Bacterial diversity analysis of coastal

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 Aquaculture 556 (2022) 738263

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Bacterial diversity analysis of coastal superficial seawaters near

aquaculture facilities, using MALDI-TOF approach and Ribopeaks database
Jorge Freitas a, Rosa Perestrelo a, Paulo Vaz-Pires b, c, *, José S. Câmara a, d
a
CQM – Centro de Química da Madeira, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal
b
ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, R. Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
c
CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Terminal de Cruzeiros de Leixões, Av. General Norton De Matos, S/N, 4450-208 Matosinhos,
Portugal
d
Departamento de Química, Faculdade de Ciências Exatas e Engenharia, Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: The upcoming environmental regulations when applied to the aquaculture sector will further require that the
Environment industry adopt routine control methodologies and results registration, to support that its activity is respecting the
Sustainability adjacent environment and to support its sustainability claims.
Monitoring
This work aimed to determine the capability of microbial analysis with the MALDI-TOF approach and Ribo­
Coastal areas
peaks database, to establish the microbial community diversity of coastal superficial seawater near aquaculture
Anthropogenic impact
facilities and the possible influence of anthropogenic pressure.
The main conclusions are the capability of the Ribopeaks database to be used for the analysis of environmental
bacterial samples, since it was able to identify a much higher diversity of marine bacteria when compared with
the MBT Compass database. The MALDI-TOF-Ribopeaks approach has a clear potential for fast and cheaper
routine analysis of the seawater microbial community, which is becoming more relevant in the progress of
quality control procedures in the aquaculture industry. The present study also reveals the need for more studies
on the interactions between anthropogenic influence in coastal waters and the effect it might have on aqua­
culture sustainable management.

1. Introduction
Coastal areas are important zones, either due to favorable habita­
tional conditions or economic relevance for the development of several
activities (i.e.: tourism). However, the combination of a growing popu­
lation and economic development increases the threats on the marine
ecosystems, due to nutrient inputs from anthropogenic activities on the
coast or land-based activities (Câmara et al., 2021). Nutrient inputs can
have different origins such as insufficient treatment of wastewater (e.g.:
primary treatment only), domestic wastes, industrial wastes, agricul­
tural runoffs, etc. All of these are sources of organic carbons and min­
erals, that once reach the marine coastal areas, can lead to
eutrophication affecting the ecosystem balance (Kalkan and Altuğ,
2020). Also, marine activities developed near the coast can impact the
quality of the proximate waters.
Aquaculture activities are one of the most scrutinized sectors,
mostily their influence on the adjacent environment (Tičina et al.,
2020). The new environmental legislation will require that the industry
adopt additional routine control methodologies and results registration,
to support that its activity is respecting the surrounding environment
and to withstand its sustainability claims (Stentiford et al., 2020). In
European Union (EU) the aquaculture exploration is regulated under
nine major legal requirements, that cover all the steps from the project
phase, implementation, and operation, to prevent conflict of interest
between all active users of coastal areas and habitat protection. The
major policies are Marine Strategy Framework Directive; The Water
Framework Directive; Birds and Habitats Directives; Natura 2007 reg­
ulations, Regulation on the use of alien and locally absent species in
aquaculture; EU Regulation on the prevention and management of the
introduction and spread of invasive alien species; Environmental Impact
Assessment; and Directive on Maritime Spatial Planning (European
Commission, 2015).
Since aquaculture exploration and land-based anthropogenic activ­
ities are both major sources of nutrients inputs, it appears that they have

* Corresponding author at: ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, R. Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
E-mail address: vazpires@icbas.up.pt (P. Vaz-Pires).

https://doi.org/10.1016/j.aquaculture.2022.738263
Received 22 October 2021; Received in revised form 10 March 2022; Accepted 12 April 2022
Available online 15 April 2022
0044-8486/© 2022 Published by Elsevier B.V.
J. Freitas et al.
direct interactions. The overflow to coastal waters, of anthropogenic
run-offs, rich in nutrients or pollutants (e.g.: storm waters, sewage), can
affect aquaculture management increasing fish mortalities, diseases
outbreaks, or product contamination (Kathijotes et al., 2015). In the case
of aquaculture, negligent practices could result in environmental
degradation (e.g.: anoxic sediments). Nevertheless, aquaculture can also
have a benefic effect on the surrounding environment, providing shelter
(Stagličić et al., 2017) and nutritional input on oligotrophic waters
(Ballester-Moltó et al., 2017).
Besides the influence on the aquatic fauna and flora, independently
from the nutrient sources, one of the major impacts will be on the mi­
crobial diversity on coastal marine waters (Wang et al., 2021). It is
known that microbial metabolism has ecological importance on the
balance of matter (organic and inorganic), energy and carbon cycle on
the marine ecosystem dynamics. Microbial diversity and structure, on
the water column, are shaped by the prevailing conditions at a specific
time, being the most common, seasons influence, inland discharges,
anthropogenic pressures, or ecological limitations (e.g.: oxygen acces­
sibility and temperature) (Satyanarayana et al., 2019).
Consequently, knowing the marine microbial community diversity
and how intereacts with water quality monitorisation, as the influence
on the pathogens life cycle, is of outmost importance. The identification
is made complementing morphological (e.g. cell shape), phenotypic (e.g.
Gram staining), and sequencing techniques (e.g. polymerase chain re­
action (PCR)) (Thangavelu et al., 2021; Wani et al., 2021). Sequencing
techniques have proven to be very important providing new insights and
understandings of marine microbial structure throughout the globe,
remaining a essential instrument in this field (Sanz-Sáez et al., 2020).
Though, high level of expertise and cost undermines the implementation
as routine analysis techniques (Santos et al., 2016). Some limitations are
attributed to the primer choice that could leave some taxonomic groups
out of the analysis (Lewis et al., 2021).
An alternative method for microorganism identification is based on
the acquisition of protein fingerprint through MALDI-TOF MS and
comparing it to reference profiles on a database or analysing the un­
known profile against known profiles of bacteria. Its capability to pro­
vide fast, high throughput analysis at a lower cost, as well the reduced
amount of sample preparation, catapulted the technique as an important
advance in the microbiology field (Santos et al., 2016).
On the other end, the main drawbacks are associated with the
capability to use only culturable bacteria as well as the quality of the
database. Even though current cultivation techniques are limited in the
number of individuals from a phylum that is possible to isolate, it still
represents a very valuable technique, to study microorganism charac­
teristics that are difficult to deduce from genetic data (e.g.: growth
characteristics, metabolism, or physiology) (Lewis et al., 2021). The
databases used on MALDI-TOF protocols, strongly influence the method
accuracy and efficiency of identification. Currently, MALDI BioTyper™
(Bruker Daltonics, Inc.) and VITEK® MS Plus (bioMérieux) are the most
used databases. They differ in the used algorithms for identification, as
well in the procedures of how the spectra are obtained, Main Spectrum
analysis, and SuperSpectrum approach, respectively (Jang and Kim,
2018). However, results provided by databases are biased toward clin­
ical and food isolates since it is the most common application for MALDI-
TOF analysis. Therefore, identification of environmental microorgan­
isms may not be accurate as for clinical or food specimens (Kraková
et al., 2018). This lead to the development of independent databases,
focused on specific microorganisms of interest, using appropriate soft­
ware (Moussa et al., 2021). Brucker developed two software modules for
analysis, the MBT compass module and the MBT compass Explorer
module. First is based on the automatic acquisition and analysis of the
acquired spectra with default options (“only analyse” software). The
second is a module that allows the creation and addition of databases, as
well as the selection of multiple parameters for raw spectra analysis, as
exemplified by Moussa and coworkers (Moussa et al., 2021). However,
the proper development of environmental databases requires the access
2
Aquaculture 556 (2022) 738263
to microorganisms collections previously characterized and well main­
tained, which is not easily accessible to smaller research centers. Even
though bacterial mass spectrums can be downloaded to complete or
complement databases, the acquisition of software and up-to-date da­
tabases could constrain the development of works.
In an attempt to overcome such limitations, Tomachewski and co­
workers developed Ribopeaks (RBP), an open-access database, which is
centered on the creation of spectral information from ribosomal proteins
data stored at GenBank and using it for bacterial taxonomic classifica­
tion, through artificial intelligence algorithms. The GenBank offers ac­
cess to the sequences of 2,807,341 amino acids belonging to 57 different
r-protein families (Tomachewski et al., 2018).
On the geographical region of this study (Madeira Island, Portugal),
microbial studies on marine coastal areas are scarce when compared
with marine macro, microfauna or flora. From the analysis of literature
published between 2015 and 2021, from the marine research center
Oceanic Observatory of Madeira (OOM), only two are relative to mi­
crobial community analysis, one associated with aquaculture nets
(Canada et al., 2020) and the other to top shell community of Phorcus
sauciatus (Sousa et al., 2021). Other existing studies are related to the
analysis of sand and seawater quality control which are focused on
Coliforms, Escherichia coli, and Enterococcus spp. (Abreu et al., 2016;
Pereira et al., 2013).
This work aims to determine if the MALDI-TOF methodology asso­
ciated with RBP database analysis, is capable to establish the diversity of
microbial communities and distinguish seasonal variability, through the
qualitative analysis of the identified microorganisms.
2. Material and methods
2.1. Sample collection and geographical area
The samples collection was performed at three locations presented in
Fig. 1. Two associated with fish farm facilities, one located at Cam­
panário, in Ribeira Brava (RB), Madeira (32◦ 39′ 38.1”N 017◦ 03′ 22.1”W)
and the other in Baía d’Abra, Caniçal (CNL), Madeira (32◦ 44′ 31.4”N
016◦ 41′ 26.7”W). The third place was chosen between farms locations at
Caniço, Santa Cruz (STCZ), Madeira (32◦ 38′ 42.8”N 016◦ 49′ 31.4”W).
The samples were taken in three different periods, September 2020,
February 2021, and between May–June 2021. The sampled volume was
3 L of superficial seawater at each location, in sterile dark glass bottles.
The samples were stored in hermetical sealed containers until the lab­
oratory arrival and processed upon arrival.
2.2. Microbial cultivation
The cultivation strategy was based on the work of Sanz-Saez (Sanz-
Sáez et al., 2020). In brief, heterotrophic bacteria that grow easily under
laboratorial cultures were retrieved using Marine Agar 2216 (Difco ™),
to recover comparable portions between different oceanographic con­
ditions or future studies. The objective was not to increase the novelty of
the isolates but to access the capability of the approach to correlate with
the ones already known.
One litter of seawater samples was filtered through 0.2 μm mem­
brane filters (Whatman ™). The filters were placed on 10 mL of Ringer
solution (Oxoid ™) and serial dilutions were performed. Colonies iso­
lates were obtained through the addition of 100 μl (10× diluted and
undiluted) on marine agar plates, incubated at 22 ◦ C for 72 h. All col­
onies were analysed.
2.3. MALDI sample preparation
The Brucker Bacterial Test Standard (BTS) was prepared in 50 μL of
standard solvent (acetonitrile 50%, water 47.5%, and Trifluoroacetic
acid 2.5%). Aliquots 5 μL of BTS solution were stored at − 80 ◦ C into
screw cap microtubes. The matrix used was α-Cyano-4-hydroxycinnamic
J. Freitas et al. Aquaculture 556 (2022) 738263

Fig. 1. Location of the sampling areas. Ribeira Brava (RB); Santa Cruz (STCZ); Caniçal (CNL).

acid (HCCA) in a concentration of 1 mg/mL, dissolved in standard
solvent.
For MALDI analysis, BTS solution (1 μL) was placed on the MALDI
target and dried at room temperature. Immediatley after drying 1 μL of
HCCA solution was added. The single colonies were extracted from the
agar plate with a 1 μL loop and directly spread on the MALDI target
plate. The same BTS procedure was repeated for each colony in tripli­
cate, and a total number of 434 colonies were analysed, with the number
of colonies analysed for sampling location being: Sumer - CNL 53; RB 22;
STCZ 24; Winter - CNL 41; RB 94; STCZ 43; Spring - CNL 70; RB 55; STCZ
32 (Supplementary Table 1).
2.4. MALDI spectra acquisition and MBT analysis
Autoflex maX device MALDI-TOF MS (Bruker Daltonic, Bremen,
Germany) and the software module MBT compass version 3.4 (Bruker
Daltonic, Bremen, Germany), was used for single colonies identification.
This module only allow the identification of the analysed colonies with
default conditions. The AutoXecute acquisition control software pa­
rameters were 240 laser shots in 40 shot steps. Each spot was measured
twice automatically. The instrument was calibrated using a BTS stan­
dard (Bruker Daltonic, Bremen, Germany). The automatic applied MSP
Lybraries were BDAL (2019–04-12 T11:52:02.118) and IVD (2019–04-
12 T11:52:02.337). Reports generated from MBT compass analyses,
comprised the the rank quality, matched pattern (microorganism ID),
score value, and the NCBI identifier.
To facilitate the comparison with the results from the Ribopeaks all
matched patterns results were considered independently the score
values (high confidence 2.00–3.00; Low confidence 1.70–1.99; uncer­
tain identification 1.00–1.69). For databases comparison the results
were rearranged in the following groups, according to NCBI Taxonomy
database: Aquatic Environment; Human associated; Animal associated;
Soil/Plant; and Others (Table 1).
2.5. Ribopeaks software and data analysis
The extraction of the peak lists was performed on the Flex Control 3.4
software. Peak lists were extracted as mzmxl files. The extracted peaks
were placed on the RBP software (http://www.ribopeaks.com) (Tom­
achewski et al., 2018).
The chosen criteria to run the program were: 30S and 50S option was
selected since the algorithm was able to search in the complete genome
database or in the 30S/50S database. The other option was the species
artificial intelligence protocol since one of the main objectives was to
establish the possible environmental origin of the microorganisms.
The retrieved information was: all ten microorganisms identification
results (mID), the number of similar peaks, and total parity percentage
for each mID. The results were transferred to Excel software for the
determination of the percentage of occurrence of the mID.
The information regarding the environment of origin of each
microorganism was retrieved from NCBI Taxonomy Browser. Statistical
analysis was performed on software STATISTICA 10.0 (Stat Soft, Inc.,
Tulsa, OK, USA).
3. Results and discussion
3.1. Ribopeaks vs MBT compass
For each peak list inserted on Ribopeaks software the best first 10
results were exhibited and registered. The information retrieved from
Ribopeaks was mID, the number of similar peaks, and the total parity
percentage for each result.
The results from the MBT analysis were automatically provided by
the software as a PDF file, with the rank quality, matched pattern
(microorganism ID), score value, and the NCBI identifier. Only the
samples that presented the results “no peaks found” were not analysed.
In both cases, the 10 results that were provided were analysed,
independently of the score value or the parity percentage. To facilitate
the comparison and analysis from both softwares, each mID result was
attributed to one of the following groups, according to the main envi­
ronment from which was first sampled (according to NCBI Taxonomy
database): Aquatic Environment – encompassing results from marine and
freshwater environment, aquatic fauna and flora; Human associated –
related with sewage, clinical or commensal do human body; Animal
associated – mostly terrestrial animals and birds; Soil/Plant – results
associated with rhizosphere, plant structures; and Others – included

Table 1
Comparison between databases results according to the formed groups, for the samples taken in the summer from the studied sampling zones. Ribeira Brava (RB); Santa
Cruz (STCZ); Caniçal (CNL) The data is presented in the form of percentage of occurrence.
Database Local Colonies (n◦ ) Genus (n◦ ) Total hits Aquatic (%) Human (%) Animal (%) Plant/soil (%) Other (%)

Ribopeaks CN 53 132 520 43.0 18.7 11.8 21.6 5.0

RB 22 76 220 33.5 25.1 9.8 28.8 2.8
STCZ 24 92 240 37.0 23.3 15.4 24.2 8.7
MBT CN 41 78 410 28.3 25.4 11.5 22.0 12.9
RB 16 58 160 16.3 33.1 15.6 18.1 16.9
STCZ 16 56 160 21.3 27.5 11.3 22.5 17.5
p-values 0.024 0.105 0.841 0.187 0.010

3
J. Freitas et al.
microorganisms with origins other than the previous groups. The in­
formation about the possible environment of origin was retrieved from
the NCBI taxonomy database. This grouping structure was used for all
sampling sites comparison.
For the comparison between RBP and MBT compass results, the
summer data were chosen in all the sampling locations and are pre­
sented in Table 1.
From the results is possible to confirm the existence of significant
statistical differences (p < 0.05) between the “Aquatic” and the “Other”
groups. The analysis done with the RBP database was able to indicate a
much more variety of mID related to the aquatic environment other than
the MBT compass analysis. Also, RBP provided fewer results with un­
related origins (e.g.: engineered origin, food fermentation, or laboratory
environment) knowing that the inputs are from an environmental
sample. On the other hand, the MBT database provided similar results (p
> 0.05) to RBP for the remaining groups “Human”, “Animal”, “Plant/
soil”.
These results seam to support the idea that MBT compass analysis, at
least the “only analyse” version, presents some bias toward the analysis
of clinical samples (Kraková et al., 2018), while RBP shows greater
potential for the analysis of environmental samples. In the case of RBP,
the use of the GenBank database allows access to a higher number of
protein amino acid sequences with diverse origins from which was
predicted the mass spectra of each protein (Tomachewski et al., 2018),
while in the case of MBT compass software the database used was a
default setting, created from the direct MALDI-TOF analysis of the
collected specimens and to which was not possible to add complemen­
tary spectras or databases (Jang and Kim, 2018; Santos et al., 2016).
Since in this case, the samples have a clear environmental origin (marine
environment), the RBP data analysis was chosen to proceed with the
study.
3.2. Genus diversity analysis
All the identified genus as well the total number of results analysed
for each location and different seasons, are presented in Supplementary
Table 1. The values used are from the analysis of the genus frequency of
the occurrence of the mID and the total number of mID. Fig. 2 is a
representation of the genera with the highest frequency of occurrence,
for better visualization of the variability between seasons and locations.
By decreasing order the selected genera were: Vibrio > Pseudoalter­
omonas > Xanthomonas > Alteromonas > Staphilococcus > Escherichia >
Pseudomonas > Photobacterium > Acinetobacter. In the following sub­
sections, each genera occurrence will be analysed, having into consid­
eration the season and the location.
3.2.1. Vibrio
Vibrio is widely distributed in marine environments and is an
important element of the aquatic ecosystem due to its role in the
biogeochemical cycle (Huang et al., 2021). The abundance and com­
munity composition of Vibrio is a complex dynamic affected by envi­
ronmental factors (e.g.: temperature, salinity, or dissolved oxygen),
chemical (e.g.: inorganic and organic nutrients), and biological (e.g.:
protozoa, marine animals, and algae) (Zhang et al., 2018). In the case of
temperate regions, due to low variance of temperature, the concentra­
tions of organic and inorganic nutrients, as well phytoplankton com­
munities, could have a significant role on Vibrio diversity. Also, Vibrio
can enter in a viable but non-culturable state under adverse environ­
mental conditions (e.g.: oligotrophic or sunlight radiation) but main­
taining a quick growth response when the conditions become favorable
(e.g.: nutrient concentration), transitioning from a small part of the
community to a dominant bacterium in response to environmental and
climate variances (Ruiz-Cayuso et al., 2021). Therefore, recognizing the
dynamic alterations of the Vibrio community is of great importance for
the sustainable improvement of aquaculture and public health control.
From Fig. 2, is possible to see that summer is the period with lower
4
Aquaculture 556 (2022) 738263
frequency (CNL 12%, RB 1%, STCZ 6%) comparing with the other sea­
sons. Its presence throughout the year could be related to the temperate
conditions associated with Madeira Island. The increase in the spring
(CNL 44%, RB 13%, STCZ 30%) and winter (CNL 28%, RB 30%, STCZ
51%), is probably due to high nutrient inputs during the more frequent
raining periods. The synergetic effect of other parameters could also
impact on its occurrence during the summer, such as the example of
oxygen, salinity, and sunlight radiation.
3.2.2. Pseudoalteromonas
Pseudoalteromonas are Gram-negative, aerobic, and heterotrophic
bacteria that are ubiquitous obligatory marine bacteria. The strains
affiliated to this genus are often described as fast-growing bacteria, able
to efficiently convert dispersed organic matter into growth substrates
(Parrilli et al., 2021). It is present in all marine habitats, such as coastal,
open, and deep-sea waters, sediments, is also frequently associated with
healthy algae, invertebrates, and marine animals (Atencio et al., 2018).
Few strains have been described as pathogenic or opportunistic being
some examples of P. piscicida, P. agarivorans, and Pseudoalteromonas
undina (Offret et al., 2016; Pujalte et al., 2007; Rosenberg et al., 2013).
Therefore, having the respective precautions, this genus could be an
indicator of the relatively good status of the marine environment. In this
case is necessary to highlight the relative occurrence in the CNL sam­
pling zone, which is the one with a significantly higher percentage
during winter (42%) and the summer (21%).
3.2.3. Xanthomonas
Xanthomonas spp. is a Gram-negative bacterial genus in the class
Gammaproteobacteria, mostly associated with plants diseases (Timil­
sina et al., 2020). Therefore, its relative abundance in the results could
most probably originate from strong runoffs originated from inland
during rainy periods. This could be verified when compared the relative
abundance during the winter (CNL 8%, RB 9.5%, STCZ 15%) and spring
(CNL 11%, RB 8.4%, STCZ 16%) while in the summer season its presence
is basically in the STCZ (<2%) sampling site, which is closer to a water
stream. This location also presented a higher occurrence during the
rainy seasons.
3.2.4. Alteromonas
The genus Alteromonas incorporates Gram-negative, aerobic, motile
bacteria living in marine tropical and temperate waters, in coastal, open
waters, or deep sea (Feng et al., 2021; Ivars-Martinez et al., 2008). This
genus may be dominant in heterotrophic conditions since is generally
described as a copiotroph organism, which means it grows very rapidly
when organic nutrients are available in an oligotrophic marine envi­
ronment (Math et al., 2012). Therefore, it has an important active role in
the circulation of biogenic mater, energy flow in the ocean and as a
growth facilitator for other species (Zinser, 2018a, 2018b).
From the present data in Fig. 2, is possible to see a specific event in
winter at CNL location, where the Alteromonas occurrence was 36%,
which is probably not due to aquaculture operation since in the spring
season the occurrence is zero in all locations. However, in the summer
the Alteromonas occur (CNL 2%, RB 6%, STCZ 8%), which may be due to
higher temperatures and the increase in the nutrient’s availability, but
not as significant as in the wintertime at CNL location. Also, interesting
is the increase in the frequency in the summer at the locations associated
with recreational zones.
3.2.5. Staphylococcus
Staphylococcus spp. is considered a commensal genus from human
skin, but also to domestic animals and wildlife (van Elk et al., 2012;
Vanderhaeghen et al., 2015). The genus comprises several opportunistic
pathogens, that have been gathering attention due to its relevant pres­
ence on clinical samples and association to diverse infections (e.g.:
S. aureaus) (Preda et al., 2021). Therefore, its occurrence in the marine
environment is normally linked with recreational bath areas as well with
J. Freitas et al. Aquaculture 556 (2022) 738263

a) Vibrio

Pseudoalteromonas

Xanthomonas

Alteromonas

Staphylococcus

Escherichia

Pseudomonas

Photobacterium

Acinetobacter

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

STCZ RB CNL
b)
Vibrio

Pseudoalteromonas

Xanthomonas

Alteromonas

Staphylococcus

Escherichia

Pseudomonas

Photobacterium

Acinetobacter

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0

STCZ RB CNL

c)
Vibrio

Pseudoalteromonas

Xanthomonas

Alteromonas

Staphylococcus

Escherichia

Pseudomonas

Photobacterium

Acinetobacter

0.0 5.0 10.0 15.0 20.0 25.0

STCZ RB CNL

Fig. 2. Most relevant bacteria genus, according to season and sampling location. Season: a) Winter; b) Spring; c) Summer. This information was added between the
lines 113 and 123: Grey-Santa Cruz (STCZ); Blue-Caniçal (CNL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

5
J. Freitas et al.
urban or rural runoffs, in high anthropogenically influenced zones
(Levin-Edens et al., 2011).
The occurrence of Staphylococcus spp. in the sampling point are
relatively constant throughout the sampling time (winter - CNL 9%, RB
4%, STCZ 3%; spring - CNL 4%, RB 3%, STCZ 4%; summer - CNL 3%, RB
6%, STCZ 4%). In the case of CNL and RB, other than runoffs sources, the
presence of aquaculture workers throughout the year could be a reason
for the relative constant presence of the genus in the marine water
samples.
3.2.6. Escherichia
The genus Escherichia and the species E. coli is one of the most studied
model microorganisms since it is referred to as one of the most important
indicators of water quality (fecal indicator) and an important pathogenic
agent (Ingle et al., 2011; Tenaillon et al., 2010). E. coli is mainly asso­
ciated with the intestinal tract of homeothermic organisms (e.g.:
humans, other mammals, and birds) reaching the environment through
feces or wastewater treatment plants (Gordon, 2013; Jang et al., 2017).
In this study its relative abundance was higher in the winter (CNL
12%, RB5%, STCZ 2%) and spring (CNL 4%, RB 5%, STCZ 6%) and,
lower in the summer period (CNL 1%, RB3%, STCZ 1%). The presence of
this genus on the samples throughout the sampling time is not unex­
pected since in aquaculture facilities, is common the presence of sea­
birds. However, is not possible to dismiss the occurrence of episodic
wastewater discharges as a major source for Escherichia occurrence
peaks, due to the proximity of treatment plants.
3.2.7. Pseudomonas
The Pseudomonas genus is one of the most ubiquitous genera, with
species isolated from very diverse ecological niches (e.g.: water, soil,
plants, animals, humans, tropics, and Antarctica environments) (Lalucat
et al., 2020; Peix et al., 2009). Its metabolic and physiological versatility
gives them tools that promote their persistence in diverse environments
(Bravakos et al., 2021).
Therefore, this is one of the genera that are not easy to determine the
most probable origin, without a specific species analysis. Its presence in
the sample’s locations is: CNL 10%, RB 6%, STCZ 1%, for the winter;
CNL 2%, RB5%, STCZ 3% in the spring; and CNL 2%, RB 3%, STCZ 6% in
the summer.
3.2.8. Photobacterium
The genus Photobacterium is very prosperous in worldwide oceans
and has considerable ecophysiological variety. The environments and
isolation sources of these species comprise coastal, open-ocean, deep-
sea, sea sediments, and saline lake waters. They can also be found in a
variety of marine organisms with which is established distinct re­
lationships, from symbiotic to pathogenic (Labella et al., 2017). Mem­
bers of this genus are also effective saprotrophs in marine habitats
(Urbanczyk et al., 2011), and more recently have been identified in meat
products, increasing its environmental influence (Fuertes-Perez et al.,
2019).
In this study its relative abundance is higher in the winter (CNL 7%,
RB3%, STCZ 3%) and spring (CNL 4%, RB3%, STCZ 7%) and, and
virtually inexistent in the summer period (CNL 0%, RB0%, STCZ 1%).
3.2.9. Acinetobacter
Acinetobacter is a bacterial genus of Gram-negative, non-motile and
aerobic microorganisms that are also referred to as commensals. Most of
the Acinetobacter spp. are commonly found in the natural environment
where they act as cleaning agents for hydrocarbons, heavy metal
contamination, and phosphorus removal (Adewoyin and Okoh, 2018).
Common environments are wastewater, sewage, healthy humans, ani­
mals, water bodies, soil, and food (Carvalheira et al., 2021; Jung and
Park, 2015). Some species have been also identified as pathogens in
clinical environments (Doughari et al., 2011).
In this study, its relative abundance is higher in the winter and spring
6
Aquaculture 556 (2022) 738263
(CNL 2%, RB 4%, STCZ 4%), while relatively low for winter (CNL 1%,
RB 7%, STCZ 3%) and summer (CNL 1%, RB 5%, STCZ 1%) with
exception of RB location, with low variability throughout the seasons.
Since RB is the site with the higher number of cages, one possible reason
for this occurrence could be due to the dominant presence of the
microorganism in the fish gut, associated with resistance to unfavorable
conditions of the environment, and the capacity to produce antibiotic
compounds against other microorganisms (Etyemez and Balcázar,
2015).
3.3. Sources of inputs pressure
Madeira waters are characterized as oligotrophic, temperate tem­
peratures and a small island platform with great depths (Torres and
Andrade, 2010). Also, oceanographic conditions have a role in the
dispersal of nutrients, temperature, salinity throughout the water col­
umn, and impact microbial diversity.). However, this is not the subject
of this work, more information about the oceanographic conditions
around Madeira Island can be found in other works (Alves et al., 2020;
Azevedo et al., 2021; Narciso et al., 2019).
The orography of Madeira Island is steep, favoring the natural cre­
ation of streams with different sizes and flow intensities. In Fig. 3b, c,
and d it is represented the main (blue lines) and secondary (light blue
lines) water streams near the sampling areas. The presence of these
streams represents one of the major sources of inputs related with soil,
plants, agriculture, or sewages. It is also indicated in the same figures the
location of wastewater treatment plants, portuary zones, and recrea­
tional areas.
RB (Fig. 3b) is the sampling site under the major influence of
freshwater streams. Seven main streams being one of them among the
majors in hydrographic basin area, located between the recreational
zone at the west and the portuary zone at the east (Caetano, 2014). Also,
the nearby port, besides given support to the aquaculture facilities, is
used by smaller fishing vessels to re-supplies and bait preparation. The
aquaculture facility is constituted of 16 cages of 25 m in diameter and is
the largest in this study.
In CNL (Fig. 3d), the sampling area near the aquaculture facility is
the most isolated in terms of anthropogenic and freshwater streams in­
fluence. The point with most pressure near the aquaculture facility is a
small pear that supports the touristic point of ‘Cais do Sardinha’, and
Madeira natural park rangers (http://www.visitmadeira.pt/en-gb/wha
t-to-do/activities/search/pr8-vereda-da-ponta-de-sao-lourenco). Is
often used by the tourists as a bathing zone. On the other hand, is closer
to the island’s main port for cargo ships and fishing vessels. The aqua­
culture infrastructure is formed by 8 cages of medium size, 2.800 m3
each, currently being the smallest cage structure on the island.
STCZ sampling zone (Fig. 3d) is characterized for being a high-
frequency recreational zone and near a wastewater treatment plant,
with a history of occasional emergency flushes. However, is necessary to
refer that the main recreational zones in STCZ and RB are regularly
examined for water quality and frequently attributed with the Blue Flag
Award (https://www.blueflag.global/; https://bandeiraazul.abae.pt/).
In the case of the aquaculture facilities, the position of the cages
respects the recommendation of the Marine Space Planning from the
Madeiran Government (Png-Gonzalez et al., 2019; Torres and Andrade,
2010). The structures are positioned between the bathymetric depth of
20-80 m, being exposed to marine currents that guarantee the proper
water flow between the structures and effective dispersion of the
nutrients.
The variety of input sources in the coastal area, plus the complexity
of the interactions between environmental conditions, increases the
challenge of the microbial community analysis. This work opens the
possibility to infer the influence of future large-scale land nutrient inputs
(episodic or seasonal) on bacterial community, like the ones studied by
Abreu et al. (2016) and Pereira et al. (2013) (Abreu et al., 2016; Pereira
et al., 2013). On the other hand, due to the oceanographic conditions
J. Freitas et al.
Fig. 3. Location of the sampling areas and possible sources of nutrient input. a) Madeira Island. b) Ribeira Brava (RB) – sampling site location and nearby sources of
nutrient inputs. c) Santa Cruz (STCZ) – sampling site location and nearby sources of nutrient inputs. d) Caniçal (CNL) – sampling site location and nearby sources of
nutrient inputs.
characteristic to the island, long-term effects of the nutrients inputs are
not expected (Png-Gonzalez et al., 2019). However, for aquaculture
activities it might have drastic impacts with an increment of mortality
events, emphasizing the need for monitorization.
4. Conclusion
This work aimed to determine the capability of microbial analysis
with the MALDI-TOF approach to establish the microbial community
diversity of coastal superficial seawaters, near aquaculture facilities and
the influence of anthropogenic pressure.
The choice of using a standard marine medium was intending to
reach the most common heterotrophic bacterial fraction. Even though
the use of standard culture media will leave out many other relevant
microorganisms, it will facilitate the comparison with future studies and
between marine environments. Also, the identification of new species
was not the objective of this study, so the purification of bacterial iso­
lates was not performed (Sanz-Sáez et al., 2020).
The main conclusions of this work are the capability of the RBP
database to be used for environmental analysis since it was able to
identify a much higher diversity of marine bacteria when compared with
the MBT Compass software (“only analyse” module). These results are
corroborated by other works that state the limitations of some current
databases for microorganism identification from environmental samples
(Kraková et al., 2018). However, in this work the full potential of MBT
Compass Explorer module was not tested; future work should be per­
formed having in consideration that this module allow the addition of
complementary databases, individual or groups of mass spectras, as well
the selection of settings for spectrum analysis.
Furthermore, the present results demonstrate another possible uti­
lization of the MALDI-TOF method, as a method for the determination of
the overall bacterial community in superficial waters. The utilization of
MBT Compass protocol and the software automatic analysis, reduce
some of the variabilities associated with the cultivation and instru­
mental parameter (Santos et al., 2016). On the other hand the automatic
analysis have some restrictions in selection of alternative settings for
spectra analysis, addition of spectras that were acquired outsied the
automatic protocol and the use of complementary databases. In this
case, the RBP database, has an important role, since it allows access to
7
Aquaculture 556 (2022) 738263
much higher variability of proteins mass spectrums to support the
analysis (Tomachewski et al., 2018). The utilization of AI methodologies
for the creation of databases from already existent data, reveals a very
promising future in the improvement of bacterial databases. Improve­
ments on the software, or the development of new ones, might expand
the information output, making it closer to the information analysis
associated with the analysis of sequencing methodologies.
Further studies are necessary to confirm the present results, as the
comparison with quantitative methodologies in order to determine the
relevance of the percentages of frequency of the identified microor­
ganism. Since the MALDI-TOF analysis presents some variability in its
results, studies for comparison with the DNA sequences might be
necessary.
The present study allowed to elucidate the need for more studies on
the interactions between aquaculture production and the anthropogenic
influence in coastal waters, and the impact it might have on aquaculture
environmental control parameters.
As an overall conclusion, the studied approach has a clear potential
for fast and cheaper routine analysis of seawater microbial community,
which is becoming more relevant on the development of quality control
protocols in the aquaculture industry.
CRediT authorship contribution statement
Jorge Freitas: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Resources, Data curation, Writing – original
draft, Writing – review & editing, Visualization. Rosa Perestrelo:
Methodology, Validation, Investigation, Resources, Data curation,
Writing – review & editing. Paulo Vaz-Pires: Conceptualization,
Writing – review & editing, Supervision, Project administration. José S.
Câmara: Conceptualization, Resources, Writing – review & editing,
Visualization, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
J. Freitas et al.
Acknowledgments
This work was supported by FCT-Fundação para a Ciência e a Tec­
nologia through the CQM Base Fund - UIDB/00674/2020, and Pro­
grammatic Fund - UIDP/00674/2020, and by ARDITI-Agência Regional
para o Desenvolvimento da Investigação Tecnologia e Inovação, through
the project M1420-01-0145-FEDER-000005 - Centro de Química da
Madeira - CQM+ (Madeira 14-20 Program). The authors also acknowl­
edge the financial support from FCT - Fundação para a Ciência e Tec­
nologia and Madeira 14-2020 program to the Portuguese Mass
Spectrometry Network through PROEQUIPRAM program, M14-
20M1420-01-0145-FEDER-000008). The authors also acknowledge
ARDITI and IlhaPeixe S.A., through the support granted under the
M1420 Project-09-5369-FSE-000001 - for PhD grant to Jorge Freitas.
The authors also acknowledge the staff from Aquabaia in the person of
Rui Gonçalves, for the help in the samples collection.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2022.738263.
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Another random document with
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in the history both of the Republicans and the republic—the name of
that noble soldier and favorite child of victory, Philip H. Sheridan.
In the spirit of these great leaders and of our own devotion to
human liberty, and with that hostility to all forms of despotism and
oppression which is the fundamental idea of the Republican party,
we add fraternal congratulation to our fellow-Americans of Brazil
upon their great set of emancipation, which completed the abolition
of slavery throughout the two American continents. We earnestly
hope that we may soon congratulate our fellow-citizens of Irish birth
upon the peaceful recovery of Home Rule for Ireland.
We reaffirm our unswerving devotion to the National Constitution
and to the indissoluble union of the States; to the autonomy reserved
to the States under the Constitution; to the personal rights and
liberties of citizens in all the States and Territories in the Union, and
especially to the supreme and sovereign right of every lawful citizen,
rich or poor, native or foreign born, white or black, to cast one free
ballot in public elections, and to have that duly counted. We hold the
free and honest popular ballot and the just and equal representation
of all the people to be the foundation of our Republican government,
and demand effective legislation to secure the integrity and purity of
elections, which are the fountains of all public authority. We charge
that the present administration and the Democratic majority in
Congress owe their existence to the suppression of the ballot by a
criminal nullification of the Constitution and laws of the United
States.
We are uncompromisingly in favor of the American system of
protection. We protest against its destruction as proposed by the
President and his party. They serve the interests of Europe; we will
support the interests of America. We accept the issue and confidently
appeal to the people for their judgment. The protective system must
be maintained. Its abandonment has always been followed by
general disaster to all interests except those of the usurer and the
sheriff. We denounce the Mills bill as destructive to the general
business, the labor and the farming interests of the country, and we
heartily endorse the consistent and patriotic action of the Republican
Representatives in Congress in opposing its passage.
We condemn the proposition of the Democratic party to place wool
on the free list, and we insist that the duties thereon shall be adjusted
and maintained so as to furnish full and adequate protection to that
industry.
The Republican party would effect all needed reduction of the
national revenue by repealing the taxes upon tobacco, which are an
annoyance and burden to agriculture, and the tax upon spirits used
in the arts and for mechanical purposes, and by such revision of the
tariff laws as will tend to check imports of such articles as are
produced by our people, the production of which gives employment
to our labor, and release from import duties those articles of foreign
production (except luxuries) the like of which cannot be produced at
home. If there shall still remain a larger revenue than is requisite for
the wants of the Government, we favor the entire repeal of internal
taxes rather than the surrender of any part of our protective system
at the joint behest of the whisky trusts and the agents of foreign
manufacturers.
We declare our hostility to the introduction into this country of
foreign contract labor, and of Chinese labor, alien to our civilization
and our Constitution, and we demand the rigid enforcement of the
existing laws against it, and favor such immediate legislation as will
exclude such labor from our shores.
We declare our opposition to all combinations of capital organized
in trusts or otherwise to control arbitrarily the condition of trade
among our citizens, and we recommend to Congress and to the State
Legislatures in their respective jurisdictions such legislation as will
prevent the execution of all the schemes to oppress the people by
undue charges on their supplies, or by the unjust rates for the
transportation of their products to market. We approve the
legislation by Congress to prevent alike unjust burdens and unfair
discriminations between the States.
We reaffirm the policy of appropriating the public lands of the
United States to be homesteads for American citizens and settlers,
not aliens, which the Republican party established in 1862, against
the persistent opposition of the Democrats in Congress, and which
has brought our great western domain into such magnificent
development. The restoration of unearned railroad land grants to the
public domain, for the use of the actual settlers, which was begun
under the administration of President Arthur, should be continued.
We deny that the Democratic party has ever revoked one acre to the
people, but declare that, by the joint action of Republicans and
Democrats, about fifty millions of acres of unearned lands originally
granted for the construction of railroads have been restored to the
public domain, in pursuance of the conditions inserted by the
Republican party in the original grants. We charge the Democratic
administration with failure to execute the laws securing to settlers
titles to their homesteads, and with using appropriations made for
that purpose to harass innocent settlers with spies and prosecutions
under the false pretence of exposing frauds and vindicating the law.
The Government by Congress of the Territories is based upon
necessity only, to the end that they may become States in the Union;
therefore, whenever the conditions of population, material resources,
public intelligence and morality are such as to insure a stable
Government therein, the people of such territories should be
permitted, as a right inherent in them, the right to form for
themselves constitutions and State Governments and be admitted
into the Union. Pending the preparation for statehood, all officers
thereof should be selected from the bona fide residents and citizens
of the territory wherein they are to serve. South Dakota should of
right be immediately admitted as a State in the Union under the
Constitution framed and adopted by her people, and we heartily
endorse the action of the Republican Senate in twice passing bills for
admission. The refusal of the Democratic House of Representatives,
for partisan purposes, to favorably consider these bills, is a willful
violation of the sacred American principle of local self government
and merits the condemnation of all just men. The pending bills in the
Senate for acts to enable the people of Washington, North Dakota
and Montana territories to form Constitutions and establish State
Governments, should be passed without unnecessary delay. The
Republican party pledges itself to do all in its power to facilitate the
admission of the Territories of New Mexico, Wyoming, Idaho and
Arizona to the enjoyment of self-government as States, such of them
as are not qualified as soon as they may become so.
The political power of the Mormon church in the Territories, as
exercised in the past, is a menace to free institutions, a danger no
longer to be suffered;
Therefore, we pledge the Republican party to appropriate
legislation asserting the sovereignty of the Nation in all Territories
where the same is questioned, and in furtherance of that end to place
upon the statute books legislation stringent enough to divorce the
political from the ecclesiastical power, and thus stamp out the
attendant wickedness of polygamy.
The Republican party is in favor of the use of both gold and silver
as money, and condemns the policy of the Democratic
Administration in its efforts to demonetize silver.
We demand the reduction of letter postage to one cent per ounce.
In a Republic like ours, where the citizen is the sovereign and the
official the servant; where no power is exercised except by the will of
the people, it is important that the sovereign—the people—should
possess intelligence. The free school is the promoter of that
intelligence which is to preserve us as a free nation; the State or
nation, or both combined, should support free institutions of
learning sufficient to afford to every child growing up in the land the
opportunity of a good common-school education.
We earnestly recommend that prompt action be taken by Congress
in the enactment of such legislation as will best secure the
rehabilitation of the American merchant marine, and we protest
against the passage by Congress of a free ship bill, as calculated to
work injustice to labor by lessening the wages of those engaged in
preparing materials as well as those directly employed in our ship
yards. We demand appropriations for the early rebuilding of our
navy; for the construction of coast fortifications and modern
ordnance and other approved modern means of defence for the
protection of our defenceless harbors and cities; for the payment of
just pensions to our soldiers; for necessary works of national
importance in the improvement of harbors and the channels of
internal, coastwise and foreign commerce; for the encouragement of
the shipping interests of the Atlantic, Gulf and Pacific States, as well
as for the payment of the maturing public debt. This policy will give
employment to our labor, activity to our various industries, increase
the security of our country, promote trade, open new and direct
markets for our produce, and cheapen the cost of transportation. We
affirm this to be far better for our country than the Democratic policy
of loaning the Government’s money without interest to “pet banks.”
The conduct of foreign affairs by the present administration has
been distinguished by its inefficiency and its cowardice. Having
withdrawn from the Senate all pending treaties affected by
Republican administrations for the removal of foreign burdens and
restrictions upon our commerce and for its extension into better
markets, it has neither effected nor proposed any others in their
stead. Professing adherence to the Monroe doctrine, it has seen with
idle complacency the extension of foreign influence in Central
America and of foreign trade everywhere among our neighbors. It
has refused to charter, sanction or encourage any American
organization for constructing the Nicaragua canal, a work of vital
importance to the maintenance of the Monroe doctrine and of our
national influence in Central and South America, and necessary for
the development of trade with our Pacific territory, with South
America and with the islands and further coasts of the Pacific Ocean.
We arraign the present Democratic administration for its weak
and unpatriotic treatment of the fisheries question, and its
pusillanimous surrender of the essential privileges to which our
fishing vessels are entitled in Canadian ports under the treaty of
1818, the reciprocal maritime legislation of 1830, and the comity of
nations, and which Canadian fishing vessels receive in ports of the
United States.
We condemn the policy of the present administration and the
Democratic majority in Congress towards our fisheries as unfriendly
and conspicuously unpatriotic, and as tending to destroy a valuable
national industry and an indispensable resource of defense against a
foreign enemy.
The name of American applies alike to all citizens of the Republic,
and imposes upon all alike the same obligation to obedience to the
laws. At the same time that citizenship is and must be the panoply
and safeguard of him who wears it, and protect him, whether high or
low, rich or poor, in all his civil rights, it should and must afford him
protection at home and follow and protect him abroad in whatever
land he may be on a lawful errand.
The men who abandoned the Republican party in 1884 and
continue to adhere to the Democratic party have deserted not only
the cause of honest government, of sound finance, of freedom and
purity of the ballot, but especially have deserted the cause of reform
in the civil service. We will not fail to keep our pledges because they
have broken theirs or because their candidate has broken his. We
therefore repeat our declaration of 1884, to wit: “The reform of the
Civil Service, auspiciously begun under the Republican
administration should be completed by the further extension of the
reform system already established by law to all grades of the service
to which it is applicable. The spirit and purpose of the reform should
be observed in all executive appointments, and all laws at variance
with the object of existing reform legislation should be repealed, to
the end that the dangers to free institutions which lurk in the power
of official patronage may be wisely and effectively avoided.”
The gratitude of the nation to the defenders of the Union cannot be
measured by laws. The legislation of Congress should conform to the
pledge made by a loyal people, and be so enlarged and extended as to
provide against the possibility that any man who honorably wore the
Federal uniform shall become an inmate of an almshouse, or
dependent upon private charity. In the presence of an overflowing
treasury it would be a public scandal to do less for those whose
valorous service preserved the Government. We denounce the hostile
spirit shown by President Cleveland in his numerous vetoes of
measures for pension relief, and the action of the Democratic House
of Representatives in refusing even a consideration of general
pension legislation.
In support of the principles herewith enunciated we invite the co-
operation of patriotic men of all parties, and especially of all
workingmen, whose prosperity is seriously threatened by the free
trade policy of the present administration.
On motion of Hon. Chas. A. Boutelle of Maine, the following was
also adopted:
“The first concern of all good government is the virtue and sobriety
of the people and the purity of the home. The Republican party
cordially sympathizes with all wise and well-directed efforts for the
promotion of temperance and morality.”
COMPARISON OF PLATFORM PLANKS ON
GREAT POLITICAL QUESTIONS.

General Party Doctrines.

 DEMOCRATIC.
1856—That the liberal principles
embodied by Jefferson in the
Declaration of Independence, and
sanctioned in the Constitution, which
makes ours the land of liberty and
the asylum of the oppressed of every
nation, have ever been cardinal
principles in the Democratic faith;
and every attempt to abridge the
present privilege of becoming
citizens and the owners of soil among
us ought to be resisted with the same
spirit which swept the alien and
sedition laws from our statute books.
[Plank 8.
1860—Reaffirmed.
1864—
1868—
1872—We recognize the equality of
all men before the law, and hold that
REPUBLICAN.
1856—That the maintenance of the
principles promulgated in the
Declaration of Independence and
embodied in the Federal Constitution,
is essential to the preservation of our
Republican institutions, and that the
Federal Constitution, the rights of the
States, and the union of the States shall
be preserved; that with our Republican
fathers, we hold it to be a self-evident
truth that all men are endowed with the
inalienable rights to life, liberty, and
the pursuit of happiness, and that the
primary object and ulterior design of
our Federal Government were to secure
these rights to all persons within its
exclusive jurisdiction.
[Plank 1.
1860—That the maintenance of the
principles promulgated in the
Declaration of Independence and
embodied in the Federal Constitution,
“That all men are created equal; that
they are endowed by their Creator with
certain inalienable rights; that among
these are life, liberty, and the pursuit of
happiness; that to secure these rights
governments are instituted among
men, deriving their just powers from
the consent of the governed,” is
essential to the preservation of our
Republican institutions; and that the
Federal Constitution, the rights of the
States, and the Union of the States
must and shall be preserved.
[Plank 2.
1864—
1868—
1872—Complete liberty and exact
equality in the enjoyment of all civil,
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MP25873.
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MP25874.
Unusual behavior patterns in fishes. Thorne Films, Inc. 4 min., si.,
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MP25875.
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MP25876.
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MP25900.
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Corporation; 15Aug73; MP25907.

MP25908.
Teenage relationships: Venessa and her friends. Encyclopaedia
Britannica Educational Corporation. 19 min., sd., color, 16 mm.
(Getting together) © Encyclopaedia Britannica Educational
Corporation; 24Aug73; MP25908.

MP25909.
Arica rhythm. Arica Institute, Inc. 15 min., sd., color, 16 mm. ©
Arica Institute, Inc.; 1Jul73; MP25909.

MP25910.
E-Z Open jar and bottle opener. E-Z Open, Inc. 60 seconds, sd.,
color, 16 mm. © E-Z Open, Inc.; 29Aug74; MP25910.

MP25911.
American tall tale heroes. Coronet Instructional Media, a division
of Esquire, Inc. 15 min., sd., color, 16 mm. © Coronet Instructional
Media, a division of Esquire, Inc.; 12Aug74; MP25911.

MP25912.
Male and female in plants and animals. Coronet Instructional
Media, a division of Esquire, Inc. 10 min., sd., color, 16 mm. ©
Coronet Instructional Media, a division of Esquire, Inc.; 13Aug74;
MP25912.

MP25913.
Germs and your body. Coronet Instructional Media, a division of
Esquire, Inc. 10 min., sd., color, 16 mm. © Coronet Instructional
Media, a division of Esquire, Inc.; 14Aug74; MP25913.
MP25914.
Mammals that gnaw. Coronet Instructional Media, a division of
Esquire, Inc. 12 min., sd., color, 16 mm. © Coronet Instructional
Media, a division of Esquire, Inc.; 20Aug74; MP25914.

MP25915.
Chassis and drive-line highlights: Trail Duster and Ramcharger.
Chrysler Corporation Training Center. Made by Ross Roy, Inc. 18
min., sd., color, Super 8 mm. © Chrysler Corporation; 19Aug74;
MP25915.

MP25916.
The New emission controls. Chrysler Corporation Training Center.
Made by Ross Roy, Inc. 18 min., sd., color, Super 8 mm. © Chrysler
Corporation; 24Sep74; MP25916.

MP25917.
The Man behind the dollar. Chrysler Coloration. Made by Ross
Roy, Inc. 18 min., sd., color, Super 8 mm. © Chrysler Corporation;
4Oct74; MP25917.

MP25918.
The Drug test. Avco Broadcasting Corporation. 55 min., sd., color,
videotape (2 inch) © Avco Broadcasting Corporation; 26Jan71;
MP25918.

MP25919.
A Time to heal. Child and family Service of Springfield, Inc. Made
by Gusfilms. 22 min., sd., b&w, 16 mm. Appl. au: Jeffrey Einstein. ©
Child and Family Service of Springfield, Inc.; 17Sep74; MP25919.

MP25920.
 Threshold: the Blue Angels experience. Gardner-Marlow-Maes
Associates. 111 min., sd., color, 35 mm. Prev. reg. 5May72, LU3636.
NM: additional cinematography & editorial revision. © Gardner-
Marlow-Maes Associates; 5Dec73; MP25920.

MP25921.
Where have all the mothers gone? Avco Broadcasting Corporation.
49 min., sd., color, videotape (2 inch) Avco Broadcasting
Corporation; 21Mar72; MP25921.

MP25922.
Calories: enough is enough. Gilbert Altschul Productions, Inc.
Released by Journal Films, Inc. 12 min., sd., color, 16 mm. Appl. au:
Joel Altschul. © Gilbert Altschul Productions, Inc.; 26Jul74;
MP25922.

MP25923.
James A. Michener. Pt. 1–2. Jaqueline Shachter. 2 motion pictures
(31 min. each), sd., b&w, videotape (1/2 inch) (Profiles in literature)
Add. ti.: Meet James Michener. © Jaqueline Shachter; 28Mar74;
MP25923.

MP25924.
Skittles filled kids. Jack Candies, Ltd. 30 sec., sd., color, 16 mm. ©
Jack Candies, Ltd.; 15Jul74; MP25924.

MP25925.
Obstetrics/gynecology health series. The Educational Division of
Milner-Fenwick, Inc. 8 motion pictures (9 min. each), sd., color.
Super 8 mm. Add. ti: Ob/gyn health series © Milner-Fenwick, Inc.;
1Jun74; MP25925.

MP25926.
 Muhammad Ali (Cassius Clay), champion, versus George Chuvalo,
challenger, for the heavyweight championship of the world. Maple
Leaf Gardens, Toronto, Canada, March 29, 1966. The Big Fights, Inc.
1 reel, sd., b&w, 16 mm. (The Big fights of the decades) Add. ti:
Muhammad Ali versus George Chuvalo for the heavyweight
championship of the world, March 29, 1966, Toronto, Canada. Appl.
au: William D. Cayton. © The Big Fights, Inc.; 15Apr66; MP25926.

MP25927.
Joe Frazier versus Oscar Bonavena, Philadelphia, Pennsylvania,
December 10, 1968. Greatest Fights of the Century, Inc. 1 reel, sd.,
color, 16 mm. (The Big fights of the decades) Add. ti: Joe Frazier
versus Oscar Bonavena for the New York State heavyweight title,
December 10, 1968, Philadelphia, Pennsylvania. Appl. au: William D.
Cayton. © Greatest Fights of the Century, Inc.; 28Jan71; MP25927.

MP25928.
Muhammed Ali versus Henry Cooper for the heavyweight
championship of the world, London, England, May 21, 1966.
Greatest Fights of the Century, Inc. 1 reel, sd., b&w, 16 mm. (The Big
fights of the decades) Appl. au: William D. Cayton. © Greatest Fights
of the Century, Inc.; 16Apr73; MP25928.

MP25929.
Floyd Patterson versus Joey Maxim, Brooklyn, New York, June 7,
1954. Greatest Fights of the Century, Inc. 1 reel, sd., b&w, 16 mm.
(The Big fights of the decades) Add. ti: Floyd Patterson versus Joey
Maxim boxing contest, June 7, 1954, New York City. Appl. au:
William D. Cayton. © Greatest Fights of the Century, Inc.; 7Jun69:
MP25929.

MP25930.
George Foreman versus Ionas Chapulos, Olympic Heavyweight
Championship, Mexico City, Mexico, October 23, 1968. Greatest
Fights of the Century, Inc. 1 reel, sd., color, 16 mm. (The Big fights of
the decades) Add. ti: George Foreman versus Ionas Chapulos for the
Olympic heavyweight title, October 23, 1968, Mexico City, Mexico.
Appl. au: William D. Cayton. © Greatest Fights of the Century, Inc.;
10Feb73; MP25930.

MP25931.
Muhammed Ali versus Oscar Bonavena, New York, New York,
December 7, 1970. Greatest Fights of the Century, Inc. 1 reel, sd.,
color, 16 mm. (The Big fights of the decades) Add. ti: Muhammed Ali
versus Oscar Bonavena boxing contest, December 7, 1970, New York
City. Appl. au: William D. Cayton. © Greatest Fights of the Century,
Inc.; 16Apr73; MP25931.

MP25932.
Muhammed Ali versus Jimmy Ellis, Houston Astrodome, July 26,
1971. The Big Fights, Inc. 1 reel, sd., color, 16 mm. (The Big fights of
the decades) Add. ti: Muhammed Ali versus Jimmy Ellis boxing
contest, July 26, 1971, Houston, Texas. Appl. au: William D. Cayton.
© The Big Fights, Inc.; 10Aug71; MP25932.

MP25933.
Official fight pictures, Muhammad Ali (Cassius Clay) versus Ernie
Terrell for the heavyweight championship of the world, February 6,
1967, Astrodome, Houston, Texas. The Big Fights, Inc. 1 reel, sd.,
color, 16 mm. (The Big fights of the decades) Add. ti: Cassius Clay
versus Ernie Terrell for the heavyweight championship of the world,
February 6, 1967, Houston, Texas. Appl. au: William D. Cayton. The
Big Fights, Inc.; 3Mar67; MP25933.

MP25934.
Muhammad Ali versus Henry Cooper, London, England, June 18,
1963. Greatest Fights of the Century, Inc. 1 reel, sd., b&w, 16 mm.
(The Big fights of the decades) Add. ti: Cassius Clay versus Henry
Cooper boxing contest, June 18, 1963, London, England. Appl. au:
William D. Cayton. © Greatest Fights of the Century, Inc.; 16Apr73;
MP25934.

MP25935.
Official motion pictures, Muhammad Ali (Cassius Clay) versus
Jerry Quarry, October 26, 1970, Atlanta, Georgia. The Big Fights,
Inc. 1 reel, sd., color, 16 mm. (The Big fights of the decades) Appl. au:
William D. Cayton. © The Big Fights, Inc.; 20Nov70; MP25935.

MP25936.
Joe Frazier versus Eddie Machen, Los Angeles, California,
November 21, 1966. Greatest Fights of the Century, Inc. 1 reel, sd.,
color, 16 mm. (The Big fights of the decades) Add. ti: Joe Frazier
versus Eddie Machen boxing contest, November 21, 1966, Los
Angeles, California. Appl. au: William D. Cayton. © Greatest Fights
of the Century, Inc.; 16Apr73; MP25936.

MP25937.
Joe Frazier versus Bob Foster, Detroit, Michigan, November 18,
1970. Greatest Fights of the Century, Inc. 1 reel, sd., color, 16 mm.
(The Big fights of the decades) Add. ti: Joe Frazier versus Bob Foster
for the heavyweight championship of the world, November 18, 1970,
Detroit, Michigan. Appl. au: William D. Cayton. © Greatest Fights of
the Century, Inc.; 28Jan71; MP25937.

MP25938.
Joe Frazier versus Manuel Ramos, New York City, New York, June
24, 1968. Greatest Fights of the Century, Inc. 1 reel, sd., b&w, 16 mm.
(The Big fights of the decades) Add. ti: Joe Frazier versus Manuel
Ramos for the New York State heavyweight title, June 24, 1968, New
York City. Appl. au: William D. Cayton. © Greatest Fights of the
Century, Inc.; 15Apr71; MP25938.

MP25939.
 Floyd Patterson versus Pete Rademacher, Seattle, Washington,
August 22, 1957. Greatest Fights of the Century, Inc. 1 reel, sd., b&w,
16 mm. (The Big fights of the decades) Add. ti: Floyd Patterson
versus Pete Rademacher for the heavyweight title of the world,
August 22, 1957, Seattle, Washington. Appl. au: William D. Cayton.
© Greatest Fights of the Century, Inc.; 15Aug72; MP25939.

MP25940.
Joe Frazier versus Jimmy Ellis for world heavyweight title, New
York, February 16, 1970. Greatest Fights of the Century, Inc. 1 reel,
sd., color, 16 mm. (The Big fights of the decades) Add. ti: Joe Frazier
versus Jimmy Ellis for the heavyweight championship of the world,
February 16, 1970, New York City. Appl. au: William D. Cayton. ©
Greatest Fights of the Century, Inc.; 3Mar70; MP25940.

MP25941.
George Foreman versus Miguel Paez, Oakland, California, May 11,
1972. Greatest Fights of the Century, Inc. 1 reel, sd., color, 16 mm.
(The Big fights of the decades) Add. ti: George Foreman versus
Miguel Paez boxing contest. May 11, 1972, Oakland, California. Appl.
au: William D. Cayton. © Greatest Fights of the Century, Inc.;
17Mar73; MP25941.

MP25942.
Joe Frazier versus Jerry Quarry, New York City, New York, June
23, 1969. Greatest Fights of the Century, Inc. 1 reel, sd., color, 16
mm. (The Big fights of the decades) Add. ti: Joe Frazier versus Jerry
Quarry for the New York State heavyweight title, June 23, 1969, New
York City. Appl. au: William D. Cayton. © Greatest Fights of the
Century, Inc.; 15Apr71; MP25942.

MP25943.
Jersey Joe Walcott versus Ezzard Charles, Philadelphia,
Pennsylvania, June 5, 1952. Greatest Fights of the Century, Inc. 1
reel, sd., b&w, 16 mm. (The Big fights of the decades) Add. ti: Jersey
Joe Walcott versus Ezzard Charles for the heavyweight
championship of the world, June 5, 1952, Philadelphia,
Pennsylvania. Appl. au: William D. Cayton. © Greatest Fights of the
Century, Inc.; 15Feb72; MP25943.

MP25944.
Archie Moore versus Yvon Durelle for the light heavyweight
championship of the world, Montreal, Canada, August 12, 1959.
Greatest Fights of the Century, Inc. 1 reel, sd., b&w, 16 mm. (The Big
fights of the decades) Appl. au: William D. Cayton. Prev. pub.
20Apr67, MP17055. NM: additional cinematographic work. Greatest
Fights of the Century, Inc.; 16Apr73; MP25944.

MP25945.
Ike Williams versus Beau Jack for the lightweight title,
Philadelphia, Pennsylvania, July 12, 1948. Greatest Fights of the
Century, Inc. 1 reel, sd., b&w, 16 mm. (The Big fights of the decades)
Add. ti: Ike Williams versus Beau Jack for the lightweight
championship of the world, July 12, 1948, Philadelphia,
Pennsylvania. Appl. au: William D. Cayton. Prev. pub. 21Jul68,
MP18573. NM: additional cinematographic work. © Greatest Fights
of the Century, Inc.; 8Sep73; MP25945.

MP25946.
Muhammad Ali versus Karl Mildenberger, Frankfurt, Germany,
September 10, 1966. Greatest Fights of the Century, Inc. 1 reel, sd.,
b&w, 16 mm. (The Big fights of the decades) Add. ti: Muhammad Ali
versus Karl Mildenberger for the heavyweight championship of the
world, September 10, 1966, Frankfurt, Germany. Appl. au: William
D. Cayton. © Greatest Fights of the Century, Inc.; 15Apr73 (in notice:
1971); MP25946.

MP25947.
Playbuoy for fun! for sport! For exercise! Gilbert Sacks
Enterprises. 4 min., si., color, Super 8 mm. Appl. au: Gilbert B.
Sacks. © Gilbert Sacks Enterprises; 13Sep74; MP25947.

MP25948.
Field interviews—interview or interrogate. Keyes Hardin
Productions & Woroner Films, Inc. Made by Woroner Films, Inc. 26
min., sd., color, 16 mm. © Woroner Films, Inc.; 4Nov74; MP25948.

MP25949.
Advanced network design techniques. Edutronics Systems
International, Inc. 10 min., sd., color, 16 mm. (Data
communications) © Edutronics Systems International, Inc.;
17Oct74; MP25949.

MP25950.
Concepts of advanced network design. Edutronics Systems
International, Inc. 10 min., sd., color, 16 mm. (Data
communications) © Edutronics Systems International, Inc.;
30Sep74; MP25950.

MP25951.
Keypunch formats. Edutronics Systems International, Inc. 14 min.,
sd., color, 16 mm. (Keypunch 1/0) © Edutronics Systems
International, Inc.; 16Oct74; MP25951.

MP25952.
Executing programs in VS. Pt. 1. Edutronics Systems International,
Inc. 10 min., sd., color, 16 mm. (Virtual storage concepts) ©
Edutronics Systems International, Inc.; 9Sep74; MP25952.

MP25953.
Data control techniques. Edutronics Systems International, Inc. 10
min., sd., color, 16 mm. (Data controls) © Edutronics Systems
International, Inc.; 15Oct74; MP25953.
MP25954.
Living river. Martin Moyer Productions. 21 min., sd., color, 16 mm.
Appl. au: Martin Moyer. © Martin Moyer Productions; 23Aug74;
MF25954.

MP25955.
A Pond. Entheos Communication Corporation. 6 min., sd., color,
16 mm. © Entheos; 15Nov72; MP25955.

MP25956.
Saint Peter’s Church: a people on the move. Saint Peter’s Lutheran
Church of New York City & George C. Stoney Associates. 29 min., sd.,
b&w, 16 mm. © Saint Peter’s Lutheran Church of New York City;
10Dec73; MP25956.

MP25957.
Season of fire. A Hamlin-Barney production. 15 min., sd., color, 16
mm. Appl. au: Jerome Hamlin & John Barney. © Jerome Hamlin &
John Barney (in notice: A Hamlin-Barney production); 30May74 (in
notice: 1973); MP25957.

MP25958.
Ambroise Pare, military surgeon. Yale University, School of
Medicine, The Department of Anatomy & Section of Orthopedic
Surgery. 19 min., sd., color, 16 mm. © Yale University; 12Dec73;
MP25958.

MP25959.
Tonometry. American College of Physicians. 7 min., sd., color,
Super 8 mm. in cassette. (American College of Physicians medical
skills library) © American College of Physicians; 1Aug74; MP25959.

MP25960.