tropical Diseases.
2018;12 12:e0006845. https://doi.org/10.1371/journal.pntd.0006845.
6. Braks M, Medlock J, Hubálek Z, Hjertqvist M, Perrin Y, Lancelot R, et al. Vector-Borne Disease
Intelligence: Strategies to Deal with Disease Burden and Threats. 2014;2.
7. Kampen H, Medlock JM, Vaux AGC, Koenraadt CJM, van Vliet AJH, Bartumeus F, et al. Approaches
to passive mosquito surveillance in the EU. 2015;8 1:9. https://doi.org/10.1186/s13071-014-
0604-5.
8. World Health O. World malaria report 2016. Geneva: World Health Organization; 2016.
9. Scholte EJ, Dijkstra E, Blok H, Vries A, Takken W, Hofhuis A, et al. Accidental importation of the
mosquito Aedes albopictus into the Netherlands: A survey of mosquito distribution and the
presence of dengue virus. 2009;22:352-8.
10. Romiti F, Ermenegildi A, Magliano A, Rombolà P, Varrenti D, Giammattei R, et al. Aedes albopictus
(Diptera: Culicidae) Monitoring in the Lazio Region (Central Italy). 2020;58.
11. Gossner C, Ducheyne E, Schaffner F. Increased risk for autochthonous vector-borne infections
transmitted by Aedes albopictus in continental Europe. 2018;23.
12. Roche B, Léger L, L'Ambert G, Lacour G, Foussadier R, Besnard G, et al. The Spread of Aedes
albopictus in Metropolitan France: Contribution of Environmental Drivers and Human Activities
and Predictions for a Near Future. 2015;10:e0125600.
13. Goiri F, González MA, Goikolea J, Oribe M, de Castro V, Delacour S, et al. Progressive Invasion of
Aedes albopictus in Northern Spain in The Period 2013–2018 and A Possible Association with The
Increase in Insect Bites. International Journal of Environmental Research and Public Health.
2020;17 5.
14. Bellini R, Michaelakis A, Petrić D, Schaffner F, Alten B, Angelini P, et al. Practical management
plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus). Travel
Medicine and Infectious Disease. 2020;35:101691; doi:
https://doi.org/10.1016/j.tmaid.2020.101691.
15. World Health O, Diseases UNUWBWSPfRaTiT. Global vector control response 2017-2030.
Geneva: World Health Organization; 2017.
16. Hunsberger S, Ortega-Villa A, Powers J, Rincón León H, Sosa S, Hernández E, et al. Patterns of
signs, symptoms, and laboratory values associated with Zika, dengue, and undefined acute
illnesses in a dengue endemic region: Secondary analysis of a prospective cohort study in
southern Mexico. 2020;98.
17. Macciocchi D, Lanini S, Vairo F, Zumla A, Figueiredo L, Lauria F, et al. Short-term economic impact
of the Zika virus outbreak. 2016;39:287-9.
78
18. Weaver SC, Reisen WK. Present and future arboviral threats. 2010;85 2:328-45.
19. Jamil Z, Waheed Y, Zeb T. Zika virus, a pathway to new challenges. 2016;9.
20. Guzzetta G, Vairo F, Mammone A, Lanini S, Poletti P, Manica M, et al. Spatial modes for
transmission of chikungunya virus during a large chikungunya outbreak in Italy: a modeling
analysis. 2020;18:226.
21. Franke F, Giron S, Jeannin C, Leparc-Goffart I, de Valk H, Grard G, et al. Emergences de dengue,
de chikungunya et de zika en France métropolitaine, 2006–2019. 21es Journées Nationales
d’Infectiologie. 2020;50 6, Supplement:S109.
https://www.sciencedirect.com/science/article/pii/S0399077X20303887.
22. Brady OJ, Godfray HCJ, Tatem AJ, Gething PW, Cohen JM, McKenzie FE, et al. Vectorial capacity
and vector control: reconsidering sensitivity to parameters for malaria elimination. 2016;110
2:107-17. https://doi.org/10.1093/trstmh/trv113.
23. Haakenstad A, Harle A, Tsakalos G, Micah A, Tao T, Anjomshoa M, et al.
24. Dara SK. The New Integrated Pest Management Paradigm for the Modern Age. 2019;10 1.
https://doi.org/10.1093/jipm/pmz010.
25. Sharma A, Kumar V, Shahzad B, Tanveer M, Sidhu GPS, Handa N, et al. Worldwide pesticide usage
and its impacts on ecosystem. 2019;1:1446.
26. Aiub CAF, Pinto LFR, Felzenszwalb I. Standardization of conditions for the metabolic activation of
N-nitrosodiethylamine in mutagenicity tests. Genetics and molecular research : GMR. 2004;3
2:264-72. http://europepmc.org/abstract/MED/15266397.
27. Krief A. Pyrethroid insecticides. ARKIVOC. 2021;2021 1:48-54.
https://doi.org/10.24820/ark.5550190.p011.328.
28. Copping L. Biopesticide Market Opportunities – Strategic Knowledge Sharing and Networking
Event. 2013;24 3:136-8.
https://www.ingentaconnect.com/content/resinf/opm/2013/00000024/00000003/art00012.
https://doi.org/10.1564/v24_jun_12.
29. European Food Safety A. Conclusion on the peer review of the pesticide risk assessment of
confirmatory data submitted for the active substance pyriproxyfen. EFSA Journal. 2014;12
8:3813. https://doi.org/10.2903/j.efsa.2014.3813.
30. Apolinário R, Feder D. Existing potentials in Insect Growth Regulators (IGR) for crop pest control.
Research, Society and Development. 2021;10 1:e35910111726.
https://rsdjournal.org/index.php/rsd/article/view/11726.
31. Arias-Castro JH, Martinez-Romero HJ, Vasilieva O. Biological and Chemical Control of Mosquito
Population by Optimal Control Approach. Games. 2020;11 4.
32. Fine JD, Mullin CA, Frazier MT, Reynolds RD. Field Residues and Effects of the Insect Growth
Regulator Novaluron and Its Major Co-Formulant N-Methyl-2-Pyrrolidone on Honey Bee
Reproduction and Development. 2017;110 5:1993-2001. https://doi.org/10.1093/jee/tox220.
Diseases. 2012;6 8:e1793. https://doi.org/10.1371/journal.pntd.0001793.
34. Shi X, Zhang F, Lu XX, Wang Z, Gong T, Wang G, et al. Spatiotemporal variations of suspended
sediment transport in the upstream and midstream of the Yarlung Tsangpo River (the upper
Brahmaputra), China. 2019.
35. Gürkan S, Philippou D, Field L. Insecticide Synergists: Good or Bad for Honey Bees? 2015;26.
79
36. Wiberg-Larsen P, Nørum U, Rasmussen J. Repeated insecticide pulses increase harmful effects
on stream macroinvertebrate biodiversity and function. 2020;273:116404.
in Function of Substratum and Humidity. 2002;97:415-20.
environment. 2019.
39. Wooding M, Naudé Y, Rohwer E, Bouwer M. Controlling mosquitoes with semiochemicals: a
review. Parasit Vectors. 2020;13 1:80; doi: 10.1186/s13071-020-3960-3.
40. Schorkopf D, Spanoudis C, Mboera L, Mafra-Neto A, Ignell R, Dekker T. Combining Attractants
and Larvicides in Biodegradable Matrices for Sustainable Mosquito Vector Control.
2016;10:e0005043.
11:1471-85. https://doi.org/10.1002/ps.4044.
42. Soko W, Chimbari M, Mukaratirwa S. Insecticide resistance in malaria-transmitting mosquitoes
in Zimbabwe: A Review. 2015;4:46.
43. Scott J. Evolution of resistance to pyrethroid insecticides in Musca domestica. 2016;73.
44. Kolaczinski J, Curtis C. Chronic illness as a result of low-level exposure to synthetic pyrethroid
insecticides: A review of the debate. 2004;42:697-706.
45. El Alfy M, Faraj T. Spatial distribution and health risk assessment for groundwater contamination
from intensive pesticide use in arid areas. 2017;39 1:231-53. https://doi.org/10.1007/s10653-
016-9825-1.
46. Achee NL, Grieco JP, Vatandoost H, Seixas G, Pinto J, Ching-Ng L, et al. Alternative strategies for
mosquito-borne arbovirus control. PLOS Neglected Tropical Diseases. 2019;13 1:e0006822.
bassiana for the control of Rhipicephalus sanguineus sensu lato.
48. Zaim M, Guillet P. Alternative insecticides: An urgent need. 2002;18:161-3.
49. Unlu I, Faraji A, Strickman D, Fonseca D. Crouching Tiger, Hidden Trouble: Urban Sources of Aedes
albopictus (Diptera: Culicidae) Refractory to Source-Reduction. 2013;8:e77999.
50. Ridha M, Hairani B, Rosanji A, Fadilly A, Meliyanie G. Dengue vector surveillance (Aedes
albopictus) with ovitrap and attractants from imperata immersion (Imperata cylindrica).
2020;9:286.
51. Gao Q, Cao H, Fan J, Zhang Z, Jin S, Su F, et al. Field evaluation of Mosq-ovitrap, Ovitrap and a CO
2 -light trap for Aedes albopictus sampling in Shanghai, China. 2019;7:e8031.
53. Govindarajan M, Sivakumar R, Rajeswary M, Veerakumar K. Mosquito larvicidal activity of thymol
from essential oil of Coleus aromaticus Benth. against Culex tritaeniorhynchus, Aedes albopictus,
and Anopheles subpictus (Diptera: Culicidae). 2013;112 11:3713-21.
https://doi.org/10.1007/s00436-013-3557-2.
54. M. Domingues P, Santos L. Essential oil of pennyroyal (Mentha pulegium): Composition and
applications as alternatives to pesticides—New tendencies. 2019;139:111534.
80
55. Damalas CA, Koutroubas SD. Current Status and Recent Developments in Biopesticide Use.
Agriculture. 2018;8 1.
56. Duarte Neto JMW, Wanderley MCdA, da Silva TAF, Marques DAV, da Silva GR, Gurgel JF, et al.
Bacillus thuringiensis endotoxin production: a systematic review of the past 10 years. 2020;36
9:128. https://doi.org/10.1007/s11274-020-02904-4.
57. Pushpanathan M. Lethal ovitrap: A cost-effective weapon to fight zika virus infection. 2017;1.
58. Marini F, Caputo B, Pombi M, Tarsitani G, della Torre A. Study of Aedes albopictus dispersal in
Rome, Italy, using sticky traps in mark-release-recapture experiments. 2010;24:361-8.
59. Degefa T, Yewhalaw D, Zhou G, Lee M-C, Atieli H, Githeko AK, et al. Evaluation of the performance
of new sticky pots for outdoor resting malaria vector surveillance in western Kenya. 2019;12
1:278. https://doi.org/10.1186/s13071-019-3535-3.
Necessity? Trends in Parasitology. 2019;35 1:85-95. https://doi.org/10.1016/j.pt.2018.10.004.
61. Barrera R, Amador M, Acevedo V, Hemme RR, Félix G. Sustained, Area-Wide Control of Aedes
aegypti Using CDC Autocidal Gravid Ovitraps. 2014;91 6:1269-76.
https://www.ajtmh.org/view/journals/tpmd/91/6/article-p1269.xml.
62. Gunawan A. Using Various Types of Lethal Ovitrap to Control Aedes sp in Endemic Dengue
Hemorrhagic Fever (DHF). 2017;05:22260-5.
63. Regis L, Acioli R, Silveira Jr J, Melo-Santos M, Souza W, Ribeiro C, et al. Sustained Reduction of
the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two
Brazilian Cities. 2013;8:e67682.
64. Paz Soldan V, Yukich J, Soonthorndhada A, Giron M, Apperson C, Ponnusamy L, et al. Design and
Testing of Novel Lethal Ovitrap to Reduce Populations of Aedes Mosquitoes: Community-Based
Participatory Research between Industry, Academia and Communities in Peru and Thailand.
2016;11.
65. Pepin K, Marques-Toledo C, Scherer L, Morais M, Ellis B, Eiras A. Cost-effectiveness of Novel
System of Mosquito Surveillance and Control, Brazil. 2013;19:542-50.
66. Kaufman P, Butler J, Nelson C. Evaluation of the Mosquito Sentinel 360 Trap in Florida Residential
Environments. 2008;24:528-33.
67. Ritchie S, Long S, McCaffrey N, Key C, Lonergan G, Williams C. A Biodegradable Lethal Ovitrap for
Control of Container-Breeding Aedes. 2008;24:47-53.
68. Day JF. Mosquito Oviposition Behavior and Vector Control. Insects. 2016;7 4:65.
https://pubmed.ncbi.nlm.nih.gov/27869724.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198213/.
69. Parker AT, McGill K, Allan BF. Container Type Affects Mosquito (Diptera: Culicidae) Oviposition
Choice. 2020;57 5:1459-67. https://doi.org/10.1093/jme/tjaa045.
70. Fernandes E, Valério H, Feltrin T, Van Der Sand S. Variability in the production of extracellular
enzymes by entomopathogenic fungi grown on different substrates. 2012;43:827-33.
Morales-Ramos JA, Rojas MG, Shapiro-Ilan DI, editors. Mass Production of Beneficial Organisms.
San Diego: Academic Press; 2014. p. 357-413.
72. Blanford S, Chan Brian HK, Jenkins N, Sim D, Turner Ruth J, Read Andrew F, et al. Fungal Pathogen
Reduces Potential for Malaria Transmission. Science. 2005;308 5728:1638-41.
https://doi.org/10.1126/science.1108423.
81
73. Blanford S, Shi W, Christian R, Marden JH, Koekemoer LL, Brooke BD, et al. Lethal and Pre-Lethal
Effects of a Fungal Biopesticide Contribute to Substantial and Rapid Control of Malaria Vectors.
PLOS ONE. 2011;6 8:e23591. https://doi.org/10.1371/journal.pone.0023591.
of a candidate fungal biopesticide for use against adult malaria vectors. 2012;11 1:354.
https://doi.org/10.1186/1475-2875-11-354.
75. Scholte E-J, Knols B, Samson R, Takken W. Entomopathogenic fungi for mosquito control: A
review. 2004;4:19.
albopictus (Diptera: Culicidae) using Metarhizium anisopliae JEF-003 millet grain. 2015;18 2:217-
21. https://www.sciencedirect.com/science/article/pii/S1226861515000254.
77. Mnyone L, Kirby M, Lwetoijera D, Mpingwa M, Simfukwe E, Knols B, et al. Tools for delivering
entomopathogenic fungi to malaria mosquitoes: Effects of delivery surfaces on fungal efficacy
and persistence. 2010;9:246.
78. Muñiz-Paredes F, Miranda Hernández F, Loera O. Production of conidia by entomopathogenic
fungi: from inoculants to final quality tests. 2017;33.
79. Pilz C, Enkerli J, Wegensteiner R, Keller S. Establishment and persistence of the
entomopathogenic fungus Metarhizium anisopliae in maize fields. 2010;135:393-403.
80. Moore D, Higgins P, Lomer C. Effects of Simulated and Natural Sunlight on the Germination of
Conidia of Metarhizium flavoviride Gams and Rozsypal and Interactions with Temperature.
1996;6:63-76.
81. Milner R, Samson P, Morton R. Persistence of Conidia of Metarhizium anisopliae in Sugarcane
Fields: Effect of isolate and formulation on persistence over 3.5 years. 2003;13:507-16.
82. Rumbos C, Athanassiou C. Use of entomopathogenic fungi for the control of stored-product
insects: can fungi protect durable commodities? 2017;90.
83. Marques E, Alves S, Marques I. Effects of the temperature and storage on formulations with
Mycelia of Beauveria bassiana (Bals.) Vuill and Metarhizium anisopliae (Metschn.) Sorok.
1998;42.
84. Hong T, Gunn J, Ellis R, Jenkins N, Moore D. The effect of storage environment on the longevity
of conidia of Beauveria bassiana. 2001;105:597-602.
85. Sandhu SS, Sharma AK, Beniwal V, Goel G, Batra P, Kumar A, et al. Myco-Biocontrol of Insect
Pests: Factors Involved, Mechanism, and Regulation. Journal of Pathogens. 2012;2012:126819.
https://doi.org/10.1155/2012/126819.
86. Keswani C, Singh S, Singh H. Beauveria bassiana: Status, Mode of action, Applications and Safety
issues. 2013;3:16.
87. Clark TB, Kellen WR, Fukuda T, Lindegren JE. Field and laboratory studies on the pathogenicity of
the fungus Beauveria bassiana to three genera of mosquitoes. 1968;11 1:1-7.
88. Lee J, Woo R, Choi C, Shin T, Gwak Ws, Woo S. Beauveria bassiana for the simultaneous control
of Aedes albopictus and Culex pipiens mosquito adults shows high conidia persistence and
productivity. 2019;9.
89. Lina BF-W, Alejandro PR, Robert WB, Ephantus JM. Characterization of Tolypocladium
cylindrosporum (Hypocreales: Ophiocordycipitaceae) and Its Impact Against Aedes
aegypti and Aedes albopictus Eggs at Low Temperature. Journal of the American
Mosquito Control Association. 2017;33 3:184-92. https://doi.org/10.2987/16-6596R.1.
82
bassiana for the control of Rhipicephalus sanguineus sensu lato. 2015;8 1:80.
https://doi.org/10.1186/s13071-015-0693-9.
91. Acharya N, Seliga RA, Rajotte EG, Jenkins NE, Thomas MB. Persistence and efficacy of a Beauveria
bassiana biopesticide against the house fly, Musca domestica, on typical structural substrates of
poultry houses. Biocontrol Science and Technology. 2015;25 6:697-715; doi:
10.1080/09583157.2015.1009872. https://doi.org/10.1080/09583157.2015.1009872.
92. Ying S-h, Feng M-G. Medium components and culture conditions affect the thermotolerance of
aerial conidia of fungal biocontrol agent Beauveria bassiana. 2006;43:331-5.
93. Vemmer M, Patel A. Review of encapsulation methods suitable for microbial biological control
agents. 2013;67:380–9.
94. Sannino A, Demitri C, Madaghiele M. Biodegradable Cellulose-based Hydrogels: Design and
Applications. Materials. 2009;2 2.
95. Li J, Mooney D. Designing hydrogels for controlled drug delivery. 2016;1:16071.
96. Hoffman AS. Hydrogels for Biomedical Applications. 2002;65:18-23.
97. Demitri C, Del Sole R, Scalera F, Sannino A, Vasapollo G, Maffezzoli A, et al. Novel superabsorbent
cellulose-based hydrogels crosslinked with citric acid. Journal of Applied Polymer Science.
2008;110 4:2453-60. https://doi.org/10.1002/app.28660.
98. Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations.
2000;50:27-46.
99. Tang S, Zhao L, Yuan J, Yu C, Leng Y. Physical hydrogels based on natural polymers. 2020. p. 51-
89.
100. Shen X, Shamshina JL, Berton P, Gurau G, Rogers RD. Hydrogels based on cellulose and chitin:
fabrication, properties, and applications. Green Chemistry. 2016;18 1:53-75.
http://dx.doi.org/10.1039/C5GC02396C.
101. Mahinroosta M, Farsangi Z, Allahverdi A, Shakoori Z. Hydrogels as intelligent materials: A brief
review of synthesis, properties, and applications. 2018;8:42–55.
102. Ghorpade VS, Chen Y. Chapter 4 - Preparation of hydrogels based on natural polymers via
chemical reaction and cross-Linking. Elsevier; 2020. p. 91-118.
103. Kono H. Carboxymethyl Cellulose-Based Hydrogels. 2015. p. 243-58.
104. Chang C, Zhang L. Cellulose-based hydrogels: Present status and application prospects.
2011;84 1:40-53. https://www.sciencedirect.com/science/article/pii/S0144861710010003.
105. Pimentel D. Amounts of pesticides reaching target pests: Environmental impacts and ethics.
1995;8:17-29.
106. Ahmad L, Mahdi SS. Precision Pest Management. Cham: Springer International Publishing;
2018. p. 119-27.
107. Tay J-W, Choe D-H, Mulchandani A, Rust MK. Hydrogels: From Controlled Release to a New
Bait Delivery for Insect Pest Management. 2020;113 5:2061-8.
https://doi.org/10.1093/jee/toaa183.
108. Roy A, Singh S, Bajpai J, Bajpai AK. Controlled pesticide release from biodegradable polymers.
2014;12.
109. Olivares A, Silva P, Altamirano C. Microencapsulation of probiotics by efficient vibration
technology. 2017;34:1-28.
83
110. Clifford D. Integrated pest management in the global arena. Ed by KM Maredia, D Dakouo and
D Mota-Sanchez. CABI Publishing, Wallingford, UK, 2003. pp 560. ISBN 0 85199652 3. Pest
Management Science. 2004;60 7:733-. https://doi.org/10.1002/ps.881.
111. Buczkowski G, Roper E, Chin D. Polyacrylamide Hydrogels: An Effective Tool for Delivering
Liquid Baits to Pest Ants (Hymenoptera: Formicidae). 2014;107 2:748-57.
https://doi.org/10.1603/EC13508.
112. Fraser KJ, Mwandigha L, Traore SF, Traore MM, Doumbia S, Junnila A, et al. Estimating the
potential impact of Attractive Targeted Sugar Baits (ATSBs) as a new vector control tool for
Plasmodium falciparum malaria. Malar J. 2021;20 1:151; doi: 10.1186/s12936-021-03684-4.
113. Traore MM, Junnila A, Traore SF, Doumbia S, Revay EE, Kravchenko VD, et al. Large-scale field
trial of attractive toxic sugar baits (ATSB) for the control of malaria vector mosquitoes in Mali,
West Africa. 2020;19 1:72. https://doi.org/10.1186/s12936-020-3132-0.
114. Noreen A, Zia KM, Tabasum S, Khalid S, Shareef R. A review on grafting of
hydroxyethylcellulose for versatile applications. 2020;150:289-303.
115. Mazur K, Buchner R, Bonn M, Hunger J. Hydration of Sodium Alginate in Aqueous Solution.
2013;47.
116. Shalapy A, Zhao S, Zhang C, Li Y, Geng H, Ullah S, et al. Adsorption of Deoxynivalenol (DON)
from Corn Steep Liquor (CSL) by the Microsphere Adsorbent SA/CMC Loaded with Calcium.
Toxins. 2020;12 4.

Pubblicazione 7 PHD Thesis Marco Friuli Unisalento Xxxiii Ciclo Ing. Materiali

Uploaded by

Copyright:

Available Formats

You might also like

Pubblicazione 7 PHD Thesis Marco Friuli Unisalento Xxxiii Ciclo Ing. Materiali

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Pubblicazione 7 PHD Thesis Marco Friuli Unisalento Xxxiii Ciclo Ing. Materiali

Uploaded by

Copyright:

Available Formats

UNIVERSITY OF SALENTO

DEPARTMENT OF ENGINEERING FOR INNOVATION

Development of macromolecular hydrogels based on natural polymers as oviposition substrates and

Table of contents ........................................................................................................................................ I

3.3.3 AEDES ALBOPICTUS OVIPOSITION ASSAY ................................................................................................... 22

 Yield stress ................................................................................................................................................... 24

 Gel viscosity ................................................................................................................................................ 24

 On-field Oviposition assay and water release .............................................................................................. 25

3.4 RESULTS AND DISCUSSION .................................................................................................................... 25

Introduction to the work

From tissue engineering to mosquitoes: Biopolymers as a tool for the development of

2.1 Chapter Highlights

2.3 Biomimetic lure and kill approach

West Africa. Malaria Journal. 2020;19 1:72; doi: 10.1186/s12936-020-3132-0.

PLOS Neglected Tropical Diseases. 2019;13 10:e0007615; doi:

nanocomposite. RSC Advances. 2017;7 42:26468-77; doi: 10.1039/C7RA02185B.

Preparation and evaluation of biopolymer-based hydrogels as biomimetic oviposition

3.1 Chapter Highlights

3.2.1 Rationale for oviposition parameters selection

3.2.2 Rationale in biopolymer and substrate composition selection

3.2.3 Aim of the work

3.3 Materials and methods

3.3.2 Hydrogels preparation

Table 3.2 Parameter variation ranges.

3.3.3 Aedes albopictus oviposition assay

3.3.4 Oviposition substrates characterization

Water release in plastic trap set-up at controlled conditions

3.4 Results and discussion

3.4.4 Water content and water releaseat controlled conditions

3.4.5 Yield stress and Shear viscosity

Shear viscosity (Pas) Yield stress (Pa)

2 0.4±0.03 0.2±0.01 9.6±2.8 8±2.7

3.4.6 Morphological analysis

3.4.7 On-field water release and oviposition

Growth and effectiveness on Aedes albopictus eggs of Beauveria bassiana added to

4.1 Chapter Highlights

 Beauveria bassiana (Bb)was embedded into some oviposition-attractive hydrogel

4.3 Materials and methods

4.3.1 Beauveria bassiana origin and infection suspension preparation

4.3.2 Preparation of Beauveria bassiana/Gel systems

4.3.3 Ades albopictus eggs

4.3.4 Bioassay 1: Evaluation of Beauveria bassiana viability

4.3.5 Bioassay 2: Effect of Beauveria bassiana/Gel systems on Aedes albopictus eggs

4.3.6 Substrate characterization

4.4.1 Bioassay 1: Beauveria bassiana growth evaluation in Bb/gel system

CS2-HEC16 CS2-SA16 CS2-HEC30 CS1-HEC16

Incubation Time (Days)

Active conidia/Total conidia Initial diameter

4.4.3 Bioassay 2: Beauveria bassiana/Gel effect on Aedes albopictus eggs hatching

5 Days 10 Days 15 Days CFU/ml at 24 Days

4.4.4 Morphological assays on Beauveria bassiana/Gel systems

4.4.5 Gel viscosity measurement

NoBb Gel Control CS2 Gel samples

Conclusions and future perspectives

The research activities have focused on the development of an innovative, environmentally

A.1 Aedes albopictus

A.1.1 Aedes albopictus: description and behaviour1