Neotropical Entomology

https://doi.org/10.1007/s13744-021-00895-x

FORUM

Next-Generation Sequencing and Its Impacts on Entomological

Research in Ecology and Evolution
Débora Pires Paula 1

Received: 17 September 2020 / Accepted: 6 July 2021

# Sociedade Entomológica do Brasil 2021

Abstract
The advent of NGS-based methods has been profoundly transforming entomological research. Through continual development
and improvement of different methods and sequencing platforms, NGS has promoted mass elucidation of partial or whole genetic
materials associated with beneficial insects, pests (of agriculture, forestry and animal, and human health), and species of
conservation concern, helping to unravel ecological and evolutionary mechanisms and characterizing survival, trophic interac-
tions, and dispersal. It is shifting the scale of biodiversity and environmental analyses from individuals and biodiversity indicator
species to the large-scale study of communities and ecosystems using bulk samples of species or a mixed “soup” of environ-
mental DNA. As the NGS-based methods have become more affordable, complexity demystified, and specificity and sensitivity
proven, their use in entomological research has spread widely. This article presents several examples on how NGS-based
methods have been used in entomology to provide incentives to apply them when appropriate and to open our minds to the
expected advances in entomology that are yet to come.

Keywords Agroecosystems . conservative biological control . ecosystem services . food webs . insect-vectored diseases . NGS .
pollination

Introduction
High-throughput next-generation sequencing (HT-NGS), of-
ten referred to simply as NGS, is a DNA sequencing method
launched in 2005 (Wheeler et al. 2008; Pareek et al. 2011), in
which multiple fragments are sequenced through a massively
parallel sequencing process. It is based on cluster emulsion or
bridge PCR amplification of DNA fragments (second gener-
ation) or single DNA molecule sequencing (SMS) (third gen-
eration), both with several commercial platforms available or
in development (see Pareek et al. 2011 and Kulski 2016 for
development history, and Shokralla et al. 2012 for how they
work and limitations). The second and third generations of
DNA sequencing differ in several respects from the first-
generation DNA sequencers (Sanger and Coulson 1975;
Sanger et al. 1977; Maxam and Gilbert 1977; Ansorge et al.
Edited by Herbert AA Siqueira
* Débora Pires Paula
debora.pires@embrapa.br
1
Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil
1986, 1987; Smith et al. 1986), such as not requiring knowl-
edge of parts of the DNA sequence to design oligos to initiate
the sequencing reaction, sequencing simultaneously several
regions of the DNA, multiplexing of dozens to hundreds of
samples in a run and generating hundreds of millions of se-
quenced DNA fragments (called reads), and producing “big
data” (Shendure et al. 2017). Due to this advanced methodol-
ogy, NGS-based methods allow the faster elucidation of ge-
nomes and large-scale screening of molecular markers (loci)
in greater numbers of individuals from model or non-model
organisms, with or without previous genomic information, as
well as estimation of genome variation within and between
populations of the same or different species. Consequently,
faster and larger advances in physiological, developmental,
ecological, and evolutionary studies are being achieved.
Initially, the main application of NGS in entomology was
the elucidation of genomes of several insects of interest for
agriculture, urban environments, conservation, and public
health. In March 2020, considering only insects, the public
databases GenBank/EMBL/DDBJ contained 552 nuclear ge-
nomes, 861 assemblies, and 9912 mitogenomes (Fig. 1), in-
cluding biological control agents, pollinators, agricultural
pests, and vectors of diseases (Table 1 and Supporting
 Pires

Fig. 1 Timeline of the insect

genomes released in GenBank up
to March 2020. A Nuclear
genomes (search key
“insecta[organism] genome”),
552 total; B mitogenomes (partial
and complete; search details: key
“insecta[organism]
mitochondrion,” custom
sequence length range from 7 k
and 35 k bp, and custom genome
filtering), 9912 total. Some
species have several releases of
mitogenome sequences. The
species and GenBank identifiers
are provided in Table S1
(Supplementary Material)

Information). More insect genomes are expected to be eluci-
dated at a faster rate in the future, as the costs of NGS have
been decreasing consistently and the technologies and bioin-
formatic pipelines are being periodically improved.
According to Li et al. (2019), 1219 insect genome-
sequencing projects were at that time in progress in
GenBank. The i5k Workspace Project by the National
Agricultural Library – USDA/NAL (https://i5k.nal.usda.
gov/) plans to elucidate and annotate (from 2018 to 2023)
the genomes of 5000 arthropod species (Poelchau et al. 2015).
The unpreceded ease in obtaining insect genomic data has
generated new understandings in insect evolution, genome
size, gene number, gene order (synteny), unique species-
specific genes, genetic, and developmental mechanisms of
evolutionary diversification in the basic body plan
(evolutionary developmental biology or evo-devo) (Cusson
2008). As exemplified further, this knowledge has contributed
to the identification of pest-specific molecules that can be
targeted for monitoring, insecticide development (biorational
target sites, i.e., reduced risk insecticides due to the targeting
of genes that encode proteins unique to insects or to specific
insect taxa, Allenza and Eldridge 2007) or genome editing.
Detailed, high-resolution patterns of phylogeography in any
organism have become feasible, regardless of the previous
existence of genomic data. More broadly, the identification
of incipient speciation and genome-wide variation in natural
populations is being revealed, enabling more accurate infer-
ence of patterns of population relatedness, adaptation to envi-
ronmental stresses, determination of species diversity, life his-
tory, and migration routes.
This review illustrates how NGS has been contributing to
entomological research through the identification, manage-
ment and conservation of insect species, populations, commu-
nities, and other processes related to the functioning of the
ecosystems. The contributions are presented in five topics:
biodiversity surveys and monitoring; population genomics,
phylogenomics, phylogeography, and dispersal routes;
deciphering adaptive evolution; untangling food webs; and
identification of insect symbionts. A number of NGS-based
methods are described in the extant literature, for example,
whole-genome sequencing (WGS, Li et al. 2010), restriction
site-associated DNA sequencing (RAD-seq, Davey et al.
Table 1 Some insect biological control agents, pollinators, agricultural pests, and vectors of diseases with the genome elucidated by NGS

Species Common name Description Genome ID

Aedes aegypti (Linnaeus) (Diptera: Culicidae) Yellow fever mosquito Vector of dengue, chikungunya, Zika, yellow fever 44
Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) Dark sword-grass Pest of seedling crop plants 15346
Anopheles gambiae Giles (Diptera: Culicidae) African malaria mosquito Vector of malaria 46
Apis mellifera Linnaeus (Hymenoptera: Apidae) Honeybee Pollinator 48
Bombus terrestris (Linnaeus) (Hymenoptera: Apidae) Buff-tailed bumblebee Pollinator 2739
Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) Medfly Pest of fruits 2738
Cimex hemipterus Linnaeus (Hemiptera: Cimicidae) Bed bug Global human ectoparasite 45190
Culex quinquefasciatus Say (Diptera: Culicidae) Southern house mosquito Vector of West Nile virus, St. Louis encephalitis 39
Diabrotica virgifera LeConte (Coleoptera: Chrysomelidae) Western corn rootworm Pest of corn 17855
Diaphorina citri Kuwayama (Hemiptera: Psyllida) Citrus psylla Pest of citrus 867
Drosophila suzukii Matsumura (Diptera: Drosophilidae) Spotted wing drosophila Pest of citrus 18317
Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) Neotropical brown stink bug Pest of soybean 72975
Fopius arisanus (Sonan) (Hymenoptera: Braconidae) - Egg parasitoid of fruit flies 35518
Harmonia axyridis Pallas (Coleoptera: Coccinellae) Multicolored Asian ladybeetle Aphidophagous predator 71559
Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) Cotton bollworm Pest of wide range of host plants 13316
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution

Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae) Mandaçaia Pollinator 12726

Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) Jewel wasps Pupal parasitoid of flies 760
Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) Diamondback moth Pest of crucifer crops 11570
Rhodnius prolixus Stål (Hemiptera: Reduviidae) Blood-sucking bug Vector of Chagas 447
Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) Fall armyworm Pest of corn 10985
Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) The red flour beetle Pest of grains 216
Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) Cabbage looper Pest of crucifer crops 18285
Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) Tomato pinworm Pest of tomato 79094

2011), genotyping-by-sequencing (GBS, Elshire et al. 2011),
multiplexed inter-simple sequence repeats (ISSR) genotyping-
by-sequencing (MIG-seq, Suyama and Matsuki 2015), target
sequence capture/hybrid enrichment (Gnirke et al. 2009),
amplicon sequencing (amplicon-seq, Wachi et al. 2018), RNA-
Seq (Wang et al. 2008), metabarcoding (Taberlet et al. 2012;
Piper et al. 2019), metagenomics (Escobar-Zepeda et al. 2015),
and direct DNA shotgun sequencing (Paula et al. 2015, 2016;
Srivathsan et al. 2015, 2016). Their mechanisms and examples
are summarized in Table 2. Unfortunately, other excellent re-
search examples and NGS-based methods were not included in
this brief overview of this rapidly evolving area. Although this
work is focused on insects, non-insect spiders and mites were
also included.
Biodiversity surveys and monitoring
Biodiversity surveys and monitoring initiatives have benefited
significantly from NGS developments. Traditionally, they were
based on the identification of species by acoustic and/or visual
surveys or by sampling and counting of individuals using mor-
phological characters (Häuser and Riede 2015). Morphological
identification is time-consuming and requires taxonomic exper-
tise in specific taxonomic groups, which is often lacking and has
been declining (Basset et al. 2012). Therefore, morphological
identification of all species for entire communities or ecosystems
is considered onerous, even though it has been necessary to
assess spatial and temporal biodiversity patterns. In addition,
insects often have several taxa with cryptic species, i.e., morpho-
logical identification is not reliable (Sha et al. 2006), and have
many taxa where identification is only feasible for a particular life
stage (e.g., only adult males of some insects), which is not nec-
essarily sampled in field expeditions (Sinclair and Gresens 2008).
Since the year 2000, species identifications started to be support-
ed by DNA barcodes (Hebert et al. 2003; Hajibabaei et al. 2006)
enabling the identification of cryptic species (Janzen et al. 2009).
However, the vanguard DNA barcode method (i.e., based on
individual Sanger sequencing) is labor-intensive for biodiversity
surveys in terms of tissue sampling of each specimen. About 10
years later, the combination of DNA barcoding and NGS, re-
ferred to as metabarcoding (Taberlet et al. 2012), shifted biodi-
versity surveys from sampling individual organisms (biodiversity
indicators) towards sampling dozens to hundreds of insect spe-
cies in bulked (i.e., mass-collected, usually containing a mixture
of co-occurring taxa) environmental samples from terrestrial
(e.g., soil meiofauna, Shokralla et al. 2015) and aquatic commu-
nities (e.g., benthic samples, Hajibabaei et al. 2011). This enabled
faster and cost-effective taxonomic identification of any life stage
and facilitated the detection of rare, endangered (Thomsen et al.
2012), elusive, secretive, and invasive species (Bohmann et al.
2014).
Pires
In addition to the taxonomic identification of mass collec-
tions of organisms, NGS can also detect environmental DNA
(eDNA) from several sources (feces, urine, epidermal cells,
scales, pieces of pupae, etc.). In this review, the definition of
eDNA is based on Thomsen and Willerslev (2015): “genetic
material obtained directly from environmental samples (soil,
sediment, water, ice, etc.) without any obvious signs of bio-
logical source material,” and on Taberlet et al. (2012) and
Deiner et al. (2017): “DNA captured from an environmental
sample without first isolating any target organisms.” The use
of eDNA is possible because, depending on the environmental
setting, DNA can persist in the environment beyond the
lifespan of an organism (Dejean et al. 2011). Such environ-
mental persistent DNA is referred to as “zombie DNA” and
enables the identification of species that belong to a commu-
nity even when the specimen was not present at the time of the
sampling. In aquatic ecosystems, it has been demonstrated that
eDNA has relatively rapid degradation (hours to days or
weeks, Dejean et al. 2011), suggesting that results are restrict-
ed in space and time. In soil, eDNA can persist significantly
longer, for decades or possibly even centuries (Thomsen and
Willerslev 2015), and therefore may not necessarily represent
the actual biodiversity of present communities/ecosystems or
the presence of a species of conservation concern.
There is an extensive literature that demonstrates the appli-
cability of using metabarcoding and other NGS methods for
biodiversity surveys or monitoring, and only the pioneer
works are mentioned here. Hajibabaei et al. (2011) used
eDNA metabarcoding of large-scale, bulk samples of benthic
macroinvertebrate larvae from two Canadian rivers, to con-
trast patterns of species occurrence in urbanized and
conservation habitats. The taxonomic identification of such
immatures would not be possible beyond the family level
using morphological methods. In the terrestrial environment,
Yu et al. (2012) were one of the first to demonstrate that
metabarcoding can reliably estimate the community composi-
tion of hundreds of terrestrial arthropods. They identified 673
unique haplotypes (mostly flying insects) caught in Malaise
traps from three locations in China and mixed the species into
seven mock communities to analyze them by metabarcoding.
Through metabarcoding, 76% of the species were iden-
tified, but metabarcoding failed to estimate species
abundance, which could not be predicted by sequence
(i.e., reads) abundance, probably due to PCR amplifica-
tion bias and reaction stochasticity.
Ji et al. (2013) compared the species composition and rich-
ness obtained by metabarcoding and three high-quality stan-
dard data sets from 55813 terrestrial arthropod and bird spec-
imens collected from a Malaysian rainforest, a forest in China,
and a woodland in the United Kingdom (UK). Both methods
exhibited statistically correlated alpha- and beta-diversities;
however, the metabarcoding data were taxonomically more
comprehensive, at least two times faster to obtain (1165
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution

Table 2 Examples of NGS-based methods applied to entomological
research. Amplicon-seq, amplicon sequencing; CCD, colony collapse dis-
order; DDSS, direct DNA shotgun sequencing; GBS, genotyping-by-
sequencing; IAPV, Israeli acute paralysis virus; ISSR, inter-simple se-
quence repeats; MIG-seq, multiplexed ISSR genotyping-by-sequencing;
NGS, next-generation sequencing; RAD-seq, restriction site-associated
DNA sequencing; RE, restriction enzyme; RNA-seq, RNA sequencing;
SNPs, single-nucleotide polymorphisms; WGS, whole-genome
sequencing

Method(s) Mechanism Application(s) Example(s) Advantage(s) Limitation(s)

Amplicon-seq PCR of specific DNA Biodiversity survey; Identification of unique Cost-effective Time-consuming and
(Wachi et al. region followed by species monitoring; symbionts in 28 labor-intensive for
2018) NGS phylogeography; families and 8 orders large sampling size as
phylogenetics; of insect (Jones et al. separate PCR is re-
mutation 2013) quired for each target
identification and locus and sample.
frequency analysis
DDSS NGS sequencing Gut content analysis Identification of species No PCR bias; Not cost-effective for
(Paula et al. without enrichment of (food webs) or preyed upon by preservation of the large sample size as
2015, the DNA community identification of predatory ladybird original composition samples cannot be
Srivathsan followed by other highly beetles (Paula et al. and abundance of the multiplexed in a same
et al. 2015) taxonomic assignment degraded eDNA 2016) DNA community; library; dependent on a
of unassembled reads samples potential for good reference data-
quantitative analysis base
Metabarcoding DNA barcode Biodiversity surveys; Identification of insects Good cost-benefit; high Detection of false
(Taberlet et al. enrichment of monitoring of in: benthic capacity to process positives and/or false
2012, Piper multiple taxa followed species (including communities large-scale or bulk negatives; no estima-
et al. 2019) by NGS of conservation (Hajibabaei et al. samples; highly sensi- tion of species abun-
concern or 2011), soil meiofauna tive dance; may not pro-
invasive); study of (Shokralla et al. 2015), vide a species-level
species Malaise traps (Gibson taxonomic resolution;
interactions/food et al. 2014); identifica- dependent on a good
webs tion of host plants of reference database
hoverflies through
identification of the
carried pollen (Lucas
et al. 2018)
Metagenomics NGS of large fragments Biodiversity surveys; Identification of the Enable study of May not return good
(Escobar-Zepeda or complete genomes study of species IAPV as a diagnostic uncultivable taxonomic resolution
et al. 2015) (nuclear and or interactions marker for CCD in microorganisms
organellar) from a honeybees
community (usually (Cox-Foster et al.
microorganisms) 2007)
without previous
isolation
MIG-seq Multiplex PCR of ISSRs Population genomics; Phylogeny of 10–23 No need for prior genetic Lower identification of
(Suyama and followed by NGS phylogenomics; years old, dried muse- information; high SNPs compared to
Matsuki 2015) (genome-wide marker phylogeography; um specimens capacity to process RAD-seq
discovery) dispersal routes Pheidole ants (Eguchi large-scale samples;
et al. 2020) require lower DNA
quantity and quality
than RE-based NGS
methods; wide range
of species
RRL, RAD-seq, DNA digestion by Population genomics, Evolution of populations No need for prior genetic Missing data; restricted to
GBS restriction enzymes phylogenomics, of the pitcher plant information; high populations or closely
(Baird et al. combined with NGS phylogeography, mosquito (Emerson capacity to process related species; not
2008, Davey (genome-wide marker and dispersal routes et al. 2010) large-scale samples suitable for old (e.g.,
et al. 2011, discovery) museum) or very small
Elshire et al. specimens as requires
2011) high amount and
quality of DNA
RNA-seq RNA extraction and Adaptation studies; Study of adaptive No need for prior genetic Not suitable for studies
(Wang et al. conversion to cDNA gene expression; evolution of social information; more involving
2008) followed by NGS gene identification; complexity among direct correlation of non-functional genes
metabolism Polistes paper wasps, genotype with
analysis; phylogeny Bombus bumblebees phenotype; sensitive

Table 2 (continued)
Method(s) Mechanism Application(s) Example(s)
Target NGS of captured (e.g., Measurement of Study of insecticide
enrichment by hybridization) adaptation; genetic
(Gnirke et al. genomic regions of diversity;
2009) functional or phylogeny
phylogenetic
significance
WGS Elucidation of a whole Several Determination of the
(Li et al. 2010) genome (nuclear and
or organellar) by
fragmentation
followed by NGS
person-hours for all taxa versus 2505 person-hours of taxo-
nomic expertise), less reliant on taxonomic expertise, and dis-
pensed with the use of biodiversity indicator taxa (e.g., moths,
ants, spiders, carabid beetles, dung beetles). Gibson et al.
(2014) had more taxa identified from a bulk sample of 1066
individuals captured in Malaise traps in a Costa Rican conser-
vation area from Guanacaste using metabarcoding (with 11
primer sets for the same barcode region of the cytochrome c
oxidase subunit 1 – COI gene) than through morphological
and Sanger-sequencing analysis combined (using one COI
primer pair). They found several phyla (including arthropods),
12 orders, 47 families, 70 genera, and 41 species using
metabarcoding versus 12 orders, 37 families, 30 genera, and
11 species using the other methods.
Piñol et al. (2014) assessed the biodiversity of prey con-
sumed by the generalist spider Oedothorax fuscus (Blackwall)
(Aranea: Linyphiidae) in Wales (England) with only 6% of the
total good quality metabarcoding reads of the predator gut
content and identified five prey species plus five prey
genera. Beng et al. (2016) used metabarcoding to conduct an
impact assessment of land use (forest versus rubber and tea
plantations) on the community of the highly cryptic and rela-
tively small-sized litter arthropods in 35 matched forest plan-
tation sites from southwestern China. They found that the
alpha-diversity of more insect groups declined significantly
from forests into adjacent rubber plantations than into tea
plantations; the diversity of Orthoptera was significantly
higher in tea; and forests had a higher species turnover.
Lucas et al. (2018) used metabarcoding to identify the
range of plant species used as a source of pollen for the non-
Hymenopteran pollinator Eristalis hoverflies in four grassland
sites in the western UK. The knowledge of the structure of
Pires
Advantage(s) Limitation(s)
and honeybee, Apis
mellifera (Dogantzis
et al. 2018)
No PCR bias Need of previous
resistance in Culex knowledge of genetic
quinquefasciatus information to design
(Kothera et al. 2019) probe(s); limited
applicable taxonomic
range
Generates genetic Cost and high expertise
relation between wing information to required
polyphenism (affects develop markers,
dispersal distance) probes, and primers
with insulin receptors
in the rice brown
planthopper
Nilaparvata lugens
(Xue et al. 2014, Xu
et al. 2015)
plant-pollinator networks is highly relevant to safeguard the
provision of the ecosystem service of pollination, particularly
in the presence of environmental stressors (climate change,
invasive species, etc.). Pollen was washed off the bodies of
192 hoverflies and they were identified as coming from 65
plant taxa, a higher richness than anticipated. In addition, the
data revealed different degrees of plant-pollinator network
specialization at the community, species, and individual
levels, as well as temporal changes along the sampling season.
Barsoum et al. (2019) compared the use of metabarcoding
and taxa- and habitat-based surrogates (i.e., biodiversity indi-
cators) to assess the effects of tree composition and manage-
ment (mono- or polyculture) on the arthropod biodiversity in
15 plantation stands (five Scots pine-oak mixtures, four Scots
pine, and six oak monocultures) in southeast England.
Although the measures of biodiversity were highly congruent
between metabarcoding and the taxa-based surrogates,
metabarcoding was more taxonomically comprehensive,
which facilitated the identification of 116 arthropod species
highly associated with stand types.
Other NGS methods, not based on barcode enrichment
through PCR, have also been used for biodiversity surveys
and monitoring. They were motivated by the criticisms on
PCR bias (including varied “universal” primer efficiency
across taxa) that can lead to false positives and false negatives
and loss of the original DNA sample composition and richness
(Clarke et al. 2014; Deagle et al. 2014). Zhou et al. (2013)
isolated the mitochondrial DNA of a community of 73 insects
from the Beishan Mountains in China and directly processed
an ultra-high Illumina sequencing (15.5 Gb data) to recover
the first 648–658 bp of the COI gene, known as the Folmer
region (Folmer et al. 1994), which is commonly used for
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution
animal species identification. They successfully identified 92–
97% of the insect species and suggested that the relative abun-
dance of each taxon could be estimated by using its total
number of nucleotides obtained (referred by them as “se-
quencing volume”) to infer biomass and thus presumably
number of individuals. In 2014, Tang et al. 2014 used a similar
method, but without mitochondrial enrichment, to recover the
whole mitochondrial DNA as a multi-locus approach to iden-
tify taxa (mostly at species level) from a pool of 49 known
taxa, mostly insects, sampled in 11 sites in China. They re-
ferred to this method as “mitogenome metagenomics” (aka
mitogenomics or mitochondrial metagenomics or mito-
metagenomics) and used it (Tang et al. 2015) to assess the
composition of wild bees collected in pan traps in southern
England. From a pooled sample of 204 bee specimens,
mitogenomics enabled the correct identification of 59 species
out of 63 identified morphologically (93.7% detection rate).
Andújar et al. (2015) combined mitogenomics and DNA
barcoding to determine the community of arthropod soil
mesofauna (1494 specimens in 69 samples from three regions
in the Iberian Peninsula) and their phylogenetic relation from
three different locations in Spain. They found an amazing
diversity of more than 300 species of soil beetles
(Coleoptera) and a gradient of community differentiation
along with the soil depth. Using a similar methodology,
Gomez-Rodriguez et al. (2015) estimated the species richness
and community dissimilarity in over 2600 specimens (171
species) from 10 communities of leaf beetles
(Chrysomelidae) in the Iberian Peninsula.
Population genomics, phylogenomics,
phylogeography, and dispersal routes
Many NGS-based methods have been developed for the im-
provement of the study of population genomics,
phylogenomics, and phylogeography in model and non-
model species (see Davey and Blaxter 2010; Davey et al.
2011, Rice et al. 2011, Lemmon and Lemmon 2013,
McCormack et al. 2013, Narum et al. 2013 and Wachi et al.
2018). For example, Emerson et al. (2010) used RAD-seq to
resolve the postglacial phylogeography of a non-model organ-
ism: the North American pitcher plant mosquito Wyeomyia
smithii (Coquillett) (Diptera: Culicidae). They analyzed six
individuals from 21 populations and identified 3741 SNPs
among 13627 loci in the genome, which were sufficient to
demonstrate that following the recession of the Laurentide
Ice Sheet, refugial populations of W. smithii dispersed
northward and then westward across North America.
Timmermans et al. (2010) reconstructed the phylogeny (at
the family level) of 30 beetle species (Coleoptera) from eight
countries with no a priori genetic information. They first elu-
cidated the mitogenomes using long-range PCR (LR-PCR)
and multiplexing the amplicons in one single library without
indexing tags. After DNA shotgun sequencing, the taxonomy
was assigned using baits (sequences from the COI Folmer
region of each beetle species). Later, Timmermans et al.
(2016) studied the phylogenetics of 35 butterfly museum
specimens stored dried for 9 to 35 years. They reconstructed
the butterfly mitogenomes using DNA shotgun sequencing,
without a shearing step before library construction. Such re-
covery of genetic information from museum specimens using
NGS has been called “museum genomics” (Rowe et al. 2011).
Using MIG-seq, Eguchi et al. (2020) were able to study the
phylogeny of 55 dry specimens of Neotropical ants from the
genus Pheidole (Hymenoptera: Formicidae) from a 10- to 23-
year-old museum collection by the successful identification of
36862 SNPs from 4849 polymorphic loci.
Soria-Carrasco et al. (2014) analyzed the whole-genome
divergence in walking sticks Timema cristinae Vickery
(Phasmatodea: Timemidae) populations one generation after
they were transplanted from their preferred host plants to an
alternative host. This species has ecotypes adapted to different
host plants. Instead of finding different gene frequencies of
particular alleles, they found genomic divergence of whole
sections of the genome between populations (some of them
unique to a population/host), many of which involved multi-
ple genes with specific molecular functions. Walking stick
genes diverged in parallel suggesting that speciation can in-
volve both repeatable and idiosyncratic divergence at the ge-
netic level and that the repeatable component involves many
genomic regions affected by divergent selection.
NGS has facilitated the assessment and monitoring of ge-
netic variation in populations of biological control agents (Li
et al. 2017b) that ultimately may reduce the biological control
agent fitness or performance, for instance by diluting locally
adapted genomes or disrupting coadapted gene complexes
built up in isolated populations (outbreeding depression), or
enhance its capacity to adapt to new environments (Li et al.
2018). In addition, recently NGS has been used to speed up
selection for improved efficacy of biological control agents
(Lommen et al. 2017), in an effort called “next-generation
biological control” (Le Hesran et al. 2019; Leung et al.
2019). The underlying idea is to use NGS-based tools and
approaches (e.g., genotype-by-sequencing-GBS, Baird et al.
2008; genome-wide association studies-GWAS, Bastide et al.
2013; large-scale SNP panels, Bruni et al. 2015) to character-
ize the genetic architecture of and select for desired traits (e.g.,
climate adaptation, attack rate, offspring sex ratio, Wajnberg
2010). This may facilitate the screening and selection of de-
sired traits, eliminating the need for laborious classical breed-
ing techniques based on artificial selection for a few optimal
phenotypes.
The knowledge about the dispersal or migration route of an
invasive species, pest, beneficial insect (e.g., biological con-
trol agents, pollinators), or species of conservation concern is

critical to developing efficient tactics for prevention or miti-
gation, conservation and pest management. Genetic diversity
analysis through NGS-based methods can be used to more
accurately determine the center of origin of species and recon-
struct invasion histories, colonization routes, and regional dis-
persal patterns to improve predictive models of mechanisms
of invasion or adaptation in new or changing environments
(Kirk et al. 2013a). This is achieved by tracking insect move-
ment across landscapes and geographic regions and es-
timating population size and rates of admixture across
different management areas, thereby assessing the effect
of management practices.
The red imported fire ant Solenopsis invicta Buren
(Hymenoptera: Formicidae) had its route of global invasion
reconstructed with the aid of NGS pyrosequencing to geno-
type 66 nuclear microsatellite loci in females collected from
2144 fire ant colonies from 75 geographic sites worldwide
(Ascunce et al. 2011). They discovered that the fire ants were
introduced in nine different events: initially twice from South
America to southern USA, where the fire ants were confined
for decades; from southern USA, it spread twice to California
and once to Australia; from California, it spreads to China;
and from southern USA and California to Taiwan. Analyses
of specimens from Trinidad and New Zealand suggested that
the fire ants came from the USA. Thus, NGS is allowing the
study of what has been called the “invasive bridgehead effect”
(Lombaert et al. 2010), i.e., an invasive population acting as
the source of subsequent invasions to remote new territories.
The worldwide introductions of oriental fruit moth Grapholita
molesta Busck (Lepidoptera: Tortricidae, a pest of a wide
range of fruits) were traced by genotyping 13 single sequence
repeats (SSR) loci using NGS, demonstrating introductions
from Asia to Australia, from North America to South Africa,
South America and the Azores, and from Brazil to Europe
(Kirk et al. 2013b). In Australia, Perry et al. (2020) used
RAD-seq to map a large number of single-nucleotide poly-
morphisms (SNPs) in diamondback moth Plutella xylostella
(L.) (Lepidoptera: Plutellidae) to estimate the structure of nine
populations and, consequently, their dispersal patterns. As an
unexpected outcome, they identified the endemic cryptic spe-
cies P. australiana Landry and Hebert.
Deciphering adaptive evolution
NGS has unraveled many cases of phenotypic innovation and
phenotypic plasticity (also called “polyphenism,” i.e., a ge-
nome expresses different phenotypes in response to environ-
mental cues). It has enabled the identification of genomic re-
gions with differential responses to selection (e.g., transcript
profiling), genes highly correlated with adaptive phenotypic
responses, and parallel (repeated) evolution in populations,
ecotypes, or species with similar or different selection
Pires
pressures, as well as characterization of patterns of adaptive
evolution (Radwan and Babik 2012; Whitehead 2012). The
contributions of NGS to the understanding adaptation of or-
ganisms to changing environments has been called “adapta-
tion genomics” (Stapley et al. 2010), and the integration of
genomic techniques in species conservation has been called
“conservation genomics” (Angeloni et al. 2012).
A famous example of phenotypic innovation and adapta-
tion is the remarkable diversity in the wing color patterns of
the passion vine butterflies in the genus Heliconius
(Lepidoptera: Nymphalidae) (Brown 1981). A conjunction
of different molecular methods, including NGS (e.g., de novo
assembly, targeted resequencing), demonstrated, surpris-
ingly, that the diversity in the wing color patterns is due
to changes in cis-regulatory regions of one or a few loci
(Kronforst and Papa 2015).
NGS has helped to clarify that many cases of polyphenism
in insects are due to two insulin receptors (InR1 and InR2)
involved in the insulin/insulin-like growth signaling pathway
(IIS), including such diverse cases as caste differentiation in
social insects (Corona et al. 2016) and wing polyphenism in
planthoppers (Xu and Zhang 2017). One example is the dis-
covery that, despite eusociality having evolved independently
in ants, bees, wasps, and termites, the control of caste
polyphenism is similar, depending on differential regulation
of lnR1 and lnR2 that leads to a cascade of regulation in other
physiological pathways (Cristino et al. 2006; Terrapon et al.
2014; Berens et al. 2015). This results in the sociality gradient
from subsocial species (small nests, no caste system, well-
developed parental care) to advanced eusocial species (large
colonies, specialized castes) (Kent and Zayed 2015). Another
example is wing polyphenism in the rice brown planthopper
Nilaparvata lugens Stål (Hemiptera: Delphacidae) determined
by the similar regulation of lnR1 and lnR2 (Xue et al. 2014;
Xu et al. 2015). They observed that nymphal knockdown of
InR2 resulted in long-winged adults, while dysfunction of
InR1 resulted in short-winged adults, which perfectly fit the
known species ecology to escape deteriorating environments
and colonize new habitats. The long-winged morph is capable
of long-distance dispersal, and in East Asia, crowding during
nymphal development during Spring and Summer, intensified
by low-quality plant hosts (nutrition), stimulates the develop-
ment of long-winged morphs (Kisimoto 1956) and migration
from tropical/subtropical areas to temperate areas where rice
becomes available (Kisimoto 1976). Offspring of the migrants
are at low density on high-quality plants and develop short-
wing forms, which have higher reproductive fitness than the
long-winged morphs (Manjunath 1977). In autumn, when
crowding occurs, they develop long wings and migrate back
to tropical/subtropical areas (Riley et al. 1991).
In the agricultural and public health sectors, NGS has fa-
cilitated the understanding of the mechanisms of adaptation to
pesticides and selection pressures on pollinators, pests, and
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution
insect disease vectors. The elucidation of the Ap. mellifera
genome (Honeybee Genome Sequencing Consortium-HGSC
2006) revealed that honeybees had two-thirds fewer proteins
implicated in innate immunity and 30–50% fewer genes relat-
ed to detoxification of xenobiotics (cytochrome P450,
glutathione S-transferase, and carboxylesterase) compared to
Drosophila and Anopheles, but it has unique genes associated
with nectar and pollen utilization. They suggested that honey-
bee has a compromised ability to circumvent pathogens and
detoxify pesticides. Grbić et al. (2011), in elucidating the ge-
nome of the two-spotted spider mite Tetranychus urticae
Koch (Acari: Tetranychidae, a pest of a wide range of plants),
observed that despite a smaller genome size (ca 90 M bases)
compared to 1342 arthropods with a genome size of 1099.2 ±
2045.8 Mb (Animal Genome Size, http://www.genomesize.
com/, Table S2 Supplementary Material), it had a higher
diversity of detoxification gene families. They suggested that
the high polyphagy of this mite is related to the high diversity
of detoxification genes, enabling the species to feed on many
host plants and consequently to develop pesticide resistance
more rapidly. Wang et al. (2013), using digital gene expres-
sion (DGE) and NGS, showed that two invasive highly po-
lyphagous whitefly species from the Bemisia tabaci Genn
species complex (Hemiptera: Aleyrodidae) had significantly
higher expression levels of detoxification genes compared to
an indigenous whitefly species from the B. tabaci complex
with a narrower host range. They suggested that this might
be related to the adaptability of the two invasive whitefly
species to environmental stress. Through the elucidation of
the genome of the soybean aphid Aphis glycines Matsumura
(Hemiptera: Aphididae, soybean pest) (Wenger et al. 2017),
Paula et al. (2020) identified a dozen detoxifying enzyme
genes potentially related to detoxification (resistance) of pyre-
throid insecticides in three resistant clonal populations of the
soybean aphid in northern USA. They observed a high corre-
lation between the number of constitutively overexpressed
detoxifying enzyme genes and the level of pesticide resistance
and that the overexpression profiles of the detoxifying enzyme
genes varied among populations. They suggested that the var-
iation among populations possibly reflects differences in the
genetic background and pyrethroid selection pressures.
Based on the genome of the triatomine Rhodnius prolixus
Stål, 1859 (Heteroptera: Reduviidae), a vector of Chagas dis-
ease, Mesquita et al. (2015) observed the uncommon absence
of several components of the immune-deficiency (IMD)-me-
diated immune response pathway. They suggested this may be
related to the ability of the protozoan parasite Trypanosoma
cruzi to evade or tolerate the immune response of the host-
vector. Benoit et al. (2016) elucidated the genome of the bed
bug Cimex lectularius and found several features that may
have enabled the bed bug to become a successful urban pest.
Three features are highlighted here: the recent expansion in
pro-resilin genes compared to other blood feeders, which
possibly enabled females to adapt to the traumatic copulation
attempts by males; the multiple putative lateral gene transfer
events from various bacteria, including Wolbachia and
Arsenophonus; and the provision by Wolbachia of the vitamin
B complex that is not available in blood meals, yet is critical
for reproduction and development of the bed bug. Kothera
et al. (2019) studied seven populations (125 individuals) of
Culex quinquefasciatus (vector of the arboviruses causing
West Nile virus disease and St. Louis encephalitis) from the
USA and found several SNPs and genes with copy number
variants (CNVs), potentially associated with pesticide resis-
tance. Comparative genomic analysis of 16 anopheline spe-
cies from nine geographical regions led Mitchell et al. (2015)
to infer that a steroid hormone, sexually transferred in the
“mating plug” by male Anopheles spp. to females, induced
higher reproductive fitness and female longevity, concomi-
tantly reducing the mosquito immune response against
Plasmodium (malaria parasite causal agent), and consequently
increasing Anopheles spp. capacity to vector malaria. They
also observed that in regions with higher malaria prevalence
(Africa and India), some mosquito species had a higher fre-
quency of “mating plug” behavior with higher steroid hor-
mone concentration. Rinker et al. (2016) provide several other
examples of how NGS helped to better understand the biology
of insect-vectored diseases.
Jaffé et al. (2019) presented a rich example of what has
been called “landscape genomics.” They investigated to
which extent habitat loss could affect population genetic di-
versity and gene flow in the stingless bee, Jandaíra, Melipona
subnitida Ducke (Hymenoptera: Apidae), native to Brazil.
Using RAD-seq on 156 bees sampled from 31 locations cov-
ering the full distribution range of Jandaíra in northeastern
Brazil (ca 950 × 500 km), they identified 3454 loci that indi-
cated a clinal change in genetic structure across the distribu-
tion range, forming four genetic clusters. Amazingly, related
colonies were found up to 300 km apart. Thermal stability was
the main factor explaining gene flow patterns and was highly
correlated with forest cover. Together, thermal stability and
forest cover were the main mediators of genetic connectivity
in Jandaíra, while the elevation was a dispersal barrier.
Although the researchers detected genomic signatures of ad-
aptation to temperature, forest cover, and altitude, habitat frag-
mentation was not significantly associated with genetic diver-
sity. Nevertheless, they pointed out that it may take a longer
time for such an association to become detectable. They sug-
gested that forested areas are valuable dispersal corridors for
Jandaíra and could facilitate its migration through higher ele-
vations. This is particularly important for this species because
it is predicted that, due to climate change, the lowlands climate
will not support their survival by 2050. Franks and Hoffmann
(2012), Stillman and Armstrong (2015), and Li et al. (2017a)
present excellent reviews on the influence of climate change
and landscape on genetic diversity and functional connectivity

and how to use NGS-based methods to identify adaptations to
the local environment, which are directly applicable to ento-
mological research.
Untangling food webs
The study of insect food webs and trophic interactions has
become one of the most active areas at the interface of biolog-
ical control and molecular techniques. There is a long-term
consensus that predation studies cannot rely only on observa-
tions of direct feeding in the field, cage experiments, patterns
of co-occurring species, expert opinion, feeding trials, or vi-
sual inspection of prey remains in the predator’s guts. The
advent of NGS was revolutionary to identify the complex diet
breath of polyphagous insects, uncovering simultaneously tro-
phic interactions of multiple species, new and unexpected
trophic links, and levels of complexity not previously
possible. Consequently, it has improved the understanding
of food webs by the better characterization of the
composition, structure, functioning, and niche partitioning of
communities in multiple habitats along with seasonal changes.
In the central French Alps, Ibanez et al. (2013) demonstrat-
ed trophic niche partitioning according to the nutritional re-
quirements of four species of grasshoppers (Orthoptera): large
mountain grasshopper Chorthippus scalaris Fischer von
Waldheim (Acrididae), large banded grasshopper Arcyptera
fusca Pallas (Acrididae), small gold grasshopper Euthystira
brachyptera Ocskay (Acrididae), and stripe-winged grasshop-
per Stenobothrus lineatus Panzer (Acrididae). Through
metabarcoding analysis of the feces of the grasshoppers,
known to be highly polyphagous, they showed that the spec-
trum of plants they each fed on were not strongly related to the
plant species abundance in the habitat, but that the grasshop-
pers selected host plants for their optimal nutrition.
A basic aspect of food webs that DNA barcoding and NGS
are helping to improve is the taxonomic resolution of the
nodes (taxa represented) and more accurate structure of the
links between nodes (representation of feeding interactions)
(Roslin and Majaneva 2016). We focus here on the trophic
interactions involved in two ecosystem services: biological
control of pests and pollination.
a) Biological control
In conservation biological control, the landscape (e.g., di-
versification of vegetation) and farming practices (e.g., estab-
lishing a strip of nectar plants in the border of a crop) are
managed to conserve and increase a community (or popula-
tion) of local natural enemies of a pest or pests. The design of
more environmentally “friendly” cropping systems requires a
better understanding of the food webs. As a given natural
enemy (predator or parasitoid) may have multiple prey/host
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species, it is a fundamental goal to increase natural enemies
that can efficiently control a pest population with minimal
non-target effects. Molecular gut content analysis is a strong
ally as it provides information that a pest and a natural enemy
share a habitat and encounter each other, that the natural ene-
my consumes the pest, and potential non-target hosts/prey,
such as species of conservation concern or other natural
enemies (intraguild predation). NGS-based methods can
help determine the diet breath of predators and the host
range of parasitoids, recognize cryptic species, and de-
tect undescribed species.
Gut content analysis by NGS-based methods has uncov-
ered a higher occurrence of intraguild predation than previ-
ously presumed among generalist predators in agricultural
systems. Gomez-Polo et al. (2016) detected by metabarcoding
intraguild predation by Orius majusculus Reuter (Hemiptera:
Anthocoridae) on other Orius species, syrphids, spiders, and
predatory ladybird beetles in lettuce (Spain). Paula et al.
(2016) detected by direct DNA shotgun sequencing (DDSS)
frequent intraguild predation in cole crops (Brazil) among
several species of predatory ladybird beetles (e.g.,
Cycloneda sanguinea L., Hippodamia convergens Guérin-
Méneville, Eriopis connexa Germar, Harmonia axyridis
Pallas), other predators (insidious flower bug Orius insidiosus
Say and earwig Doru luteipes Scudder (Dermaptera:
Forficulidae)) and parasitoids of coccinellids (Dinocampus
coccinellae Schrank (Hymenoptera: Braconidae)),
Homalotylus mirabilis Brethes (Hymenoptera: Encyrtidae)),
and coccinellid prey (Aphidius ervi Haliday (Hymenoptera:
Braconidae)). DDSS is a recent barcode-free enrichment
method in which a DNA community is sequenced directly
after its extraction (no barcode primer pair or probe needed)
and the generated reads are assigned to taxa, without previous
assembly, by alignment with a reference database using high
similarity (> 95%) above a minimum overlap length (> 150
bp) (Paula et al. 2015, 2016; Srivathsan et al. 2015, 2016).
Palomo et al. (2016) determined the contribution of a com-
munity of carabid beetles (14 species, gut content
metabarcoding analysis of 513 individuals) to the biological
control of pests in six arable landscapes in Britain and France.
They found intense niche overlap among the carabids for the
consumption of arachnids, collembolans, insects, plants, and
earthworms (functionally categorized as natural enemies,
pests, plants, and detritivores). While the carabid community
preyed on several agricultural pests, contributing to the eco-
system service of biological control, it also consumed other
natural enemies of pests at a similar rate and, to a lesser extent,
detritivores, providing dis-services to the ecosystem services
of biological control and nutrient recycling. However, the
authors suggested that carabid beetles should not be
dismissed as good biological control agents, because the diet
consumed by the community is not an intrinsic property of the
predator species. Instead, it is dynamically modulated by
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution
extrinsic environmental factors, such as the cropping system
or management intensity, so in some circumstances, the
carabids might be effective biological control agents.
Molott et al. (2014) used metabarcoding to characterize the
gut contents of eight common ground-dwelling predators in
banana plantations in Martinique (Bahamas): wolf spiders
(Araneae: Lycosidae), the earwig Euborellia caraibea
Hebard (Dermapthera: Anisolabididae), the carpenter ant
Camponotus sexguttatus F. (Hymenoptera: Formicidae), the
trap-jaw ant Odontomachus baurii Emery (Hymenoptera:
Formicidae), the tropical fire ant Solenopsis geminata F.
(Hymenoptera: Formicidae), the little fire ant Wasmannia
auropunctata Roger (Hymenoptera: Formicidae), rove beetles
(Coleoptera: Staphylinidae), and centipedes
(Scolopendromorpha: Scolopendridae). They aimed to identi-
fy the predators consuming the banana weevil Cosmopolites
sordidus Germar (Coleoptera: Curculionidae) and evaluate if
the predator diets would be affected by a cover crop of signal
grass, Brachiaria decumbens, in banana plantations. Cover
cropping is a tactic to control weeds and improve physical soil
properties, reducing the use of agricultural herbicides and fer-
tilizers, while potentially increasing the community of alter-
native prey to attract generalist predators to control the banana
weevil. The gut analysis of 572 individuals revealed that
E. caraibea, C. sexguttatus, and S. geminata consumed the
banana weevil and the frequency of consumption of the pest
did not differ between bare soil and cover-crop plots.
However, the cover crop reduced the consumption of other
pests because the predators switched to forage on alternative
non-pest prey. They demonstrated that plant diversification
(introduction of B. decumbens) in the banana cropping system
altered the arthropod food webs, which potentially could ad-
versely affect the populations of two other secondary pests.
They suggested that the cover crop provided food,
shade, and resting areas for the alternative non-pest
prey, increasing their abundance, inadvertently reducing
predation on the secondary pests.
b) Pollination
NGS has been used to identify the plant species composi-
tion of the pollen present in honey (Valentini et al. 2010;
Bruni et al. 2015; Hawkins et al. 2015; Richardson et al.
2015a), bee host plant use from the analysis of the pollen loads
on pollinator bodies (Galimberti et al. 2014; Richardson et al.
2015b), and pollination networks over time and geographical
areas, i.e., plant-pollinator interactions (Wilson et al. 2010;
Pornon et al. 2016, 2017; De Vere et al. 2017). The identifi-
cation of plant pollen species in honey is important for
assessing its nutritional value, safety (e.g., poisoning by
“mad honey,” Koca and Koca 2007), and the geographic or-
igin of the product, which affects the honey price (Bruni et al.
2015). Conventional taxonomy of the pollen is performed by
microscopic inspection and classification according to mor-
phological keys, which is difficult for some plant taxonomic
groups, has a low taxonomic resolution (often only to the
family level), and requires a high level of expertise that is
not readily available (Rahl 2008). Metabarcoding allows the
analysis of a larger number of pollen samples and gives a
higher taxonomic resolution of the plants from which the pol-
len originated (Bell et al. 2016). Bell et al. (2016) presented
detailed information about barcodes/markers used to identify
host plants and gave other methodological guidelines.
Bruni et al. (2015) identified 38 host plant species in four
commercial honey samples available in Italy during June-
July, 2012. Richardson et al. (2015b) collected pollen from
honeybees entering hives (USA) using pollen traps during a
15-day period and identified the plant species more efficiently
using metabarcoding (19 plant families) than microscopy
(eight plant families). Sickel et al. (2015) identified 650 plant
taxa with 95% at the species level from 384 pollen samples
from solitary bees, Osmia spp. (Hymenoptera: Megachilidae),
at various sites in Germany. Pornon et al. (2016) used
metabarcoding (trnL and ITS1 barcodes) to analyze pollen
loads from 402 insect pollinators (e.g., honeybee, bumble-
bees, wild bees, Syrphidae, Empididae, other Diptera,
Coleoptera, Lepidoptera) captured in the alpenrose shrub
community in the French Central Pyrenees. Metabarcoding
detected 2.5 times more plant-pollinator interactions than by
direct observation of pollinator visits to host plant flowers. In
2017, Pornon et al. then presented the resulting plant-
pollinator networks and the pros and cons of metabarcoding
compared to visitation surveys. On the one hand,
metabarcoding might inflate the number of species and inter-
actions due to its high sensitivity and therefore higher likeli-
hood of false positives. On the other hand, metabarcoding
improves visitation surveys and palynology diversity assess-
ments by the detection of rare or uncommon interactions,
thereby improving the accuracy of the floral constancy and
specialization and connectivity patterns of pollinator-plant
communities. In addition, it also enables the investigation of
the whole pollen load in individual insects and of evaluation
of interactions over larger areas. De Vere et al. (2017) used
metabarcoding to study honeybees foraging during spring in
Wales and observed that despite the high availability of floral
resources (native and horticultural plants), honeybees only
foraged on a few of them and an even smaller number of plant
species were recorded from the pollen in the honey. They
suggested that honeybees during the spring had a staple
diet of pollen from a small number of major host plants
that are supplemented at low levels with pollen from
horticultural plants.
In a reverse approach, Clare (2014) reported in a review
that traces of salivary DNA left on chewed seeds or fruits
could be used to identify the dispersing animal to construct
unobserved trophic interactions. Ushio et al. (2015) used

metabarcoding to identify pollinators of a dominant plant spe-
cies in Japan, tall goldenrods (Solidago altissima), by the
identification of the microorganisms the pollinators left on
flowers and leaves. They found that species-specific insect
microbial communities can be transferred from the insect
body to a flower surface, and these microorganisms can serve
as a “fingerprint” of the visiting insect. In the same line of
thinking, Vamosi et al. (2017) suggested that this tactic could
reduce sampling effort, construct individual and community-
level metrics of pollinator specialization, and survey if a pop-
ulation of a certain pollinator species is declining. Thomsen
and Sigsgaard (2019), through the use of metabarcoding (COI
and 16S barcodes), were also able to detect trace amounts of
arthropod DNA on 56 flowers from seven wild plant species
in 2017 in Denmark. They identified 135 arthropod species in
67 families and 14 orders (for instance bumblebees, ground
beetle, ladybirds, braconid wasps, thrips, aphids, plant bugs)
and observed arthropod community clusters associated with
plant species. Nonetheless, there are potential limitations to
the use of these NGS-based tools (e.g., metabarcoding, ge-
nome skimming) to study pollination networks that were
raised by Li et al. (2015) and Hollingsworth et al. (2016).
Identification of insect symbionts
NGS, mainly through metagenomics, has contributed tremen-
dously to the discovery and study of insect symbionts, which
are most often uncultivable (Chandler et al. 2012; Yun et al.
2014; Miller et al. 2016; Boccazzi et al. 2017; Deutscher et al.
2018; Mohammed et al. 2018; Ziganshina et al. 2018). The
identification of symbionts in insects has revealed their impor-
tance to the provision of ecosystem services such as biological
control and pollination through defense against abiotic and
biotic stressors (Oliver et al. 2005, Perlman et al. 2006,
Kaltenpoth and Engl 2014, Käch et al. 2018, Gabriel 2018,
Vorburger 2018, Postic et al. 2020) and reproductive regula-
tion (Pan et al. 2020). Using metagenomics, Cox-Foster et al.
(2007) determined that Israeli acute paralysis virus (IAPV) is
prevalent in colony collapse disorder (CCD) in honeybees,
establishing it as a diagnostic marker for CCD. Also using
metagenomics, Engel et al. (2012) showed evidence of sym-
biont niche specialization in the hindgut of 150 honeybees
(adult workers). They found that the symbiont community,
although composed of only a few species, had high
symbiont genetic diversity conferring to the honeybee
distinct functional capabilities for pathogen defense, im-
munity, and nutrient utilization.
The elucidation of whole symbiont genomes can reveal the
underlying mechanisms driving adaptation and evolution in
insects. Using available genomes of 994 prokaryotes, many of
them sequenced by NGS, and the predicted gene sets of 142
eukaryotes (insects, other animals, plants, fungi, and protista),
Pires
Li et al. (2011) found that a large number of the detoxification
genes in lepidopteran genomes originated from lateral gene
transfer (LGT), mostly from pathogenic bacteria. They sug-
gested that LGT may have contributed significantly to the
ability of lepidopteran herbivores to detoxify the secondary
defense metabolites produced by host plants, facilitating lep-
idopterans to become one of the most varied and dominant
groups of agricultural pests worldwide. Metagenomic data
enabled Adams et al. (2013) to determine that gut symbiont
communities (Pseudomonas, Rahnella, Serratia, and
Burkholderia) enable the digestion of host pine tree polymers
and detoxification of terpenes for the mountain pine beetle
Dendroctonus ponderosae Hopkins (Coleoptera:
Curculionidae).
Brucker and Bordenstein (2013), through metagenomics
and transcriptome analyses, found that the species-specific
gut microbiota in closely related species of the wasp genus
Nasonia caused lethality in interspecific hybrids. They pro-
posed that the gut symbionts and the host wasps were a co-
adapted complex and that hybridization would break down
this complex leading to lethality, possibly contributing to the
speciation process. They found that hybrids between
N. vitripennis and N. giraulti disrupted the gut symbiont com-
munity. When this community was not allowed to establish by
rearing on a bacteria-free diet, hybrid survival increased
significantly, and when the hybrid diet was inoculated with
bacterial symbionts, lethality was restored.
Wang et al. (2017) using amplicon-seq found four bacterial
isolates (two from the genus Enterobacter and one each from
the genera Pantoea and Rahellathat) that increased glucose
oxidase (GOX) activity in the labial glands of corn earworm
Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) feeding
on tomato. GOX is an elicitor in herbivore saliva for the pro-
duction of jasmonic acid (JA), which regulates plant defenses.
In addition, they observed that tomato plants damaged by
H. zea larvae inoculated with Enterobacter ludwigii had sig-
nificantly higher expression of three JA-responsive genes
(Pin2, CysPI, and PPOF). JA is a volatile compound produced
by wounded or herbivore-injured plants that triggers several
plant defense mechanisms, such as induction of higher expres-
sion of defensive proteinase inhibitors in the leaf tissue or
attractants of natural enemies of the herbivore.
Pascar and Chandler (2018) developed a bioinformatics
approach to detect Wolbachia in insect NGS data, enabling
the detection of multiple Wolbachia species, and used se-
quencing depth information to distinguish real infection from
LGT to the host nuclear genome. Jones et al. (2019), using
amplicon-seq, determined that herbivores (fall armyworm and
corn earworm) feeding on the same host plant (e.g., corn) did
not necessarily have similar midgut bacterial communities,
and individuals of the same species collected from different
sites also had different bacterial communities. They also found
that in the same insect, the composition of the bacterial
Next-Generation Sequencing and Its Impacts on Entomological Research in Ecology and Evolution
symbiont community varied in different parts of the digestive
system (e.g., foregut, midgut) of the host, suggesting differen-
tial spatial bacterial colonization (niche partitioning). Sugio
et al. (2015) and Giron et al. (2017) presented excellent re-
views about the enhanced characterization of the insect micro-
bial diversity by NGS, as well as the modulation of plant-
insect interactions by symbionts.
Challenges and perspectives
Three major challenges are considered here related to the use
of NGS in entomological research: the technical problem of
false-positive and false-negative detections, cost of NGS, and
capacity building to enable its use in developing countries,
especially in those with low investment in science.
Despite all the unprecedented advantages of NGS-based
methods, they all have the common methodological problem
of inaccurate detections with failures in both false positives
(type I error) and false negatives (type II error), which can lead
to incorrect ecological conclusions or inconclusive results.
False-positive detections can have several causes and the most
usual are due to DNA contamination, PCR errors during am-
plification of DNA, sequencing errors, assembly errors (in-
cluding production of chimeras), insufficient species-level
variation among sequences, and taxonomic overclassification
(i.e., erroneous detection of related species when the true spe-
cies is absent in the reference database) (Richardson et al.
2017). False-negative detection can arise from inhibition of
PCR (e.g., by humic acids), lack of representative sampling,
or incomplete DNA reference database. Several reports illus-
trate the occurrence of both false positives and false negatives.
Hajibabaei et al. (2011) reported a taxonomic bias for their
COI primer set as well as biases due to varying abundances of
species in bulk samples. Yu et al. (2012) reported a low de-
tection rate of hymenopterans and suggested that this bias is
associated with COI “Folmer” barcoding primers (Folmer
et al. 1994). Clarke et al. (2014) also suggested bias of COI
primers towards Lepidoptera and Diptera with certain orders
failing to amplify. Although false positives and negatives re-
main a problem, there is developing literature discussing how
they may be avoided or minimized (Liu et al. 2013; Ficetola
et al. 2015, 2016; Ruppert et al. 2019), for example, through
the use of negative field controls, validation of results by
qPCR (Paula et al unpublished), and field sampling replicates
to allow the use of site occupancy models. Site occupancy
models are statistical models developed to estimate the prob-
ability of imperfect detections based on the number of sites
with the presence/absence of a species (Schmidt et al. 2013).
An approach used in transcriptomics (e.g., Bi and Liu 2016) or
population genomics (e.g., Farrer et al. 2013) studies to deal
with false positives is through estimation of false discovery
rates (FDR, Benjamini and Hochberg 1995) and the
subsequent use of algorithms to control FDR at a certain level,
for example, at 0.05 (or 5%). FDR is the proportion of false
positives in the total number of positives.
Regarding the NGS cost, countries with low economic
power and investment in science have only marginally partic-
ipated in the progress provided by NGS. Molecular biology
supplies/equipment and NGS services usually are provided by
developed countries in which the monetary conversion rates to
developing countries are significantly unbalanced. In a
PubMed search for publications using “NGS AND (insect*
OR entomol*)” in the title or abstract from 2012 to 2020, 151
best matches were found. Considering the country of the last
author (or corresponding author when not available), 88 pub-
lications were from 21 developed countries (58% of all devel-
oped countries, United Nations 2020), and 62 publications
were from 20 developing countries (16% of all developing
countries, United Nations 2020), of which 22 were from
China and 10 were from Brazil, two countries with significant
investments in science. Although this search probably
underestimated the number of existing NGS publications re-
lated to entomology, the disproportional amount of literature
from developed countries brands this technology as the priv-
ilege of an “elite.” In contrast, developing countries comprise
some of the biologically richest natural areas for entomologi-
cal research. Their budgetary limitations lead also to the stag-
nation of capacity-building efforts to train experts to use NGS
methods and to analyze the data. Although the library con-
struction and sequencing services can be provided by third
parties, as long as research groups can pay for it, the subse-
quent steps of data curation and bioinformatic analysis are
usually and preferentially done by the researchers.
Hopefully, the difference in the access of the NGS technol-
ogies between developed and developing/least developed
countries will continually decrease in the future as the price
of the NGS services of library construction and sequencing
have continuously declined due to technological improve-
ments and higher competition among providers. In addition,
cheaper and portable NGS platforms are proving efficient for
use in several areas, for example, the lower-cost Oxford
Nanopore’s MinION technology (Laver et al. 2015;
Mikheyev and Tin 2014), also called nanopore DNA sequenc-
ing (e.g., Castro-Wallace et al. 2017) or real-time (nanopore)
DNA sequencing (e.g., Johnson et al. 2017) or portable ge-
nome sequencing (e.g., Quick et al. 2016). MinION is a por-
table device of the size of a flash stick that connects to a
computer through a single USB port. Srivathsan et al. (2019)
demonstrated the efficient use of the 1D MinION sequencing
to assess the diversity of Diptera from the Phoridae in a sample
containing 8699 specimens (7059 sequenced) collected in one
Malaise trap during 8 weeks (2010–2011) in the Kibale
National Park, Uganda. Through a metabarcoding approach
(COI Folmer barcode), they accurately identified ca 650 spe-
cies, which according to them exceeds the phorid species

described for the entire Afrotropical region. They tested the
reliability of the MinION performance by comparing it with
Illumina sequencing (in 6251 of the 7059 specimens) and
morphology-based taxonomic identification (in 1500 of the
7059 specimens) and MinION had ca 99% of congruence of
the results with both. The increasing use of this portable and
lower-cost DNA sequencing technology is a recent trend
(Pomerantz et al. 2018, Schilthuizen et al. 2019) and will
accelerate the “democratization” of the NGS worldwide while
promoting local research capacity building.
As a future perspective, it is expected that NGS methods
will successfully address the problem of false-positive and -
negative detections, continue to decrease associated costs to
become more equitable throughout the world, reducing the
current bias of benefits mostly accruing to developed coun-
tries in the global north, and improve the production of data
for absolute quantification of entomologically relevant param-
eters by converting the amount of reads to a desired biological
unit (e.g., biomass, number of species).
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s13744-021-00895-x.
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