Neotropical Entomology

https://doi.org/10.1007/s13744-022-01011-3

BIOLOGICAL CONTROL IN LATIN AMERICA

DNA High‑Throughput Sequencing for Arthropod Gut Content

Analysis to Evaluate Effectiveness and Safety of Biological Control
Agents
Débora Pires Paula1   · David Alan Andow2

Received: 27 June 2022 / Accepted: 20 November 2022

© Sociedade Entomológica do Brasil 2022

Abstract
The search for effective biological control agents without harmful non-target effects has been constrained by the use of
impractical (field direct observation) or imprecise (cage experiments) methods. While advances in the DNA sequencing
methods, more specifically the development of high-throughput sequencing (HTS), have been quickly incorporated in
biodiversity surveys, they have been slow to be adopted to determine arthropod prey range, predation rate and food web
structure, and critical information to evaluate the effectiveness and safety of a biological control agent candidate. The lack
of knowledge on how HTS methods could be applied by ecological entomologists constitutes part of the problem, although
the lack of expertise and the high cost of the analysis also are important limiting factors. In this review, we describe how the
latest HTS methods of metabarcoding and Lazaro, a method to identify prey by mapping unassembled shotgun reads, can
serve biological control research, showing both their power and limitations. We explain how they work to determine prey
range and also how their data can be used to estimate predation rates and subsequently be translated into food webs of natural
enemy and prey populations helping to elucidate their role in the community. We present a brief history of prey detection
through molecular gut content analysis and also the attempts to develop a more precise formula to estimate predation rates,
a problem that still remains. We focused on arthropods in agricultural ecosystems, but most of what is covered here can be
applied to natural systems and non-arthropod biological control candidates as well.

Keywords  Diet breath · Food web · Predation rates · Prey biodiversity · Risk assessment · Trophic interaction

Introduction literature; cage or barrier experiments (exclosures or enclo-

Gut content analysis has played a significant role in advanc-
ing the understanding of the feeding relationships of arthro-
pod natural enemies, and recent advances in DNA detection
place these investigations on the cusp of delineating critical
details of species interactions in natural communities. In
the past, prey range was done by direct field observation
(live observations or video surveillance, Jones 1979; Hol-
mes 1984; Frank et al. 2007); compilation of the scientific
Edited by Marcos R de Faria.
* Débora Pires Paula
debora.pires@embrapa.br
1
Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF,
Brazil
2
University of Minnesota, St. Paul, USA
sures) with non-choice or choice tests (van Lenteren et al.
2006); prey baits through sentinel or artificial prey (Gei-
ger et al. 2010); indirect inference by correlating prey and
natural enemy abundance (Furlong 2015); and prey remains
by visual inspection under a microscope (Ingerson-Mahar
2002). Several limitations were intrinsically related to each
of those methods. Host-feeding, plant consumption (such as
leaves and pollen), and predation are ephemeral events and
often cryptic, especially for small organisms such as arthro-
pods (insects and mites), so direct field observations are
laborious and hard to monitor, especially for mobile natural
enemies. The literature may be incomplete regarding prey
and other food species of a natural enemy, especially for less
studied natural enemies or those released in a new environ-
ment. Cage experiments may introduce bias that ultimately
influences the natural enemy behavior and, consequently, its
parasitism or predation (van Lenteren et al. 2006). In addi-
tion, they are limited to the number of trophic interactions

13
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that can be tested or predicted artificially (van Driesche and
Hoddle 1997). Correlations of prey and natural enemy abun-
dances do not demonstrate a causal predatory relation as sev-
eral biotic and abiotic factors can influence their abundances
(Brandon and Ives 2014). Visual analysis of prey remains
under a microscope is limited to prey with hard body parts
(unsuitable for fluid feeding predators), which often are
not consumed by predators (Greenstone 1996), and by the
degree of the sample digestion and skill of the taxonomist
(Dennison and Hodkinson 1983). Rarely, in this case, does
the taxonomic resolution go beyond family level.
Molecular gut content analysis is being applied to cir-
cumvent the limitations of the previous methods by analyz-
ing prey remains in natural enemy gut contents (Symondson
2002). The evidence for predation is obtained after preda-
tion has occurred. It has been critical for assessing potential
biological control efficacy (Greenstone et al. 2010; Peterson
et al. 2018) and could be used to characterize environmental
risks on non-target species of candidate biological control
agents. The former is particularly important to target appro-
priate natural enemies in conservation biological control,
and the latter to not harm non-target species in classical bio-
logical control. Molecular gut content analysis has also pro-
vided various estimates of predation rates (Dempster 1960;
Nakamura and Nakamura 1977; Lister et al. 1987; Sopp
et al. 1992; Andow and Paula, submitted), which can allow
assessment of the potential impact of the natural enemy on
the prey population. Finally, it is revealing the interactions
of natural enemies with multiple prey species in food webs
(Paula et al. 2016; Lefort et al. 2017), which may allow
evaluations of impacts in a community context, and more
generally, the contribution of natural enemies to commu-
nity stability. In this review, we demonstrate how DNA-HTS
(high-throughput sequencing) gut content analysis can be
used for prey range determination, estimation of predation
rates, and characterization of natural enemy food webs.
Within conservation and classical biological control,
molecular gut content analysis is often overlooked. In con-
servation biological control, it can be used to determine the
main natural enemies of key agricultural or medical pests in
a habitat or ecosystem (e.g., Greenstone et al. 2010; Peter-
son et al. 2018). There is a tendency to rely on monitoring
population fluctuations of the key pests and local natural
enemies to identify the natural enemies that control the key
pests. This is based on the assumption that the significant
natural enemies occur in the same niche and habitat as
the key pests(s) and are those with high populations when
key pest(s) populations are low and vice versa. Although
potentially indicative of a significant trophic interaction, the
negative association of predator and prey populations does
not provide sufficient cause-effect evidence that a natural
enemy can control or even consume another population (Fur-
long 2015); not all co-occurrences represent true trophic
13
D. P. Paula, D. A. Andow
interactions. Only direct examination of predation, such
as with direct field observation and molecular gut content
analysis, can accurately determine which natural enemies
were frequently preying on the key pests and, therefore,
which natural enemies have the potential to control the pest
populations. In classical biological control, molecular gut
content analysis could be used to determine the efficacy of
controlling target pests and assess the potential of a natural
enemy to harm non-target species.
Along with the determination of prey range, estimation of
predation rates is vitally important to determine the overall
effect of the natural enemy on the prey population. Ideally,
predation rate is the number/biomass of prey consumed
by an individual natural enemy in a specified unit of time
(Dempster 1960; Hagler and Naranjo 1994). This requires
that molecular gut content analysis provides quantitative
information about the amount of prey consumed by a natural
enemy. As will be seen below, this is controversial for some
methods of molecular gut content analysis.
Most natural enemies interact with multiple species,
both as consumers of prey and as the consumed prey. To
fully understand their role in a community, it is important to
characterize these interactions. Molecular gut content analy-
sis can contribute to a deeper understanding of the web of
trophic interactions in which a natural enemy is embedded
and can begin to reveal the role of natural enemies in sta-
bilizing community structure and possibly providing long-
term suppression of prey populations. While molecular
gut content analysis will not provide information on non-
consumptive interactions, such as many mutualistic or trait-
mediated interactions (Glossary Supplementary Information,
SI), it has the power to unravel complex food webs, through
determination of the many density-mediated interactions (SI
Glossary) in a food web. It has contributed to the identi-
fication of considerable intraguild predation (SI Glossary)
among arthropod natural enemies within an agroecosys-
tem (Gagnon et al. 2011a, b; Davey et al. 2013; Hagler and
Blackmer 2013; Raso et al. 2014; Paula et al. 2016). It was
also used to verify that alternative prey are important to con-
serve the natural community of generalist natural enemies
in the absence of pest prey species in an agroecosystem and
that alternative prey can, in high abundance, disrupt preda-
tion on a pest prey (Kuusk and Ekbom 2010).
Within the molecular gut content analysis techniques,
DNA high-throughput sequencing (HTS) enables analyses
of a large number of samples and increases considerably the
probability of detection of prey and other foods that were
consumed a long time ago (days) or were rare or small. This
provides a more complete prey range with a finer taxonomic
resolution (Ji et al. 2013; Stein et al. 2014). This review
focuses on prey range determination through gut content
analysis by HTS DNA-based methods and on the construc-
tion of food webs from such data. We begin by providing
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
a brief history of the development of molecular gut con-
tent analysis to identify prey range and trophic interactions
of arthropod natural enemies. Following this, we present
an overview of important issues that must be considered
prior to starting DNA-based molecular gut content analysis.
Then, we describe the most recent HTS molecular methods
used for arthropod gut content analysis, metabarcoding and
Lazaro, a method to identify prey by mapping unassembled
shotgun reads, considering their strength and limitations. We
then discuss how gut content analysis can be used to char-
acterize prey range, estimate predation rates, and generate
natural enemy food webs.
Prey range
Historical Overview of Molecular Gut Content
Analysis
The history of molecular gut content analysis of arthropods
has been described extensively (e.g., Sunderland 1988;
Greenstone 1996; Symondson 2002; Harwood and Obrycki
2005; Sheppard and Harwood 2005; King et al. 2008; Fur-
long 2015; Birkhofer et al. 2017; Hagler 2019), so here it
will be mentioned briefly. It started nearly 80 years ago using
proteins as markers for the detection of prey, mostly by sero-
logical methods: precipitin test with polyclonal antibodies
(Brook and Proske 1946; Hall et al. 1953; Fox and MacLel-
lan 1956; Dempster 1960), agglutination (Greenstone
1977), complement fixation, and immunoassay (Boreham
and Ohiagu 1978; Sunderland 1988). The most commonly
adopted method was enzyme-linked immunosorbent assays
(ELISA, polyclonal or monoclonal antibodies) (Fichter and
Stephen 1981; Miller 1981; Ragsdale et al. 1981; Symond-
son et al. 1999, 2000; Naranjo and Hagler 2001; Hagler
2006). Non-serological methods were radio-isotope labe-
ling (Pendleton & Grundman, 1954), paper chromatography
(Putman 1965), and isoenzyme analysis (usually esterase)
through electrophoresis (Murray and Solomon 1978).
Although prey detection by protein-based methods has the
advantage of allowing detection of stage-specific conspecific
prey, therefore enabling study of cannibalism (Sigsgaard
et al. 2002), after the development of the polymerase chain
reaction (PCR) in the 1980s by Mullis (1990), these methods
were largely replaced by methods based on DNA (Agustí
et al. 1999; Zaidi et al. 1999; Chen et al. 2000; but see Hagler
2019 on universal food immunomarking technique-UFIT).
This was because the DNA-based methods were less expen-
sive, less time-consuming and labor-intensive, and more sen-
sitive and reproducible (Symondson 2002; Sheppard et al.
2005; although see Fournier et al. 2008). Examples of prey
detection by PCR-based methods are random amplified poly-
morphic DNA (RAPD) (Agustí et al. 1999), microsatellite
(Torr et al. 2001), PCR followed by temperature or denatur-
ing gradient gels (TGGE and DGGE) (Harper et al. 2006;
Martin et al. 2006), ligase detection reaction (LDR) PCR (Li
et al. 2011), terminal restriction fragment length polymor-
phism (tRFLP) (Juen et al. 2012), and prey-specific primers
to detect prey DNA in their natural enemy species using
PCR (Zaidi et al. 1999; Agustí and Symondson, 2001; Foltan
et al. 2005; Juen and Traugott 2005; Lundgren et al. 2009;
King et al. 2011; Davey et al. 2013) and qPCR (Lundgren
et al. 2009; Weber and Lundgren 2009).
Stable isotope analysis ( 15   N/ 14   N = δ 15 N and
13 12
C/ C = δ13C; Prasifka et al., 2004; Raso et al. 2014) and
fatty acid analysis (FAs) (Traugott et al. 2013) were also
developed and are particularly suitable for studying sea-
sonal changes in natural enemy diets and trophic material
flows as they address linkages between natural enemies and
groups of prey from different trophic levels or different basal
resources. 15 N isotope accumulates up the food chain, while
13
C does not, allowing the identification of the trophic level
of the natural enemy (Post 2002; Layman et al. 2007). The
fatty acid analysis is performed by comparing the FA pro-
files from the different trophic levels or groups of organ-
isms (e.g., bacteria versus fungi), mostly for soil food webs
(Ruess et al. 2002; Ferlian et al. 2012). These methods are
unable to identify prey species, but can complement protein-
or DNA-based methods for gut content analysis.
All these pioneering molecular methods provided impor-
tant contributions for the detection of prey in predator gut
contents as they demonstrated through feeding bioassays that
prey detectability is influenced by several factors, including
biomass of prey consumed (or meal size) (Sopp and Sun-
derland 1989; Hagler and Naranjo 1997; Agustí et al. 1999;
Hoogendoorn and Heimpel 2001); elapsed time after prey
consumption (Sopp and Sunderland 1989; Chen et al. 2000;
Weber and Lundgren 2009); predator species (Sunderland
et al. 1987; Sopp and Sunderland 1989; Symondson and
Liddell 1993; Chen et al. 2000; Hosseini et al. 2008; Lun-
dgren et al. 2009), stage/age (Rothschild 1966; Barazzoni
et al. 2000; Cotterill et al. 2013) and sex of the predator
(Symondson et al. 1999; Harwood et al. 2001); prey spe-
cies (Foltan et al. 2005; Harper et al. 2005); predator feed-
ing mode (chewing versus sucking, Greenstone et al. 2007);
predator starvation period or hunger level (Lövei et al. 1985;
Sunderland 1996; Weber and Lundgren 2011); subsequent
food consumed (chaser diet, Weber and Lundgren 2009);
temperature (Sopp and Sunderland 1989; Hosseini et al.
2008); sample preservation (Weber and Lundgren 2009);
taxon-specific variation in DNA copy number per cell (in the
case of comparison of quantity of different species of prey
consumed (Deagle et al. 2013); primer choice (Engelbrekt-
son et al. 2010); amplicon length (Agustí et al. 1999; Zaidi
et al. 1999; Chen et al. 2000; Hoogendoorn and Heimpel
2001; Deagle et al. 2006; Hajibabaei et al. 2006; King et al.
13

2008), with smaller amplicons having a higher chance of
detection (preferentially < 300 bp, Yu et al. 2012; although
the minimum length for taxonomic discrimination should
be determined, for example, Hajibabaei et al. 2006 reported
it to be 109 bp for COI); and sequencing direction (Deagle
et al. 2013); read quality filtering threshold (Thomas et al.
2014; Deagle et al. 2013; Nguyen et al. 2015). Later, it was
demonstrated that methodological choices also affect prey
detection, such as choice of metabarcode primer (Alberdi
et al. 2018), sequencing platform, sequencing depth (Smith
and Peay 2014), and the technique (Paula et al. 2022a). It is
clear that generalizations cannot be drawn reliably from the
analysis of a few samples. The lack of detection of a prey
might only mean that the detection was not possible due to
at least one of the aforementioned factors, as opposed to
lack of predation.
These older molecular methods were used to detect
specific and known prey. Prey detection and identification
relied on knowing some unique particularities of the known
prey, which were used to design the molecular method for
that prey. For example, immunodetection methods require
development of a prey species-specific antigen; PCR-based
methods require identification of species-specific primers to
amplify a unique DNA template sequence region; RAPD,
DGGE, and SSCP require characterization of a species-spe-
cific DNA fragment length separation profile. While some
of these methods can be extended to detect multiple species
(e.g., multiplex-PCR, De Barba et al. 2014), they are still
limited to detecting known species relying on the unique
particularities of those species.
More recently, methods for determining the biodiversity
of a community from environmental samples were developed
that enable simultaneous identification of multiple unknown
species in a sample and these methods have been applied to
gut content analysis. One method relies on the concept of
DNA barcoding (Hebert et al. 2003a), in which species have
a DNA region with sufficient sequence divergence to resolve
species taxonomy, flanked by highly conserved priming sites
where “universal” primer pairs (in reality: the most general
primer available, Clare 2014) could match and amplify the
region for a wide range of taxa (Simon et al. 1994; Valentini
et al. 2009a). A standard barcode marker for animals is a
fragment of 648–658 bp of the cytochrome c oxidase I gene
(COI) (Hebert et al. 2003b), known as the Folmer region
(Folmer et al. 1994). This sequence is available for many
species at The Barcode of Life Data System (BOLD-Ratnas-
ingham & Hebert 2007) (http://​www.​bolds​ystems.​org). The
main DNA barcoding primers used to amplify various parts
of the Folmer region can be found in Pompanon et al. (2012).
DNA barcoding enables amplification of DNA for a wide
range of taxa without prior knowledge of any part of the
genome. Barcode sequences can be classified into molecular
operational taxonomic units (MOTUs, Floyd et al. 2002), or
13
D. P. Paula, D. A. Andow
if species identifications are desired, matched to a reference
database of taxonomically identified barcode sequences.
Barcode-based methods have been applied in gut content
analysis to determine the biodiversity of prey consumed by
a natural enemy, first in small scale studies through Sanger
sequencing (Sanger and Coulson 1975) (Zaidi et al. 1999),
and later expanded to large scale analyses with the advent
of HTS. The latter is called metabarcoding and explored in
more detail in a subsequent section.
Recent methods rely on analysis of DNA shotgun
sequencing data without PCR amplification of the environ-
mental samples to characterize the community structure
from a DNA bulk sample taken from mock or field com-
munities (Li et al. 2015). These methods are based on HTS
of the naturally occurring multicopy genomic segments in
the sample (e.g., mitochondrial DNA, plastids, and nuclear
ribosomal DNA clusters), which facilitate species identifi-
cations without the need for amplification. These sequences
(called reads or query sequences) are matched to one or
more reference databases to identify species and have had
a high rate of correct species identifications (e.g., ≥ 95%,
Paula et al. 2022a). Some of these methods (e.g., mito-
metagenomics, genome skimming) assemble the reads into
longer contigs, which are matched to one or more reference
databases (Zhou et al. 2013; Tang et al. 2014; Crampton-
Platt et al. 2015; Linard et al. 2018). The longer sequence
of the contigs is expected to improve species identifications.
However, these assembly based methods are not suitable
for gut content analysis, because the prey DNA community
in the gut of a natural enemy is degraded and very difficult
to assemble error free. Other methods based on DNA shot-
gun sequencing data are assembly free (Zhou et al. 2013;
Ji et al. 2020; Sarmashghi et al. 2019; Paula et al. 2015,
2022a, b) making them suitable to be applied to gut content
analysis. As these PCR-free methods preserve the original
amount of DNA in the sample, they have demonstrated a
positive correlation between the biomass of each identi-
fied species in the sample and the proportion of identified
reads (Gómez-Rodríguez et al. 2015; Bista et al. 2018; Ji
et al. 2020; Paula et al. 2022b). So far, the only PCR-free,
assembly-free method demonstrated for gut content analysis
of arthropod natural enemies was developed by Paula et al.
(2015, 2016,2022a,b), called Lazaro, as it represents the
“resuscitation” of prey identifications from degraded DNA.
The application of metabarcoding and Lazaro for gut content
analysis is detailed below.
DNA‑HTS‑Based Gut Content Analysis
Several choices and precautions need to be taken before
starting any HTS gut content analysis. Most of these are
guided by experience acquired from biodiversity surveys,
as the use of HTS for gut content analysis in arthropods is
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
not yet widespread. The following choices and precautions
were compiled to prepare the samples to maximize detection
of true positive (TP) prey and non-detection of true nega-
tive (TN) prey, and minimize detection of false-positive (FP)
prey non-detection of false-negative (FN) prey. A TP is the
correct detection of a prey that was consumed by the natural
enemy and present in the gut contents. A FP (type I error)
is the erroneous detection of a prey that in reality was not
consumed by the natural enemy. These are usually biological
contaminants (called “pervasive exogenous contaminants”
by Taberlet et al. 2018) occurring in any step of the proce-
dure, but for DNA-HTS gut content analysis, they can also
be generated by “internal” sample artifacts, e.g., chimeras or
amplification errors during PCR (Schloss et al. 2011; Taber-
let et al. 2018), index jumping during library preparation
and sample missassignment in the bioinformatics process-
ing (Schnell et al., 2015), and sequencing errors (Quince
et al. 2011; Taberlet et al. 2018). A TN is the correct non-
detection of a species that was not consumed by the natural
enemy. A FN (type II error) is the erroneous non-detection
of a species that was actually consumed by the predator and
present in the gut contents (Ficetola et al. 2008, 2015). Rec-
ommendations of good practices for sample preparation can
be found in, e.g., King et al. (2008), Pompanon et al. (2012),
Ficetola et al. (2016), and Taberlet et al. (2018). Estimation
of performance measures, such as sensitivity, specificity,
false discovery rate (FDR), false omission rate (FOR), and
accuracy, can be found in Paula et al. (2022a). We highlight
specific issues here:
• Decide on using non-invasive (e.g., analysis of regur-
gitates or feces) or invasive means (post-mortem
analysis) of sample collection. Non-invasive means are
preferred when one wants to release the natural enemy
specimen back into nature; deposit it in an entomologi-
cal collection/museum; investigate the diet of rare or
protected species; track changes in individual dietary
preferences over time; or not affect population densities
and species interactions by taking large numbers of speci-
mens out of a habitat (Waldner and Traugott 2012). For a
non-invasive method, one will need to keep the specimen
alive to collect regurgitates or feces without giving it a
food supply or to collect regurgitates or feces in the wild.
Regurgitation (oral fluids, crop, and/or midgut contents)
is common in many arthropods as a defense mechanism,
such as carabid beetles and other ground beetles (For-
sythe 1982; Waldner and Traugott 2012), spiders (Kaes-
tner 1993) and grasshoppers (Sword 2000), and it can be
provoked during handling or by gently pressing anterior
abdominal sternites of the predator (Forsythe 1982; Wald-
ner and Traugott 2012). Waldner and Traugott (2012)
demonstrated that the prey detection in regurgitates was
similar or significantly enhanced compared to whole
specimen homogenization. Advantages of regurgitates are
less predator genetic material and higher relative “con-
centration” of prey DNA compared to invasive methods,
and reduced degradation of prey DNA compared to gut
contents or feces, which probably improves the accuracy
of prey detection. Conversely, regurgitates only represent
the most recent meal or fraction of the meal (Kamenova
et al. 2018). Invasive (post-mortem) gut content analysis
includes dissection of the natural enemy gut (Foltan et al.
2005) or whole body homogenization (e.g., Lundgren
et al., 2009) and is usually preferred for small arthropods
or ones with cryptic behavior. Most of the literature uses
post-mortem analysis. In this case, the natural enemies
will need to be killed and preserved immediately after
collection to slow down or stop the digestion process.
This can be done by immediately immersing the specimen
in 70–95% ethanol followed by storage at – 20 °C or, pref-
erably, − 80 °C (King et al. 2008). However, in our experi-
ence, adult ladybird beetles frequently regurgitate when
immersed in ethanol. So, one may prefer to kill a natural
enemy first by other means (e.g., killing jar with ethyl
acetate) before immersing it in ethanol. Alternatively,
one may extract the regurgitated DNA from the alcohol
inside the collection tube. Dissection of the gut provides
the advantage of reducing the ratio of predator:prey DNA
compared to whole body homogenates, consequently
reducing the sequencing depth required for prey detection
(please see further discussion on sequencing depth for gut
content analysis). Indeed, Krehenwinkel et al. (2017) rec-
ommend extracting prey DNA from the predator midgut
and hindgut. However, gut dissection is difficult, time-
consuming, especially for a large number of samples and/
or tiny specimens (e.g., predatory mites, microhymenop-
teran parasitoids), and may increase the chance for DNA
cross-contamination among samples.
• Wash potential external DNA from the natural
enemy bodies. This is especially important for bulked
samples, e.g., natural enemies sampled by sweeping,
beating, vacuum, malaise traps, or any other way that
mixes taxa (see Greenstone et  al. 2012 for a recom-
mended method to remove external DNA).
• Decide whether to pool or not pool specimens into
a single sample (an experimental unit or replication).
Pooling reduces a number of downstream steps (e.g.,
PCR reactions, purification, quantification, library prepa-
ration), time, and budget requirements. Pooling may be
preferable when the purpose is to characterize the average
diet of a population rather than individual variation. In
our experience, in either case (pooling or not pooling),
it is advisable to extract the DNA individually for each
specimen and retain individual aliquots for follow-up
work, such as confirming FPs and FNs as in Paula et al.
(2022b). Generally, and whenever feasible, gut content
13

analysis on unpooled individual specimens is better as
this retains all ecological information associated with
individual natural enemies and increases the number of
biological replicates.
• Use biological replicates at the sampling step and
technical replicates in the molecular methods (Taberlet
et al. 2018; Ji et al. 2020). Biological replicates enable
the use of ecological site occupancy models (SOMs) to
estimate detection probabilities for each species, which
can then be used to filter “suspicious” FP prey (Royle and
Link 2006; Miller et al. 2011; Schmidt et al. 2013; Fice-
tola et al. 2015, 2016; Lahoz-Monfort et al. 2016). The
range of biological replicates used in the literature has
been 2 to 10 (Ficetola et al. 2008; De Barba et al. 2014).
Technical replicates have been used in metabarcoding to
filter out FP species based on the relative Euclidean dis-
tances of the number of reads (i.e., fragments of DNA
sequenced) of the identified species between the PCR rep-
licates versus among the treatments (Zinger et al. 2019;
Neby et al. 2021). The higher the number of replicates,
the better the prey diversity coverage (but see Smith and
Peay 2014), but the higher the cost and workload. In addi-
tion, FN rates may increase if the detection probability of
the TP is low (Ficetola et al. 2015). Ficetola et al. (2015)
suggested a minimum 8 PCR technical replicates in meta-
barcoding when the probability of species detection is not
high as in ancient DNA studies, and this may be the case
for gut content analysis as well. Nichols et al. (2018),
using rarefaction curves, verified that more than 10 PCR
replicates would be necessary to sample the breath of taxa
in their experiment.
• Plan sequencing depth of coverage. Sequencing
depth of coverage is the number of times that a nucleo-
tide position is sequenced (Sims et al. 2014). It is com-
monly called only “depth” or “redundancy,” often used
as a synonym of “coverage,” although coverage can also
refer to “breadth of coverage,” which is the proportion
of genome sequenced. For genomic samples, the depth
of coverage is equal to the number of reads times the
average read length divided by the genome length (Sims
et al. 2014). There is no standard equation for the depth
of coverage for samples containing a mix of genomes
or fragments of genomes, such as gut content samples
and there is no widely accepted depth that recovers all
taxa in such a sample. For metabarcoding, a common
measure of depth is reads/amplicon/sample. Braukmann
et al. (2019) constructed a mock community of 374 taxa
and compared recovery of the taxa by metabarcoding
in relation to coverage depth and sequencing platform
(IonTorrent PGM, S5, and Illumina MiSeq). Their data
show that about 80% of the taxa are recovered when there
were 10,000 reads (post-filtering)/amplicon/sample and
they suggested that 95% of the taxa would be recovered
13
D. P. Paula, D. A. Andow
with 100,000–500,000 reads/amplicon/sample for all of
the platforms. Although the three platforms provided
similar recovery of taxa, MiSeq produced higher qual-
ity reads that facilitated bioinformatics analysis and was
recommended over the other two platforms. Singer et al.
(2019) conducted a meta-analysis of the 20 most indexed
metabarcoding biodiversity survey studies in Google
Scholar in 2018 and observed that 70% used MiSeq
(probably due to lower error rates and cost compared
to other technologies at the time) with a median depth
of coverage of 60,000 ± 55,000 reads/amplicon/sample,
ranging from 10,000 to ca. 900,000 reads. Using 8 meta-
barcoding samples with 3 technical replicates each, they
demonstrated that increasing sequencing depth of cover-
age in MiSeq increased to some extent the detection rate
of low-abundance taxa. But, they also demonstrated that
the Illumina NovaSeq platform detected 32–40% more
taxa than MiSeq when controlled for equal sequencing
depth (100,000 reads/amplicon/sample) using the exact
same PCR products (so no stochastic PCR biases could
justify the difference in detection). Even increasing the
sequencing depth, MiSeq did not reach the level of diver-
sity detected by NovaSeq, especially for low-abundance
taxa, due to its reduced capacity to detect exact sequence
variants (ESVs) in samples. Moreover, taxon accumula-
tion curves did not plateau in the NovaSeq analysis until
there were 1­ 07 reads/amplicon/sample. They attributed
this discrepancy in metabarcoding to the over-clustering
(SI Glossary) of low diversity reads in the MiSeq flow
cell, while in NovaSeq, this problem is alleviated by the
patterned flow cells.
Mapping of shotgun reads has also been successfully
used to characterize the biodiversity in communities.
Studies with mock communities have demonstrated 94.6–
97.9% accuracy in species determinations (Gómez-Rod-
ríguez et al. 2015, Tang et al. 2015, Bista et al. 2018, Ji
et al. 2020, Table S1). For these studies, we define cover-
age depth as the number of reads potentially hitting target
genetic material times the read length divided by the aver-
age length of the target genetic material. For example, if
the target genetic material is the mitogenome, then depth
is the total number of reads filtered to be mitogenome
reads times the read length divided by the average mitog-
enome size in the reference database. Gómez-Rodríguez
et al. (2015) tested 10 samples containing a total of 171
chrysomelid species. Using a mitogenome reference data-
base, they identified the species with 97.7% accuracy with
an estimated depth of 3233.3. Tang et al. (2015) tested
10 bee samples containing a total of 33 species and using
a mitogenome reference database they had 97.9% accu-
racy with an estimated depth of 268.4. Bista et al. (2018)
evaluated 10 samples with 13–14 freshwater hexapods
each, and using a mitogenome reference database, they
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
had 94.6% accuracy with an estimated depth of 308.7. Ji
et al. (2020) examined 14 samples with 19–20 species
of Arctic arthropods each against a mitogenome refer-
ence database and had 97% accuracy with an estimated
126.8 depth.
These findings from biodiversity studies can be applied
to gut content analysis by taking into account that as prey
DNA is less abundant than the predator DNA in the pred-
ator gut contents and is degraded or is being degraded,
the coverage depth per sample may need to be higher
than for biodiversity surveys. Although there is no stand-
ard sequencing depth recommendation for gut content
analysis, sufficient depth can be evaluated by rarefaction
curves (Nichols et al. 2018). When targeting mitochon-
drial barcodes, one should consider that the proportion of
mitochondrial genetic material sequenced corresponds to
0.1 to 5% of the total amount of reads sequenced (Taberlet
2012, Gómez-Rodríguez et al. 2015, Tang et al. 2015,
Bista et al. 2018, Ji et al. 2020), even though mitochon-
drial DNA is a natural multicopy material. Also, limit-
ing the sequencing and reducing the quantity of preda-
tor DNA will provide greater sequencing depth of prey
DNA. This can be accomplished with blocking primers
(Vestheim and Jarman 2008) and primers biased against
the predator (Krehenwinkel et al. 2019b) for metabarcod-
ing or dissection of the predator gut (Paula et al. 2022a)
and DNA size selection for Lazaro. Some examples of
sequencing depth reported in gut content analysis stud-
ies are given in Table 1. The median sequencing depth
of these predator gut content studies for metabarcoding
studies is 22,846 reads/amplicon/sample (range 1536 to
2,964,430 reads/amplicon/sample) and for Lazaro studies
is 307.4 (range 164.4 to 803.6). For metabarcoding, this
is smaller than the values used in biodiversity studies, but
for Lazaro, it is a similar sequencing depth.
• Use a focal or comprehensive reference database.
A focal reference database (e.g., Ji et al. 2020) populates
the database only with the sequences of species that are
expected to be found in the sample, while a comprehen-
sive reference database (Paula et al. 2022a,b) populates
the database with all available sequences of the target
taxon (e.g., all arthropods, all invertebrates, all plants,
or all bacterial symbionts). A focal reference database
would probably reduce FPs to a minimum, because no
unexpected species could be detected. However, it would
also probably increase FNs, because prey could be pre-
sent in the natural enemy gut that were not thought to
be consumed and therefore would not be included in the
reference database. Conversely, a comprehensive refer-
ence database may increase FPs, but reduce FNs. In any
event, if it is not certain which species are expected to be
found in the sample, a comprehensive database may be
a better choice. One should be aware that reference data-
bases typically need to be supplemented with reference
sequences of local species known to be possible prey that
are not already available in public sequence repositories,
such as GenBank or BOLD. For example, Dopheide et al.
(2019) and Liu et al. (2020) reported the lack of hundreds
of OTU representative sequences in GenBank, which
precluded them from identifying these species. For bar-
codes, this may require amplicon sequencing of positive
control samples of potential prey species. For organellar
genomes, this can be accomplished by including a library
of each positive control sample of a potential prey species
as a part of an HTS project. For all DNA-HTS methods,
the taxonomic resolution of prey species identification is
determined by the taxonomic resolution and accuracy of
sequences in the reference database (Bridge et al. 2003).
That is why it is recommended, but not always the case,
to populate the reference database with sequences from
morphologically curated specimens.
• Chose the taxonomic classifier for taxon assignment.
These classifiers include alignment of the sequenced
DNA using BLAST (Altschul et al. 1990), phylogeny-
based methods (Munch et al. 2008a), lowest common
ancestor methods (Huson et al. 2011), naïve Bayesian
classifier (NBC) (Porter et al. 2014), or an alignment-
free method, such as k-mer-based methods (Breitwieser
et al. 2018).
Prey detection by metabarcoding
Species biodiversity assessment through DNA barcoding
coupled with HTS is called “DNA metabarcoding,” first
coined by Taberlet et al. (2012), but the method had already
been used for species identification in environmental sam-
ples (e.g., Valentini et al. 2009a). Taberlet et al. (2018)
described the method in detail in the book “Environmental
DNA: For Biodiversity Research and Monitoring.” Briefly,
metabarcoding starts by selecting one or more “universal”
or group-specific primers to amplify a barcode region of
the set of species in the sample. Considering the Illumina
sequencing platform, sample DNA is amplified and tagged,
preferentially with unique forward and reverse tags, during
PCR amplification so that they can be pooled (multiplexed)
in the same library without losing the sample identity. For
library preparation, the pooled tagged samples have their
ends extended by a few PCR cycles to include a library
index and sequencing adaptors (sequencing priming sites
and flow cell binding sites P5 and P7). Libraries are quanti-
fied (Harris et al. 2010) and, if desired, multiplexed in the
same sequencing lane (usually up to 96 libraries with dual
13


Table 1  Sequencing depth used in prey range determination or diet analysis by metabarcoding or Lazaro of predator gut contents. For metabarcoding, sequencing depth was estimated as
the total reads/amplicon/sample. For Lazaro, sequencing depth was estimated as the number of reads associated with the target genome × read length/target genome length (for mitoge-
nomes, ~ 15,000 bp); *inferred from Dryad data archive; **fecal and spider web samples; †mitogenome reads with BlastN query match with ≥ 100 b, 90% identity; ‡mitogenome reads only. QC,
quality control

13
Average Average Sequencing HTS prey Depth Number Number Number of Number of Predator taxa Identity Number of % Predator Number Number of Number of Reference
read prey target parameters detection of indi- of sam- reads before reads after threshold reads used/ reads of prey prey reads prey reads
length length method vidual ples QC/ sample QC/ sample sample species per speci- per sample
(bases) (bp) speci- or OTU’s men
mens detected

150* 300 HiSeq- Metabar- 12,046 156 52* n/a 12,046 Spider 97% OTU 12,046 61 434 OTUs 1349 12,046 Saqib et al.
Illumina coding families (2021)
300 211 MiSeq-Illu- Metabar- 21,650 333 333 n/a 21,650 Spiders n/a 21,650 20.9, 99.4, n/a 65, 130, 65, 130, Krehenwin-
mina, v3 coding 99.7 17,125 17,125 kel et al.
(2019b)
300 201–296 MiSeq-Illu- Metabar- 6382 (1798– 333 333 n/a 31,911 Spiders 3% OTU 31,911 45.5 (2.5– Order- 3478 (815– 3478 (815– Krehenwin-
(5) mina, v4 coding 21,700) radius 72.1) level 10,177) 10,177) kel et al.
OTUs (2019b)
250 150 MiSeq-Illu- Metabar- 117,357 n/a 107** n/a 141,121 Fish, bats, 3% diver- 141,121 0-n/a 3.00–6.76 n/a n/a Corse et al.
mina, v5 coding spider gence OTUs (2019)
300 350 MiSeq-Illu- Metabar- 24,043 210 210 48,220 24,044 Spiders 97% OTUs 14,155 99.0 50 145 145 Toju and
mina, v3 coding hexapod Baba
OTUs (2018)
300 130, 350 MiSeq-Illu- Metabar- 28,970 27 n/a n/a n/a Spider, 10−3 n/a 97.32 Order- 1552 n/a Krehenwin-
mina, v3 coding Hololena e-value (92.57– level kel et al.
adnexa 99.07) OTUs (2017)
250 313 MiSeq-Illu- Metabar- 113,128 6 3 n/a 113,128 Spider, n/a 113,128 90.85 144 OTUs 0 0 Macías-
mina, v2 coding Dysdera Hernán-
verneaui dez et al.
(2018)
300 332 MiSeq-Illu- Metabar- 15,052 505 505 n/a 15,873 Spiders, 97% 15,053 n/a 3 (1–17) n/a n/a Verschut
mina, v3 coding Lycosidae taxa/ et al.
sample (2018)
150 69, 157 MiSeq-Illu- Metabar- 4146, 1536 670 670 8667, 3664 4608, 1707 Spider, Par- 98%  ~ 4146, 1536 n/a 25 families n/a n/a Eitzinger
mina, v2 coding dosa sp. et al.
(2018)
150 190 HiSeq Metabar- 2,964,430 3968 27 3,801,666 2,964,430 Ants, der- 98% 2,964,430 71.76 14 species 11,339 83,7057 Paula et al.
4000-Illu- coding mapteran, (114– (5244– (2022a)
mina, v1 carabid 39,900) 1,940,400)
150 150 HiSeq Lazaro 447.7 3968 27 5,509,048 5,504,578 Ants, der- 100%, 44,769† 97.92 13 species 10.8 (1.3– 933 (355– Paula et al.
4000-Illu- mapteran, 130 bp 29.2) 2040) (2022a)
mina, v1 carabid
250 250 HiSeq Lazaro 803.6 340 34 n/a 2,750,640 Ladybird 99%, 48,213† 99.10 7 species 33.5 335 Paula et al.
2500-Illu- beetles 150 bp (2022b)
mina
Rapid
Mode
250 250 MiSeq-Illu- Lazaro 164.4 22 4 n/a 2,777,244 Ladybird 98%. 9863‡ 99.39 12 spe- 51.0‡ 60.5‡ Paula et al.
mina, v2 beetles, 225 bp cies‡ (2016)
der-
mapteran
D. P. Paula, D. A. Andow
 DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…

indexing sequencing) to proceed the sequencing. Depending

Paula et al.
Reference
on the desired sequencing depth per sample, the number of

(2015)
samples per lane can be calculated. For example, suppose
one lane of an Illumina MiSeq sequencer can provide 30
million reads and you want to have at least 100,000 reads
per sample
Number of
prey reads

per sample and 10% of the reads will be from the calibration
11.5‡

control library PhiX. This means that you can load up to 270
samples into the lane.
Number of
prey reads
per speci-

After amplicon HTS, the raw sequences pass through a

1.2‡
men

quality control and then are analyzed using a bioinformat-

ics workflow or a combination of them, available in several
1 species‡
or OTU’s

open source packages, initially for biodiversity surveys of

detected
Number

species
of prey

microbial communities, such as MOTHUR (Schloss et al.

2009) and QIIME (Caporaso et al. 2010), later also for diet
% Predator

analyses, such as OBITools (Boyer et al. 2016). These pack-

99.89
reads

ages basically will perform: (a) quality control of the data-

set sequencing quality and trimming sequencing adapters;
(b) reconstitute the full amplicon sequences by pairing the
reads used/
Number of

10,019‡

paired-end reads and removing non-overlapping reads; (c)

sample

sorting the amplicons from different samples to their original

sample by identification of their tags (i.e., demultiplexing);
threshold

225 bp

clustering identical sequences and reducing them to only one,

Predator taxa Identity

98%.

while preserving their occurrence count (dereplication); and

classifying the amplicons by similarity of their sequences
Harmonia
axyridis

with thousands of sequences from the barcode region pre-

Ladybird
beetle,

sent in a reference database of several species (presumably

including those from the sampled habitat) with a predefined
taxonomy (supervised taxa classification) according to their
QC/ sample
Number of
reads after

3,654,005

similarity to a reference database; or classifying them accord-

ing to their similarity to one another within a cluster, without
a taxonomic reference (unsupervised classification), i.e., clas-
of indi- of sam- reads before
QC/ sample
Number Number Number of

sifying the reads into molecular operational taxonomic units

(MOTUs). In this last case, only sample richness and not
n/a

sample composition is characterized. In supervised classifica-

tion, taxon assignment is based on sequence similarity (match
ples

of query to the reference database) with a minimum percent

2

identity (usually > 98%) and overlap length (usually > 100 bp)

vidual
speci-
mens

threshold, usually set arbitrarily by the researcher (Reeder

20

and Knight 2010; Quéméré et al. 2013; De Barba et al. 2014).

Metabarcoding is a tremendous advance over previous
HTS prey Depth

167.0

methods for gut content analysis (e.g., Piñol et al. 2014a, b).
Unlike these previous methods, it does not rely on a priori
detection

knowledge or assumptions that a species is, in fact, a prey.

method

Lazaro

Most significantly, instead of screening for a single target

species (prey-specific PCR, King et al. 2011; Davey et al.
MiSeq-Illu-
Sequencing
parameters

mina, v2

2013), it screens for the entire community of prey, with an

indefinite number of species that can be detected (Varennes
et al. 2014). Nonetheless, the Achilles heel in metabarcod-
Table 1  (continued)

ing is the use of PCR to amplify target barcode(s) and tag

prey target
Average

length

samples due to problems related to variation in primer effi-

(bp)

250

ciency of the “universal” barcode primers across a broad

range of taxa (Clarke et al. 2014; Deagle et al. 2014; Elbre-
Average

(bases)
length
read

cht and Leese 2015, 2017), DNA polymerase errors, PCR

250

13

stochasticity (mainly in the first cycles of PCR), and tem-
plate switches (generation of hybrid sequences) (Kebchull
and Zador 2015). Kobayashi et al. (1999) reported that errors
originated in PCR amplification steps can be present in as
much as 2% of all amplicons for a 250-bp amplicon.
Primer efficiency is reduced when the primer does not
anneal perfectly with its template and is related to mis-
matches between the primer sequence and its template.
In part, this happens in template protein-coding regions
because of the degeneracy of the third nucleotide in a codon
(Taberlet et al. 2012). Another source of mismatches is the
natural variation (haplotype polymorphisms) in the nucleo-
tide sequences (e.g., SNPs) within and between individuals
from the same species or heteroplasmy, presence of different
organellar genomes in the same cell or individual (Rubinoff
et al. 2006). No matter the source, position, or type (Kwok
et al. 1990), mismatches can result in the absence or poor
amplification of the barcode for a species in the DNA mix-
ture of a sample. Beside mismatches, Pan et al. (2014) dem-
onstrated that primer efficiency is influenced by the DNA
polymerase due to its preference for certain sequence motifs
in the six nucleotides at the primer 3’-end and four nucleo-
tides downstream of the priming site in the template DNA.
DNA polymerases errors are related to single-base sub-
stitutions, indels (insertions/deletions causing frameshift
errors) (Cline et al. 1996), and errors derived from PCR
stochasticity (Kebschull and Zador 2015) that lead to the
formation of chimeras and heteroduplexes (Qiu et al. 2001).
Chimeras may occur among closely related or abundant
sequences in complex samples (Haas et al. 2011; Schloss
et al. 2011; Elbrecht and Leese 2015; Taberlet et al. 2018)
and are generated when incomplete extension occurs during
the elongation step and the resulting fragment acts as primer
in the next cycle of PCR (Quince et al. 2011). Schnell et al.
(2015) discussed the different mechanisms for chimera for-
mation and suggested that it can be avoided by using emul-
sion PCR as each template is amplified separately inside a
microdroplet. Heteroduplexes are the result of recombination
between dissimilar PCR products.
Regarding template switches, according to Pääbo et al.
(1990), lesions in the template DNA, such as breaks, apu-
rinic sites, and UV damage may cause the extending primer
to “jump” to another template during the PCR. Considering
this, it is reasonable to assume that the problem of template
switches might happen also for gut content samples, and
therefore, metabarcoding could be less appropriate for gut
content analysis, as the prey DNA might be damaged due to
the predator’s digestion process.
Additional potential sources of errors occurring down-
stream of the PCR step may arise during library construc-
tion, such as tag jumps (Amend et al. 2010; Harris et al.
2010; Porazinska et al. 2010; Carlsen et al. 2012; Schnell
et al. 2015); sequencing, such as miscounted homopolymeric
13
D. P. Paula, D. A. Andow
extensions (Kunin et al. 2010; Quince et al. 2011; Schloss
et al. 2011); bioinformatic processing, such as (a) incorrect
assembly of reads due to low coverage/sequencing depth
(Smith and Peay 2014), (b) missorting of the reads to the
correct sample (Amend et al. 2010), and (c) taxonomic over-
classification (i.e., detection of a closely related species of
the prey as opposed to the actual prey, which was missing
from the reference database) (Richardson et al. 2017); and
errors in the sequences in the reference database and mis-
taken taxonomy assignment of deposited sequences, both
due to lack of sequence quality and taxonomic curation in
most public databases (e.g., GenBank, Harris et al. 2003).
Sequencing errors and taxonomic overclassification are com-
mon problems in any HTS method, not only for metabarcod-
ing (e.g., Martin-Laurent et al. 2001; Dopheide et al. 2019).
Most of the mentioned errors can be mitigated, at least
in part, by the use of algorithms specifically designed to
identify and remove them (e.g., UCHIME for chimeras,
Edgar et al. 2014; Edgar and Flyvbjerg 2015). If errors not
removed from the datasets, they might generate FPs and/or
FNs. This has created suspicion that species identified with
a low number of reads are artifacts of one or more of these
errors (Reeder and Knight 2010). According to Pommier
et al. (2010), these potential artifacts can substantially inflate
diversity estimates as they can account for more than 50% of
the MOTUs after data quality control. Consequently, many
authors arbitrarily remove all species identified by a small
number of reads, usually < 100 reads (e.g., De Barba et al.
2014). Champlot et al. (2010), Ficetola et al. (2016), and
Taberlet et al. (2018) provide guidance to avoid or minimize
the occurrence of FPs and FNs. Their recommended proce-
dures are summarized as follows:
A. Employing multiple metabarcode primer pairs for the
same or different barcodes (Dupuis et al. 2012; De Barba
et  al. 2014; Deagle et  al. 2014; Krehenwinkel et  al.
2017);
B. Using metabarcode primers tailored for specific taxo-
nomic groups (Harper et al. 2005; Jarman et al. 2005;
Piñol et al. 2015), designed by, e.g., ecoPrimer (Riaz
et al. 2011);
C. Using IUPAC degenerate nucleotide codes in the meta-
barcode “universal” primer. While this probably reduces
bias caused by mismatches of the primer with the tem-
plate, it most likely also decreases primer efficiency
(Jaric et al. 2013; Gibson et al. 2014);
D. Using blocking primers (Vestheim and Jarman 2008)
that exclude or minimize amplification of natural enemy
DNA (Deagle et al. 2009; De Barba et al. 2014) or prim-
ers biased against the natural enemy (Krehenwinkel
et al. 2019b), resulting in relatively more prey DNA for
sequencing. This is recommended only when the natural
enemy and the expected prey are phylogenetically dis-
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
tant, as blocking or biased primers could also block or
bias the amplification of prey species closely related to
the predator;
E. Pretesting in  vitro and/ or in silico (e.g., through
ecoPCR, Ficetola et al. 2010) the metabarcode primer
pairs to verify the taxonomic coverage of the expected
prey and, if possible, reduce amplification of the natural
enemy DNA (Clarke et al. 2014; Alberdi et al. 2018;
Taberlet et al. 2018);
F. Adopting good laboratory practices to minimize the risk
of contamination among samples (cross-contamination)
and PCR reactions (PCR product carryover) (King et al.
2008; Taberlet et al. 2018);
G. Including biological and technical replicates to be able
to use site occupancy models (Schmidt et al. 2013; Fice-
tola et a. 2015, 2016; Lahoz-Monfort et al. 2016; both
in Taberlet et al. 2018) to infer the probability of detec-
tion, define a threshold to eliminate FPs, evaluate if the
level of replication is appropriate to control FPs, and
reduce the likelihood of FNs (Alberdi et al. 2018). For
this purpose, biological and technical replicates should
be sequenced separately (Smith and Peay 2014), and
some advocate for sequencing separate PCR replicates
for each barcode per sample (Robasky et al. 2014; Ji
et al. 2020). The number of PCR replicates per sample
have varied from 2 to 24 (De Barba et al. 2014; Smith
and Peay 2014; Willerslev et al. 2014; Ficetola et al.
2015; Lahoz-Monfort et al. 2016; Alberdi et al. 2018;
Dopheidi et al. 2019; Shirazi et al. 2021). Several studies
have reported that the higher the number of PCR repli-
cates, the higher the alpha diversity detected (specially
for rarer taxa) (e.g., Alberdi et al. 2018; Dopheide et al.
2019; Shirazi et al. 2021). However, there is no general
recommendation for the number of PCR replicates, as
it seems to be case specific depending on the expected
number of rare taxa and sequencing depth (Smith and
Peay 2014; Alberdi et al. 2018). For example, for Shi-
razi et al. (2021), 24 PCR replicates were not enough
to reach a species saturation in the rarefaction curves.
Therefore, ideally a preliminary test could be conducted
to estimate the number of replicates, e.g., using site
occupancy models (Ficetola et a. 2015; Lahoz-Monfort
et al. 2016) and rarefaction curves (Hsieh et al. 2016;
Shirazi et al. 2021), both based on predictions of taxon
abundance;
H. Using negative DNA extraction controls, negative
and positive PCR controls, unique tags and tagging
system controls (Schloss et al. 2011; De Barba et al.
2014; Taberlet et al. 2018), and even a mock com-
munity (i.e., known set of organisms with quanti-
fied amounts of DNA) (Amend et al. 2010; Nguyen
et al. 2015). These would enable evaluation of some
sources of contamination and the efficacy of the
PCR, to allow identification of tag jumps, choose
objectively the appropriate sequence quality filtering
threshold and calibrate the clustering threshold for
MOTUs (i.e., 95%, 97%, 97.5%) to best recover the
actual number of MOTUs or species in the dataset.
Instead of a mock community, Shirazi et al. (2021)
made a positive control PCR and library with one
species known not to occur in the sampling area to
be able to detect index hopping (SI Glossary) dur-
ing sequencing (index hopping is also discussed in
Singer et al. 2019; van der Valk et al. 2020);
I. Evaluating carefully the choice of the DNA polymerase
between non-proofreading versus proofreading (high
fidelity) polymerases. Proofreading DNA polymerases
correct for DNA amplification errors (single-base sub-
stitution errors and frameshift errors due to indels) dur-
ing the elongation step in PCR because it has 3’ → 5’
exonuclease activity that removes a mismatch at the
3’-end of the new DNA strand being synthesized and
replaces the incorrect nucleotide with the correct one.
Sze and Schloss (2019) tested the influence of different
proofreading DNA polymerases (AccuPrime, KAPA
HiFi, Phusion, Platinum, and Q5) and number of PCR
cycles in chimera production. They demonstrated that
fewer chimera were formed using DNA polymerases
with the highest fidelity and minimizing the number of
PCR cycles. On the other hand, Nichols et al. (2018)
evaluated two non-proofreading DNA polymerases
(AmpliTaq Gold and Qiagen Multiplex Master Mix)
and four proofreading DNA polymerases (KAPA HiFi,
Phusion, Platinum HiFi, and Q5) to accurately estimate
species occurrence and relative species abundance and
the proofreading DNA polymerases did not have the
best results. They reported that Platinum HiFi had a
preference to amplify templates with 34–38% of GC
content (polymerase GC bias) that was sufficient to
distort the final result of species relative abundance.
Qiagen Multiplex Master Mix had the least GC bias
and, therefore, resulted in the most accurate prediction
of sample species relative abundance, although it also
had the highest sequence amplification errors. In addi-
tion, in complex DNA mixtures, proofreading DNA
polymerases can also remove mismatches at the 3’-end
of the primers, leading to non-specific amplifications
(loss of specificity) (Taberlet et al. 2018) (see Taber-
let et al. (2018) for a discussion of the choice of non-
proofreading versus proofreading DNA polymerases);
J. Optimizing PCR components and parameters to mini-
mize PCR chimera formation, such as the use of a hot-
start Taq polymerase, an appropriate number of PCR
cycles (Sze and Schloss 2019), increased elongation
time during library index PCR, decreased template
concentration (Qiu et al. 2001; Schnell et al. 2015), and
13

increased duration of the denaturation step (initial and
in each cycle) to reduce GC bias (Aird et al. 2011). A
higher number of PCR cycles might increase the likeli-
hood of detection of rare taxa, especially in gut content
analysis where the target DNA is scarce and degraded;
however, it could also skew abundance estimates by
amplifying the biases. Murray et al. (2015) recommend
the use of qPCR to determine the optimal number of
PCR cycles in the sample barcode amplification and
Schnell et al. (2015) recommend the use of qPCR to
determine the minimum number of PCR cycles during
library preparation (tag incorporation) to minimize risk
of tag jumping. Taberlet et al. (2018) recommended
the use of at least 1-min elongation time to avoid the
creation of artifactual sequences generated by single-
stranded DNA during the downstream steps of library
preparation and sequencing;
K. Investing in improving the comprehensiveness, accuracy,
and redundancy of the reference database for the studied
ecosystem (true for any DNA-HTS-based method), pref-
erentially with sequences from specimens in which the
species had its taxonomy verified and curated. This is a
special challenge for generalist natural enemies, which
may have many unknown or scarce prey;
L. Increasing sequencing depth per sample to reduce the
possibility for FN and increase alpha diversity detected
(Krehenwinkel et al. 2017; Alberdi et al. 2018; Taberlet
et al. 2018). On the other hand, Shirazi et al. (2021)
observed that, generally, higher sequencing depth
requires a higher number of PCR replicates to reach
species saturation in the rarefaction curves;
M. Removing “uncertain” taxa detected only once out of the
number of independent replicates (Nr) (Giguet-Covex
et al. 2014; Willerslev et al. 2014; Ficetola et al. 2015).
N. Calculating relative Euclidean distances of the number
of reads of the identified species among the PCR repli-
cates (not pooled) versus among the treatments (Zinger
et al. 2019; Neby et al. 2021). This assumes that the
distance among replicates is smaller than the distance
among treatments;
O. Testing species assignment using Bayesian phylogenetic
analysis and provide a measure of statistical confidence
in the assignment (Munch et al. 2008b);
P. Filtering by minimum amplicon sequence count to
remove sequences with low frequency of occurrence,
either in the whole dataset or observed in a limited
number of samples (Shehzad et al. 2012; Quéméré et al.
2013; De Barba et al. 2014);
Q. Removing from the detection results species very
unlikely to be preyed upon by a predator, either due to
anatomical reasons, separation by geographic distances
or seasonal patterns (De Barba et al. 2014; Taberlet et al.
2018);
13
D. P. Paula, D. A. Andow
R. Using another method to confirm the metabarcoding
results, such as melting curve analysis (MCA) in qPCR
(Paula et al. 2022a).
For arthropods, the most common universal barcode
primers are for the Folmer region (Folmer et al. 1994) of the
COI mitochondrial gene, but many others are also used (see
Valentini et al. 2009b; Pompanon et al. 2012; Taberlet et al.
2018). The COI barcode has a large reference database, but
it has some important limitations, such as insufficient con-
servation of primer sites to allow similar primer efficiency
across taxonomic groups (Deagle et al. 2014; Elbrecht et al.
2016; Sousa et al. 2019), lower taxonomic coverage than
16S (Clarke et al. 2014), and bias in amplifying lepidop-
terans and dipterans, while failing to amplify other insect
orders (e.g., hymenopterans) (Clarke et al. 2014). A good
metabarcoding primer has the combination of providing
high taxonomic coverage and high taxonomic resolution,
but none so far fit all the criteria (Ficetola et al. 2010; Riaz
et al. 2011; Valentini et al. 2009b). One important consid-
eration about using mitochondrial metabarcoding primers is
that non-functional copies of mitochondrial genes might be
transposed to the nuclear genome, creating NUMTs (nuclear
mitochondrial DNA sequences). Because they lose their
function (pseudogenes), they can rapidly accumulate muta-
tions (Leite 2012) and, therefore, generate FPs.
Mapping Unassembled Shotgun Reads (Lazaro)
Lazaro, in its preliminary version called DDSS (direct DNA
shotgun sequencing), is a HTS detection method developed
for gut content analysis in which the prey DNA community
is not enriched by PCR before sequencing and there is no
read assembly after sequencing (Paula et al. 2022a). Briefly,
after DNA extraction, concentrations are normalized across
samples. Individual libraries are created for each sample,
as there is no PCR step to tag and enable sample multi-
plexing in a same library, and sequenced. The raw reads are
passed through quality control and, without assembly due
to the degraded DNA of the prey, taxa are assigned right
in the beginning of the bioinformatic workflow by match-
ing to a single or multiple reference databases (Paula et al.
2016) using local BlastN with an E-value < 1e-30, specifying
an output format XML and removing matches with over-
lap length < 100 bp or identity < 90% (Paula et al. 2022a).
Next, a customized BlastNToSNP script is used to print the
relative positions of the mismatches. Using R scripts, mis-
matches falsely generated by IUPAC degenerate nucleotide
codes are eliminated and matches that are under a thresh-
old of minimum percent identity (95%) and overlap length
(100 bp) are discarded. A threshold is used to clean the most
FPs and retain the most TPs. For 250 paired-end reads, 99%
identity in 150-bp overlap was optimal (Paula et al. 2022b),
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
and for 150 paired-end reads, 100% identity in 130 bp over-
lap was optimal (Paula et al. 2022a). Single-end reads are
eliminated, as well as reads not mapping to coding sequence
regions, e.g., of the target genome. Finally, the number of
reads of species identified in the blank library are subtracted
from the sample datasets. The reads of the remaining hits are
then considered to identify the prey species. More details on
the Lazaro methodology can be seen in Paula et al. (2022a,
b) and at the GitHub repository: https://​github.​com/​molec​
ular-​ecolo​gy/​DDSS.
Lazaro shares the same advantages of metabarcoding over
previous methods for DNA-based gut content analysis. It
does not require a priori knowledge or assumptions that a
species is a prey, and most importantly, it screens for the
entire community of prey in the gut of the natural enemy
(Paula et al. 2016, 2022a). However, because it does not
require sample DNA amplification, it eliminates all of the
limitations of metabarcoding associated with PCR, and the
absence of amplification enables any part or multiple parts
of the prey genome (nuclear or organellar) to be used to
detect and identify prey, including any barcode sequence
(Paula et al. 2016). In addition, parasites, symbionts, and
plant species can also be identified in the gut contents of the
natural enemy (Paula et al. 2015, 2016). These characteris-
tics mean that the original composition of the sample DNA
is preserved throughout the sequencing process and the num-
ber of reads of a prey is proportional to the amount of that
prey in the original predator gut content, enabling quantita-
tive interpretation of the results (Paula et al. 2015, 2022a,
b). Moreover, this allows the samples to be reanalyzed at any
time in the future when reference databases or the bioinfor-
matic workflow have improved. Its assembly-free charac-
teristic was designed to be suitable for applications involv-
ing degraded DNA. Instead of using assembled genomes or
barcodes and sequence similarity for taxa assignment, one
can use the Skmer method (Sarmashghi et al. 2019) so that
unassembled reads are used in the reference database and the
taxa assignment is based on counting unique k-mers. How-
ever, one should note that the absence of PCR amplification
is also the source of its major limitation; samples cannot be
multiplexed in the same library, because individual samples
cannot be uniquely tagged. At present, this means that each
sample must be made into its own library, increasing the
cost of sequencing.
Some of the factors that can generate FP and FN in the
species detections of metabarcoding are shared with Lazaro.
For example, natural sources of mismatches (e.g., SNPs,
heteroplasmy), the integrity of the DNA fragment to be
sequenced, contamination in the DNA extraction, errors
in the library construction, sequencing and bioinformatics
steps, including taxonomic overclassification and low rep-
resentativeness, and the accuracy and redundancy of spe-
cies in the reference database. Based on our experience, we
recommend using some of the same procedures mentioned
for metabarcoding to maximize TP detection and minimize
FP and FN detections with Lazaro: adopt good laboratory
practices to minimize the risk of sample contamination (item
F of the metabarcoding list); include biological and techni-
cal replicates (item G); use negative DNA extraction con-
trols, a positive spike-in control community (Ji et al. 2020),
and unfed predator controls (Paula et al. 2022b) (equivalent
to item H); improve the comprehensiveness, accuracy and
redundancy of reference databases for the studied ecosystem
(item K); increase sequencing depth per sample (item L);
remove “uncertain” taxa detected only once out of the num-
ber of independent replicates (item M); calculate relative
Euclidean distances of the number of reads of the identified
species among the replicates versus among the treatments
(item N); remove from the results species very unlikely to be
preyed upon by a predator (item Q); use additional method
to confirm the results, as in Paula et al. (2022a) (item R).
To our knowledge, metabarcoding and mapping unas-
sembled shotgun reads, such as Lazaro, have only been
compared in two studies. Srivathsan et al. (2015) compared
metabarcoding and read mapping (using BlastN) to identify
diet composition by fecal analysis (host plant chloroplasts)
of two red-shanked doucs langurs (Pygathrix nemaeus) fed
with a known diet. While metabarcoding detected 34% of the
diet composition, read mapping detected 50% of the known
diet plus an unexpected species that was later confirmed to
be in the diet. Paula et al. (2022a) compared metabarcod-
ing and Lazaro prey detection in a coccinellid predator fed
a mock community of prey, and in field-collected samples
where detections were confirmed by qPCR-MCA. In the
mock community, Lazaro detected 57% of expected prey,
while metabarcoding detected none of them. In the field
samples, metabarcoding and Lazaro had similar sensitiv-
ity, specificity, false discovery rate, false omission rate, and
accuracy. However, prey detection was partially complemen-
tary, and while the methods shared 87% of the confirmed
prey, the resulting food webs would be quite different if
only one method had been used. Thus, it is not clear which,
if either, of metabarcoding or Lazaro provides better prey
detection in gut content analysis, but there is a slight indica-
tion that under some conditions, Lazaro may be better.
Scavenging, Secondary Predation, and Cannibalism
Even with the considerable advances in the molecular tech-
niques for gut content analysis, the area still struggles to
identify scavenging (Sunderland 1988, 1996; Calder et al.
2005; Foltan et al. 2005; Juen and Traugott 2005), sec-
ondary predation (Harwood et al. 2001; Hoogendoorn and
Heimpel 2001; Sheppard et al. 2005; Paula et al. 2022b),
and cannibalism (except for serological tests, Sigsgaard
et al. 2002), all of which cause errors in the determination
13

of prey range or predation rates with profound ecological
implications (King et al. 2008). They are not uncommon
among arthropods (they have been noted in nearly every
order) and may grossly overestimate the biological con-
trol services rendered by a predator species (see several
examples in Sunderland 1996). For example, intraguild
predation, which can lead to secondary predation, has been
observed quite commonly (e.g., Vance-Chalcraft et  al.
2007; Gagnon et al. 2011a; Davey et al. 2013). An example
of how secondary predation can cause a predation error is
the study of Sheppard et al. (2005), who readily detected
aphid DNA remains in carabid beetles that had consumed
spiders (the true aphid predator). Carabid beetles, the sec-
ondary predator, had a positive detection of aphids as prey
when in fact a lower trophic level predator had previously
consumed the aphids. So, the actual aphid predator is not
considered while the secondary predator is falsely credited
with providing the biological control service.
Several attempts have been made to identify cannibal-
ism. For example, Lövei (1986) suggested that because
isoenzyme electrophoresis relies on active enzymes which
may alter after death, predation could be separated from
scavenging by using a combination of electrophoretic and
serological tests, i.e., if serological methods detect prey
but electrophoresis does not, then carrion feeding may be
suspected. Sunderland (1996) proposed marking living and
dead prey to study predation relative to scavenging: if liv-
ing and dead prey are marked with different labels, their
relative rates of consumption by predators can be studied
in laboratory and field. Hagler (2019) suggested that uni-
versal food immunomarking technique (UFIT) is an ideal
tool for examining arthropod scavenging and cannibalism
activities. They conducted field cage studies to detect the
frequency of cannibalism and intraguild predation occur-
ring in a cotton predator assemblage. They marked early
instar Chrysoperla carnea larvae with rabbit IgG, and
late instars with chicken IgG. The two larval life stages
(which are known to be cannibalistic) were then intro-
duced into field cages containing other generalist predator
species. The UFIT data revealed a very low frequency of
cannibalism and a relatively high frequency of intraguild
predation (i.e., the other generalist predators fed on the
protein-marked C. carnea larvae), respectively. However,
as C. carnea larvae generally do not ingest the cuticle
of their prey, cannibalism may have been underestimated.
A study on secondary predation by Paula et al. (2022b)
using Lazaro to detect a secondarily consumed extraguild
prey, Myzus persicae (Aphididae, Heteroptera), which was
preyed upon first by Chrysoperla externa and secondarily
by Harmonia axyridis, found that there was no significant
difference in the decay rate of the M. persicae DNA as a
primary or secondary prey of H. axyridis. In addition, the
previous feeding history of the predator C. externa on M.
13
D. P. Paula, D. A. Andow
persicae did not alter its DNA decay rate in the gut of H.
axyridis.
Predation Rates
The previous topics addressed the challenges of accurately
detecting true prey (avoiding FPs and FNs) to determine
which natural enemies are preying on key pests or non-target
species. For robust decision-making for biological control
and risk assessment, it is necessary to go beyond prey range
and determine how much predation a natural enemy may
provide. Quantification of consumed prey is not easy to
measure, as detected prey biomass in a predator gut is a
result of meal size and time since consumption, parameters
that are difficult to disentangle. In addition, it is affected
by a number of aforementioned uncontrolled biotic and
non-biotic factors in the field and laboratory. Estimation
of predation rates is further complicated because they typi-
cally depend on the density of the prey and natural enemy.
When prey are scarce, they are harder to find and predation
rates are lower. When prey are highly abundant, predation
rates can saturate and become independent of prey density
as occurs in a type II and type III functional response (Hol-
ling 1959). When natural enemies become highly abundant,
they can interfere with each other (e.g., through competition
or intraguild predation), reducing the predation rate (Has-
sell and May 1973). These complications have yet to be
addressed with molecular gut content analysis.
Quantification of prey consumption can estimate the
number of prey consumed, the biomass of prey consumed,
or both. The number of prey consumed is a measure of the
impact of the natural enemy on the prey population, while
the biomass consumed measures the impact of consumption
on the natural enemy population. Sunderland (1988, 1996)
argued that prey detection by molecular methods of natural
enemy gut contents measures the amount of prey biomass
consumed rather than the number of prey consumed. How-
ever, he pointed out that prey biomass consumed can be
converted to the number of prey consumed by measuring
biomass per prey and prey-size preference. He also argued
that prey detection measures consumption rather than pre-
dation, because some food items can be partially consumed
without killing them, and conversely, the natural enemy
can kill prey but fail to ingest any prey material. Here, we
use predation rates (biomass or number of prey eaten per
unit of time by the entire natural enemy population) as a
synonym of consumption rates (Dempster 1960; Hagler and
Naranjo 1994). Some authors refer to per capita predation
rates (biomass or number of prey eaten per unit of time for
an individual natural enemy), such as attack rates (Lister
et al. 1987). Others have used relative predator efficiency
(Ragsdale et al. 1981) or predation index (Sunderland et al.
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
1987) as a surrogate for predation rate. There have been
various terms used for the time that prey can be detected in
a natural enemy gut content. Sunderland et al. (1987) clari-
fied this by defining the maximum detectability period as
the time from prey consumption to when it can no longer be
detected. Implicitly, this definition is related to the limit of
detection (LOD) for the detection method, and therefore, the
maximum detectability period can depend on the detection
method used. The terms “digestion rate,” “rate of digestion,”
and “decay rate” have been used to refer to the rate at which
prey decline in the natural enemy gut. We prefer “decay rate”
because the rate of prey decline is related to both digestion
and elimination (egestion and excretion). Greenstone et al.
(2013) pointed out the difference between the decline of prey
contents in a natural enemy gut (decline of analyte concen-
tration = decay rate) and the decline in the detection of prey,
such as by PCR (decline of proportion of natural enemies
testing positive for prey). They proposed to call the latter
a “detectability half-life,” the time when only 50% of the
natural enemies test positive for prey. We will use this term
to contrast with the “decay rate.” In separate work, we show
that the detectability half-life is equivalent to the maximum
detectability period (Andow and Paula, unpublished).
Attempts to quantify prey consumption to estimate pre-
dation rates started back when predator gut contents were
analyzed by serological methods (Dempster 1960; Fichter
and Stephen 1981; Sopp and Sunderland 1989; Greenstone
1996). The serological methods demonstrated that, except
for Fichter and Stephen (1981), the antigen mass in the pred-
ator gut declined exponentially and the decay rate could be
used to estimate biomass of ingested prey (Sopp and Sun-
derland 1989) and the detectability period of the biomass.
Models employing the Poisson distribution (Nakamura and
Nakamura 1977; Lister et al. 1987) were efforts to estimate
predation rates from the frequency of predation. Several of
the early works for estimating predation rates from gut con-
tent analysis in arthropods were based on a combination of
predator density, proportion of predators positive for prey
detection, and detection period of the prey remains in the
predator gut determined experimentally in feeding trials.
In the pioneering work of Dempster (1960), predation
rate was related to the proportion of the predator population
(p) that fed on the prey (in his case, the chrysomelid beetle
Phytodecta olivacea Forster, detected by the precipitin test)
within a certain number of hours. He sampled 11,286 arthro-
pod predators from 19 taxa to estimate predation rates (k)
for each predator species. He first determined the detection
period (which he called rate of digestion) for each preda-
tor (about 1 day) and estimated the likelihood of a preda-
tor preying on multiple prey during 1 day by observing the
mobility of the predators in an insectary and the mean dis-
tance between the prey eggs and larvae. He argued that pred-
ators were highly unlikely to prey on multiple prey during
a day because prey were far apart compared to the mobility
of the predators and estimated the daily rate of predation for
each predator in the community with k = (P × p)/D, where
P is the predator density, and D is detection period. How-
ever, his predation rate equation was criticized because it
was only applicable to predators consuming a single prey
during the prey detectability period, which was considered
unusual, except when the prey is bigger than the predator or
rare. This predation rate equation was considered, in most
cases, to underestimate predation rates.
Rothschild (1966), studying predation rates on Conome-
lus anceps (Germar) (Homoptera: Delphacidae) by 91
predator species by precipitin tests (only 29 species tested
positive), adjusted Dempster’s (1960) equation by intro-
ducing what he called the rate of feeding (kl), i.e., average
number of prey consumed during the detectability period
(D), measured in the laboratory. So k = (P × p × kl)/D (in
Dempster 1960 kl = 1). Later, Kuperstein (1979) estimated
predation rates on Eurygaster integriceps Puton (Hemiptera:
Scutelleridae) by five species of carabids and independently
developed the same equation for predation rate as Rothschild
(1966).
Nakamura and Nakamura (1977) studied predation on the
small chestnut gall wasp Dryocosmus kuriphilus Yasumatsu
(Hymenoptera: Cynipidae) by precipitin test in numerous
taxa of spiders (1063 individuals). They assumed a random
distribution of the number of prey consumed over time and,
therefore, the distribution of the number of prey consumed
would be Poisson. It is well known that the zero term of the
Poisson distribution is equal to the value p × kl in Rothschild
(1966), so k = P × [ln(1-p)]/D, where in their case D was
4 days. Their equation was criticized because it can lead to
overestimated predation rates if the predators feed on a few
large prey or have a long detection period, although neither
criticism applied to their case.
Ragsdale et al. (1981) assessed predation by 11 preda-
tors of the soybean pest Nezara viridula (L.) (Hemiptera:
Pentatomidae) using ELISA. They used only relative preda-
tor densities and the proportion of the predator population
giving a positive detection to generate a surrogate predation
rate: relative predator efficiency = [(P × p)/∑(P × p)] × 100.
They did not include the rate of consumption of prey by the
various predators or the detection period of the prey antigen
in their equation.
In 1983, Hance and Rossignol (cited by Sunderland 1988)
used ELISA to quantify predation by Bembidion quadri-
maculatum (L.) (Coleoptera: Carabidae) on Megoura viciae
(Kaltenbach, 1843) (Hemiptera: Aphididae). They proposed
that the effects of the meal size (M) and time since feeding
(t) could be separated by a regression of a dilution series of
the predator gut content (x) on the absorbance reading (y)
as they thought absorbance reading at the y-axis intercept
was related only to the meal size. However, in a subsequent
13

work, they did not observe the same relationship and could
not separate M and t (Sunderland 1988).
Sunderland et al. (1985), cited in Sunderland 1988) pro-
posed a method to improve estimates of predation rates.
They suggested that any method that quantified biomass in
a predator could be applied to field-collected predators. They
suggested measuring this over a period of days and taking
an average over those days (they called this average x/y).
They then proposed that predation rate could be estimated by
k = (Px)/(yzD), where z is a constant that adjusts the effect of
D on the predation rate. They suggested that D needed to be
adjusted by z because prey biomass declines exponentially
with time in the predator. They further proposed that z = 0.5
may be a reasonable value.
Sunderland et  al. (1987) examined which of several
polyphagous predators (1,275 individuals from the field
distributed in Diptera, Carabidae, Coleoptera, Linyphi-
idae, Staphylinidae, Dermaptera, etc.) were preying on
the cereal aphids Sitobion avenae (F.), Metopolophium
dirhodum (Wlk.), and Rhopalosiphum padi (L.) and which
had higher predation indexes using quantitative data from
ELISA. They combined the percentage of predators positive
for prey detection (p) and predator density (P) with labora-
tory estimated detectability periods. They were the first to
use the terminology Dmax for detectability period, i.e., the
maximum period over which prey antigens could no longer
be detected in the gut of any predator individuals in feeding
trials in the laboratory. Predation indexes were obtained by
the equation: Predation index = (P × p/Dmax). In this work,
they demonstrated that variations in Dmax of a prey among
predators can be significant, so they suggested that Dmax
should always be estimated to compare which predators
were controlling aphid populations as a longer detectability
period could lead to an overestimate of the predation rate
by that natural enemy. Greenstone et al. (2010) agreed that
variation in the detectability period should be incorporated
in estimates of predation rate, but suggested an alterna-
tive to Dmax, which they called the detectability half-life.
They reported large variation in the detectability half-life
of a single egg of the prey Leptinotarsa decemlineata (Say)
(Coleoptera: Chrysomelidae) by conventional PCR. In larval
Coleomegilla maculata (DeGeer) (Coleoptera: Coccinelli-
dae), it was only 7.0 h while in nymphal Perillus bioculatus
(Fabricius) (Hemiptera: Pentatomidae) it was 84.4 h. Pro-
viding a correction of the predation rate with the detectabil-
ity half-life or detectability period can be used to rank the
potential of predator species as biological control agents as
done in several other studies (e.g., Chen et al. 2000; Hosseini
et al. 2008; Gagnon et al. 2011b).
Lister et al. (1987) estimated predation rates using two
methods. They studied the amount of predation on Crypto-
pygus antarcticus Willem (Collembola: Entomobryidae)
and several other prey groups by the predaceous mite
13
D. P. Paula, D. A. Andow
Gamasellus racovitzai (Trouessart) (Mesostigmata: Ologa-
masidae) (about 2000 individuals from 14 localities in Ant-
arctica, quantitative electrophoresis). They quantified the
amount of prey in a predator by measuring the content of
an esterase band (quantified by scanning transmission den-
sitometry using green light 510 nm on an RFT Transidyne
2955 scanning densitometer, in arbitrary units) in an isoen-
zyme electrophoresis profile. They noted that a proportion,
b of the predators did not search for prey, and generalized
the Nakamura and Nakamura (1977) method to take this
into account to estimate predation rates. Here we convert
their formula using the previously defined parameters,
including their b, to estimate biomass of prey eaten for the
predator population: k = P × (1–b) × -ln[(1–p–b)/(1–b)]/D.
More importantly, they were the first to provide a method
to calculate predation rates from quantitative data of prey
biomass in predator gut contents using decay rate instead
of detectability period. They showed that Qj(t), the quan-
tity of esterase stain in band j declined exponentially with
time, t, after prey consumption, and specifically Qj(t) = bj
M exp(-djt), where t is the time after ingestion of a meal of
size M (measured in μg), bj is a constant that converts μg
ingested prey into electrophoresis band units, and dj (> 0)
characterizes the decay rate of the band. The quantity of prey
consumed was calculated as the difference in live weight of
the predator before and after being allowed to feed on the
prey for 12 h. The predation rate, using previously defined
parameters is k = P × (dj × p × Qj*)/(bj × M), where M is the
mean size of meal ingested, Qj* is the average quantity of
prey in predators with positive traces of prey measured in
electrophoresis band units and Qj*/(bj × M) is the proportion
of initial prey biomass remaining in the predator.
Sopp and Sunderland (1989) used quantitative ELISA to
detect the aphid Sitobion avenae (F.), in the guts of 12 preda-
tor species in the Linyphiidae, Carabidae, and Staphylinidae
in feeding trial experiments. The absorbance values of posi-
tive wells were converted to prey biomass (mg) using a cali-
bration curve. The mean consumed prey biomass was then
estimated for each predator species and time interval at each
temperature. To enable direct comparison between predator
species and temperatures in the rate of antigen decay, the
biomass values for subsequent time intervals were divided
by those for immediately after feeding (t = 0) to give the
proportion of the biomass present at t = 0 remaining at each
subsequent time interval. They showed that the size of the
meal has a positive relation with the prey absorbance, the
detection period did not depend very much on the initial
meal size, and that the decay rate did not depend on the
amount of prey initially consumed. Moreover, in 35 of 36
experiments, decay was exponential. They did not estimate
predation rates. These results supported a similar finding
by Fichter and Stephens (1984), also using ELISA, for an
insignificant effect of meal size on the detection period.
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
In 1992, Sopp et al. published another method to esti-
mate predation rates using prey biomass detected by quan-
titative ELISA (and a calibration curve) in the predator
gut. The predation rates of biomass of prey (mg/day) were
calculated from the formula: k = P × Q0 /(f × Dmax), where
Q0 is the average quantity of prey measured in the predator
gut content (including those with no prey detected), f is a
constant related to the way the prey biomass decays over
time and is equal to the average amount of prey left in the
gut of a random predator at the end of the detection period.
They studied predation on the cereal aphid Sitobion ave-
nae (F.) by Agonum dorsale (Pont.) and Bembidion lampros
(Herbst) (Coleoptera: Carabidae), Tachyporus hypnorum
(F.) (Coleoptera: Staphylinidae), and Erigone atra Black-
well (Araneae: Linyphiidae). They compared the predation
rates obtained by their equation with the observed predation
rates in laboratory and insectary experiments, and compared
its performance with the predation rate estimates using the
methods proposed by Dempster (1960), Rothschild (1966),
Kuperstein (1979), and Nakamura and Nakamura (1977).
They found that predation rates based on their method were
close to those measured in the laboratory experiments, while
all of the other methods underestimated predation rates.
Greenstone and Hunt (1993) introduced the concept of
detectability half-life and the importance of determining it,
as opposed to Dmax, to enable more accurate comparison
of predation rates among predators with different digestion
rates. Later, Chen et al. (2000) provided a simple, robust
method for estimating the detectability half-life using probit
regression, and demonstrated that the detectability half-life
can be significantly different for different predator species
feeding on the same prey species. They advocated for the
determination of detectability half-life to adjust estimated
predation rates to enable “fair” comparisons of predation
rates among different predator species preying on the same
prey species. They advocated the use of detectability half-
life as a correction factor (e.g., Greenstone et al. 2010; Hos-
seini et al. 2008; Gagnon et al. 2011b) as opposed to Dmax
(Sunderland et al. 1987) for the cases where prey DNA
detectability decays exponentially during digestion.
Naranjo and Hagler (2001) built on these methods and
attempted to add a functional response to prey density to
enable estimation of predation for varying prey densities.
They studied predation by Geocoris punctipes (Say) (Hemip-
tera: Geocoridae) and Orius insidiosus (Say) (Hemiptera:
Anthocoridae) on eggs of Pectinophora gossypiella (Saun-
ders) (Lepidoptera: Gelichiidae) in greenhouse cages using
qualitative ELISA. They used an empirical functional
response model based O’Neil and Stimac (1988):
( [ ] )
Na = (pP∕D[𝜃])N C1 exp −C2 N + C3
where Na is the predation rate (number of prey attacked by
the predator population density P), p is the proportion of
predators testing positive for the prey, N is the prey density,
D is the detectability period, which may depend on tempera-
ture, θ, and the parameters C1, C2, and C3 describe details
of the searching behavior of a natural enemy (the interested
reader is referred to O’Neil and Stimac 1988). Using an
independent experiment, they compared their functional
response equation to the Dempster (1960) and Nakamura
and Nakamura (1977) predation rate equations. They found
that their equation fit the observed data better than the oth-
ers. This is not surprising as their model uses 4 additional
parameters than the others, which use only 3 parameters
to fit the data. Overall, Naranjo and Hagler’s (2001) result
suggests that the functional response needs to be considered
when estimating predation rates.
Attempts have been made to quantify arthropod pre-
dation rates using quantitative PCR (qPCR) (Zhang et al.
2007; Durbin et al. 2008; Lundgren et al. 2009; Weber and
Lundgreen 2009; Lundgren and Fergen 2011). Zhang et al.
(2007) estimated the number of the red-eyed Bemisia tabaci
(Homoptera: Aleyrodidae) B-biotype preyed upon by several
field-collected predators (coccinellids Propylaea japonica,
Harmonia axyridis, and Scymnus hoffmanni, the chrysop-
ids Chrysopa pallens and C. formosa, the hemipteran Orius
sauteri, and the spiders Erigonnidium graminicolum and
Neoscona doenitzi) with absolute quantification by qPCR.
This involves creating a standard curve to relate Cq values
(quantification cycle: the cycle number at which the fluores-
cence signal intercepts the baseline threshold) to the quantity
of B. tabaci DNA and using the standard curve to convert
sample Cq values to DNA quantity. Following best practices,
the standard curve was amplified in the same qPCR run as
the samples. This is an absolute estimate of the quantity of
B. tabaci DNA in the predator gut, as opposed to the relative
estimates described above.
Lundgren et al. (2009) and Lundgren and Fergen (2011)
used the Cq values obtained in a qPCR to estimate rela-
tive predation indices of consumption of western corn root-
worm Diabrotica virgifera (Coleoptera: Chrysomelidae)
by the natural enemy in the community. The relative prey
consumption index = P × p × (100/Cq), where 100/Cq is an
index of the amount of prey DNA in the natural enemies.
While they were correct to say that there is a negative cor-
relation between the concentration of prey DNA in a sample
and the Cq, the relationship between them is highly non-
linear, so the index is not directly related to predation rates.
Although not with arthropods, Deagle and Tollit (2007)
used qPCR to test if they could estimate the relative quan-
tity of DNA of three prey fish species (50%, 36% and 14%
by mass in the food) in the feces of captive sea lions. They
observed that, although the absolute amount of prey DNA
varied considerably, the percent composition of fish mtDNA
13

roughly corresponded to the mass of fish in the food mixture
(57.5 ± 9.3%, 19.3 ± 6.6%, and 23.2 ± 12.2%, respectively).
They inferred that there was variation in the digestion rate
of the different prey species in the predator gut.
The previous methods to estimate predation rates typi-
cally aimed to quantify predation on only one or a few prey
species. More challenging is the simultaneous quantifica-
tion of the biomass of prey consumed for the diversity of
prey present in a predator gut to estimate predation rates
on multiple prey. Metabarcoding and Lazaro are promising
methods for doing this using relative read abundance (RRA)
as a proxy for species quantity in a predator gut; however,
they are presently potentially limited by some biological and
technical factors that influence RRA, as mentioned previ-
ously section, including taxon-specific variation in DNA
copy number per cell (in the case of comparison of quantity
of different species of prey consumed) and the read quality
filtering threshold (Thomas et al. 2014; Deagle et al. 2013;
Nguyen et al. 2015).
Metabarcoding has been used to estimate species relative
biomass or numerical abundance in two ways: by counting
the frequency of occurrence of each detected species in a set
of samples or by relative read abundances (e.g., Kowalczyk
et al. 2011; Rayé et al. 2011; Thomas et al. 2014; Deagle
et al. 2009; Soininen et al. 2009; Murray et al. 2011; Brown
et al. 2012; Pinto & Raskin 2012; Deagle et al. 2019). The
first is uncontroversial and provides the same information
as used by Dempster (1960) and others mentioned above.
The second is based on an expectation that metabarcod-
ing RRA would correlate positively with prey biomass in
predator guts. Relative differences in RRA has been used
to quantify two closely related plant species in sheep diets
(Willerslev et al. 2014), the taxonomic composition of mam-
malian herbivore diets (Kartzinel et al. 2015) and bacterial
gut communities combined with digital PCR (dPCR) (Bar-
low et al. 2020) at the family level, and the eDNA of fish
populations (Di Muri et al. 2020). Although these might be
special cases, they provide hope that metabarcoding RRA
can be related quantitatively to prey biomass in predator
guts. As metabarcoding relies on PCR and read assembly,
RRA is prone to have quantification biases related to vari-
ation in species-specific primer efficiency, amplicon length
(shorter amplicons may artificially increase species richness
and evenness), primer tag jumps, and barcode primers pref-
erence to amplify certain taxonomic groups (Amend et al.
2010; Engelbrektson et al. 2010; Berry et al. 2011; Ihrmark
et al. 2012; Pinto & Raskin 2012; Deagle et al. 2013, 2014;
Clarke et al. 2014; Elbrecht and Leese 2015; Alberdi et al.
2018), which leads to lack of or misrepresentation of taxa or
over-/underestimation of interaction strength (Yu et al. 2012;
Leray et al. 2013; Deagle et al. 2014; Elbrecht and Leese
2015; Piñol et al. 2015; Bista et al. 2018; Lamb et al. 2018;
Piñol et al. 2018; but see Willerslev et al. 2014; Kartzinel
13
D. P. Paula, D. A. Andow
et al. 2015; Thomas et al. 2016; Krehenwinkel et al. 2017).
Perhaps the most critical bias for quantification is species-
specific variation in primer efficiency. For example, suppose
two prey were equally abundant in the sample, but prey 1
had an amplification efficiency of 2.0, the theoretical max-
imum, and prey 2 had an amplification efficiency of 1.9.
After 25 amplification cycles, prey 1 would have 3.6 times
more read than prey 2. Thus, it would be a mistake to con-
clude that there was more prey 1 than prey 2 in the original
sample. As a second example, suppose prey 1 was 1/10 the
abundance of prey 2, but its amplification efficiency was
2.0 and prey 2 had an acceptable efficiency of 1.8. After 25
cycles, prey 1 would have 1.4 more reads than prey 2, and
it would be a serious mistake to conclude that prey 1 was
more abundant than prey 2 in the original sample. Therefore,
there remains controversy about the use of metabarcoding
RRA for quantitative analysis (Valentini et al. 2009a; Piñol
et al. 2015, 2018; Thomas et al. 2016; Bista et al. 2018;
Deagle et al. 2019; Lamb et al. 2018), i.e., to estimate host
preference, predation rates, or population-level interaction
strengths (Deagle et al. 2019; Lamb et al. 2018).
To control for the many biases mentioned previously,
Thomas et al. (2014) proposed the use of sequencing in par-
allel “control materials” (similar to “spike-in standards”)
with a diet composition matched to the sample diets to
generate correction factors for the RRAs related to species-
specific biases originating from biological differences in
DNA extraction, PCR amplification, and sequencing. They
called these correction factors “tissue correction factors”
(TCFs), which are calculated as the proportion of a prey
in the control material mixture divided by the proportion
of reads of that prey in the amplicon pool of the control
material mixture. They applied these TCFs to a diet of
known composition of three fish species consumed by har-
bor seals determined from scat samples. The use of TCFs
reduced the average difference between the prey propor-
tions in the diet provided to the seals and the proportion
of reads of the respective prey from 28 to 14%. However,
they did not produce the same rank order of prey as in the
scat samples. Later, Thomas et al. (2016), recognizing the
impracticality of matching the control material to the diet
of a field-collected individual, proposed using a series of
relative correction factors (50/50 RCFs) to correct RRAs,
which are calculated from 50/50 mixtures of a control prey
species and a target prey species. However, they found that
RCFs were not constant and were strongly dependent on
the proportional composition of the mixture of control and
target species. In the worst case observed, RCF varied from
0.4 to 2.0 as the proportion of the target species increased
from 20 to 80%. The methods of Thomas et al. (2014, 2016)
have also been criticized as they seem applicable only for
a single environmental sample or replicates from the same
location involving phylogenetically similar prey taxa. Lamb
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…
et al. (2018) conducted a meta-analysis of metabarcoding
RRAs and found a significant, but weak, relation between
the input material for each species present and the propor-
tions of reads and suggested the inclusion of mock commu-
nities to facilitate the assessment of the quantitative metrics.
Murray et al. (2011) compared the performance of qPCR
and HTS (454 GS-Junior) to quantify the relative amount of
fish present in the fecal material of the little penguin Eudyp-
tula minor and found that the proportional composition of
the four most abundant food were correlated. However, they
had only an average of 290 reads per sample, which greatly
limited primer bias, and the use of proportions eliminates
variation related to the relative amounts of food consumed,
i.e., RRA. Together, however, these studies suggest that it
may become possible to use RRA from metabarcoding to
quantify prey biomass in predator gut contents.
Quantification of multiple prey using Lazaro RRAs has
shown promise. Paula et al. (2022b) tested it to quantify the
amount of prey, the aphid Myzus persicae, consumed by the
coccinellid predator Hippodamia convergens and found that
the number of prey reads detected was directly and quantita-
tively related to the number of prey consumed and time since
consumption (r2 = 0.932). Moreover, the number of prey
consumed did not influence the prey DNA decay rate in the
predator gut. The higher the number of prey consumed, the
longer was its Dmax in the predator guts. They demonstrated
how to predict the number or biomass of prey consumed and
how long ago consumption occurred for predator samples
from the field using an inverse regression, when the number
of prey reads and prey DNA decay rates are known. While
it might be expected that digestion would be slower in a full
gut (Weber & Lundgren 2009), their result suggests that the
rate of decay of prey reads was not significantly affected
by the amount of prey consumed. Paula et al. (2022a) also
showed that the number of prey reads was correlated with
the probability of prey detection by melting curve analysis
(MCA) and that the number of reads was proportional to
the relative template concentration measured by qPCR, i.e.,
proportional to the amount of prey DNA in the predator gut.
These are promising results enabling quantitative interpreta-
tion of RRA using Lazaro, however, additional studies are
needed in a variety of ecological contexts before quantitative
interpretation of RRA can be confidently done.
We present in the following the information necessary to
quantify per capita predation rates on a prey. To facilitate this
discussion, we use two parameters that have been measured by
molecular gut content analysis: p, the proportion or frequency
of natural enemies that test positive for a prey, and Q*, the
quantity of the prey detected in a natural enemy given that prey
was detected (i.e., this excludes the individuals without prey).
Consequently, Q = pQ* is the average prey quantity in a natural
enemy, where Q includes individuals without prey). If pQ*
can be assumed to be in a steady state, i.e., staying about the
same over time, then pQ* is the balance between intake of prey
DNA via predation (per capita predation rate, kpc) and the loss
of prey DNA from the natural enemy by decay (which includes
digestion and excretion, d). Andow and Paula (unpublished)
show that per capita predation rate is
kpc = d × p × Q∗ = d × Q
and predation rate (by the natural enemy population, P) is
k = P × d × p × Q∗ = P × d × Q
Thus, estimating the decay rate, d, of Q is essential for esti-
mating a predation rate. A high predation rate or a low decay
rate can generate a high Q, and conversely, a low predation rate
or a high decay rate can generate a low Q.
To evaluate the impact of the natural enemy on the prey
population, it is necessary to estimate the predation rate per
prey, N, and per natural enemy, P. This is because impact is
estimated using population models, the simplest of which
is the Lotka-Volterra predator–prey model (which is related
to the Nicholson-Bailey parasitoid-host model). The Lotka-
Volterra model is
dN∕dt = rN − kpc NP;dP∕dt = ckpc PN − 𝛾P
where kpc is the per capita predation rate of the natural
enemy P, r is the intrinsic rate of increase of the prey popu-
lation N, c is a coefficient converting prey into natural enemy
offspring, and γ is the natural enemy death rate. For discrete
populations, it is
( ) ( )
Nt+1 = Nt exp r − kpc Pt ;Pt+1 = Pt exp ckpc Pt
where k = kpcP is the predation rate as defined in this paper.
Most of the molecular gut content methods estimate
k = kpcP as the impact of the natural enemy on the prey. In the
Lotka-Volterra model, it can be seen that kpcP is a good esti-
mate of the impact of the natural enemy on the prey. However,
this simple Lotka-Volterra model is considered too unrealistic
to estimate natural enemy impact on a prey, because it assumes
that predation rate is a constant proportion of available prey.
Specifically, it assumes that predation is a type I functional
response, which means that the natural enemy never becomes
saturated. Elaborations of the Lotka-Volterra model allowing
a non-linear predator functional response are considered more
realistic for evaluating the impact of the natural enemy on a
prey. Nearly all functional response models take the form
�
Na = kpc Ng(N)
where Na is the number (density) of prey attacked, N is the
number (density) of prey available, kpc is the per capita pre-
′
dation rate by a natural enemy when prey are scarce, and
g(N) is a function describing the non-linear effect of prey
13

number (density) on the predation rate. This formulation of
the functional response allows the Lotka-Volterra model to
be elaborated as follows
dN dP
� �
= rN − kpc PNg(N); = ckpc PNg(N) − 𝛾P
dt dt
and
� ( ) �
Nt+1 = Nt exp(r − kpc Pt g Nt );Pt+1 = Pt exp(ckpc Pt g(Nt ))
Thus, it can be seen that the method used by most molec-
ular gut content analysis (kpcP) estimates the linear part of
the functional response, and a challenge that remains is to
estimate the non-linear part, specifically, kpc g(N) , instead
�
of just kpc. This requires estimation of per capita predation,
kpc, at several prey densities and fitting the non-linear func-
tion to the result. This has only been attempted by Naranjo
and Hagler (2001) as discussed in detail above. In any case,
predation rates determined by molecular gut content analysis
alone are not a sufficient indicator to rank natural enemies
for pest suppression. Other contemporaneous prey mortal-
ity methods should also be used as an additional metrics of
predation, for example, predation on sentinel prey (Lundgren
and Fergen 2011).
Food Webs
Predation rates alone are not a sufficient indicator to rank
natural enemies for pest suppression. This is because natural
enemies interact with each other as competitors and intragu-
ild predators, and interact with other non-pest prey species
and plants as omnivores, and pollen and nectar feeders.
These interactions are needed to better understand the pest
suppression potential of natural enemies and their potential
to adversely affect non-target species. Food webs are consid-
ered to be essential for understanding the dynamics of these
complex interactions. A food web is a network of popula-
tions connected by their trophic interactions. Food webs can
be used to evaluate how predation on a prey may resonate
through the community, which has profound implications
for biological control of pests and risk assessment analysis
of non-target species. To our knowledge, there are only two
publications using DNA-HTS for food web construction in
the context of biological control. Paula et al. (2016) using
Lazaro were able to identify several direct and indirect spe-
cies interactions and trophic levels, including intraguild
predation, in the ladybird beetle community in a brassica
agroecosystem. Lefort et al. (2017) using metabarcoding
of aphid mummies were able to identify the aphid species
and the parasitoids and hyperparasitoids associated with the
mummies and compare the structure of the food webs in the
native and invaded range of the aphid.
13
D. P. Paula, D. A. Andow
There are different kinds of food webs (e.g., community
food webs, energy flow webs, functional food webs, Cohen
1978, Dunne 2006), but the one that DNA-HTS gut con-
tent analyses are eminently suitable for is the construction
of consumer centric food webs (so-called sink food webs),
which begin by identifying the consumed prey. An alter-
native approach is resource centric food webs (so-called
source food webs, Cohen 1978). These focus on a particular
food resource and identify all the consumers feeding on the
resource and the consumers of those consumers (e.g., Mem-
mott et al. 2000; Muller et al. 1999). As gut content analysis
does not reveal all consumers of a resource, source food
webs will not be considered further. Here, we address how
sink food webs can be constructed from molecular gut con-
tent analyses and explore how they can be used to address
questions of indirect interactions and community stability
as well as new questions associated with network analysis.
Food webs comprise populations (nodes) and trophic
linkages, which are lines connecting prey and consumer
populations. Food webs can either be qualitative or quantita-
tive. In a qualitative food web, the trophic relations between
predator and prey are indicated by presence or absence and
they are more common because the data requirements are
much less than for a quantitative web. However, only limited
inferences can be drawn from qualitative webs. For exam-
ple, rare feeding events are given equal weight as common
events, information about relative food preferences is not
included and communities are more likely to be considered
unstable.
In a quantitative food web, the trophic linkages are quan-
tified. There are several ways to quantify (Berlow et al.
2004), but we consider two that are relevant to molecular
gut content analysis: the probability of prey consumption (p)
and the amount of prey consumed per predator (Q). We do
not address the estimation of population dynamic interaction
strengths here, such as per capita change in prey population
growth rate, as these require additional information on the
change in prey population densities over time. These trophic
linkages are per capita effects of an individual predator on
the prey. The probability of prey consumption is estimated
from the proportion or frequency of natural enemies that
test positive for the prey, p, which is a per capita estimate.
The biomass of prey consumed per predator is estimated
from d × p × Q*, as detailed previously. Both the probability
and the amount will likely vary with the prey and predator
densities, as measured by the functional responses. Thus,
while a quantitative food web provides considerably more
information than a qualitative web, it is considerably more
challenging to construct and will be contingent on the exist-
ing community composition.
Once the feeding linkages have been characterized either
qualitatively or quantitatively, they can be assembled into an
adjacency matrix (for qualitative linkages) or a flow matrix
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…

(for quantitative linkages) (SI Glossary). These matrices

can be used to graph the food web and analyze food web
structure, as detailed below. An adjacency matrix comprises
0’s (predation not confirmed, i.e., prey not detected) and
1’s (predation confirmed, i.e., prey detection), while a flow
matrix comprises real numbers or functions ≥ 0. Both matri-
ces are square matrices, meaning that they have the same
number of rows and columns. Each population is arrayed
in the columns and appears in the same order in the rows.
When in a row, the population is the prey, and when in a col-
umn it is the predator. This means that any column i and row
i indicates the consumption of the ith population by itself,
i.e., cannibalism. Table 2 indicates feeding relations between
some hypothetical predators and prey, and Fig. S1 is the
associated adjacency matrix. A graph of this food web can
be constructed using igraph in R (Figs. 1 and S2) showing
the trophic level of each population (TL, SI Glossary) and
the trophic linkages among populations.
Community stability is an important property that can be Fig. 1  Graph of a qualitative food web, plotted using igraph in R.
Lines are linkages and arrowheads indicate the direction of consump-
evaluated using food webs. One significant form of stability tion or the flux of energy. Circles are nodes and represent populations
is that all populations in the community can persist without (orange are basal species that do not consume other populations in
going extinct, i.e., community richness is stable. This would the food web; blue are consumers). Height on the y-dimension is pro-
imply that any non-target effects of a natural enemy would portional to the trophic level. Trophic level for basal populations is
set to 0. The trophic level of consumers is the average of all the prey
not cause the loss of a non-target species. A stronger, more consumed by a population plus 1. For example, Pop7 consumes Pop5
restricted form of stability is that all populations will tend and Pop6, which are both trophic level 0. The average trophic level of
to some long-term steady state. This means that the popula- Pop5 and Pop6 is 0, so the trophic level of Pop7 is 1. Pop 1 consumes
tion densities will tend to go to some value specific to that Pop5 and Pop7, which have a trophic level of 0 and 1. Thus, the
trophic level of Pop1 is 1.5. Pop3 is cannibalistic and therefore has
population. These values would allow estimation of the bio- an arrow that loops to itself. Its trophic level is 1.5 and involves math-
control service of a natural enemy as well as its impact on ematically complex calculations (see trophic level in SI Glossary).
non-target species. Here we examine some properties of food Pop4 is a top predator of the food web because it is not consumed by
webs that appear to be associated with community stability. any other population. Its trophic level is determined from the average
of the trophic levels of Pop1 (1.5), Pop7 (1), and Pop2 (0), and is 1.83
Elton (1927) hypothesized that natural enemies play
a key role in stabilizing animal food webs in both senses
of stability. He suggested that because they switch prey to compartmentalization/modularity and coherence, and
feed on the most common prey, they will tend to keep her- greater generality in natural enemies coupled with greater
bivore populations in check. This idea lent support to the vulnerability of their prey (SI Glossary). In addition, the
broader diversity-stability hypothesis that greater diversity growing body of research on general interaction networks,
resulted in more stable communities. Theoretical studies, of which food webs are an example, has opened questions
however, showed that greater diversity would result in less about the connectedness and clustering of populations and
stable communities (Levins 1970; May 1972), and present- substructuring among a small number of populations. These
day researchers are concerned about how food web prop- will be discussed in turn in the following.
erties, such as diversity, may stabilize food webs. Several Early theoretical investigations suggested that omnivory
properties have been proposed to stabilize food webs, the would destabilize food webs, and therefore, omnivory should
most prominent of which are lack of omnivory, greater be rare (Pimm 1979). Omnivory is the feeding on multiple

Table 2  Hypothetical Prey
qualitative feeding relations
between four predator and five Pop 1 Pop 2 Pop 3 Pop 5 Pop 6 Pop 7
prey populations, including
cannibalism and intraguild Predator Pop 1 0 0 0 1 0 1
predation. 0’s indicate no prey Pop 3 0 1 1 0 1 0
detected and 1’s prey detected Pop 4 1 1 0 0 0 1
Pop 7 0 0 0 1 1 0

13
 D. P. Paula, D. A. Andow

trophic levels and is estimated with an omnivory index (OI),
which is equal to the variance of the trophic levels of a con-
sumer’s food groups. The OI is zero when all feeding occurs
from the same trophic level and increases with the variety of
trophic levels consumed. Contrary to the theoretical predic-
tion, empirical work has shown that omnivory is common in
food webs (e.g., Polis 1991). In studies of arthropod natural
enemies, intraguild predation, which is a form of omnivory,
has been commonly found (Vance-Chalcraft et al. 2007) and
has also been common in natural enemy sink webs based
on molecular gut content analysis (e.g., Paula et al. 2016,
2022a). More recent theoretical research, stimulated by the
contradiction, has found multiple conditions under which
omnivory is stabilizing, especially in complex food webs
(Kratina et al. 2012).
Food web compartmentalization/modularity is suggested
to confer stability. Compartmentalization is the organization
of the populations into groups that interact more commonly
within groups than between groups. A fully compartmental-
ized community has small groups of populations interacting
with each other with no interactions between populations in
different compartments. A community with no compartmen-
talization has random interactions among populations. Pimm
(1979) suggested that an intermediate level of compart-
mentalization may be most stable. This may be because at
intermediate levels, cross-compartment interactions reduce
the variation within compartments (McCann et al. 1998;
Rooney et al. 2006). Recently, formal methods for detect-
ing compartmentalization in food webs have been developed
(Guimarà et al. 2010). These methods have revealed signifi-
cant compartmentalization in empirical food webs (Fig. 2),
indicating that empirical food webs are organized into inter-
mediate levels of compartmentalization, which may enhance
stability (McCann et al. 1998; Rooney et al. 2006).
Food web coherence is the variation in the trophic levels
of the different prey consumed by a natural enemy and is
measured by the standard deviation of these values (q, John-
son et al. 2014). In a perfectly coherent food web, all con-
sumption occurs on prey exactly one trophic level removed
from the predator. In other words, all herbivores feed only
on plants, and all predators feed either only on herbivores
or only on other predators, and there is no omnivory, can-
nibalism, or intraguild predation. In this case, q is 0. All per-
fectly coherent food webs are stable (Johnson et al. 2014).
As maximum coherence occurs for q = 0, q is a measure of
food web incoherence. Although several simple food webs
are nearly coherent (Johnson et al. 2014), some simple sink
webs constructed from molecular gut content analysis have
larger q (Paula et al., unpublished), suggesting that in some
cases these sink food webs may not be stable.

Fig. 2  Adjacency matrix show-

ing compartmentalization in
an empirical food web. Prey
compartments (different colors
among rows) and predator
compartments (different colors
among columns) correspond to
groups of species with similar
predators (for prey) or prey (for
predators). Black squares indi-
cate the existence of a predator–
prey relation and white squares
its absence. The adjacency
matrix was rearranged to reveal
the block structure of the
compartments. Redrawn from
Guimerà et al. (2010)

13
DNA High‑Throughput Sequencing for Arthropod Gut Content Analysis to Evaluate Effectiveness…

Vulnerability and generality are food web properties

that have been examined for some time (Goldwasser and
Roughgarden 1993). Vulnerability is related to the number
of natural enemies for a given prey, and food webs with
high vulnerability mean that prey are susceptible to preda-
tion from many predator populations. Generality is related
to the number of prey populations for a given natural enemy,
which is the ecological prey range and is useful for assessing
the potential adverse effects of a predator. Gross et al. (2008)
showed that higher generality in the top natural enemies cou-
pled with higher vulnerability in the intermediate trophic
levels, such as herbivores and the prey of the top natural
enemies leads to greater food web stability. Molecular sink
webs may provide unbiased estimates of generality but may
underestimate vulnerability, because some of the predators
of the prey may be missing from the samples. In a molecular
sink food web, vulnerability varied with prey population and
time of the season, while generality varied with predator
population and slight variation over time of season (Fig. 3).
These results indicate that most of the prey populations have
high vulnerability to the predator populations and most of
the predator populations have high generality for the prey Fig. 3  A Vulnerability of six populations of extraguild prey to four
predator populations and b generality of four populations of predators
populations, suggesting that the food web may be stable. on six species of prey from May to September (Paula, unpublished
Many general interaction networks have “small world” data). A.cit, Aphis citricidus; A.sol, Aphis solanella; B.bra, Brevi-
topologies. In a small world network, nodes (populations) coryne brassicae; L.pse, Lipaphis pseudobrassicae; M.per, Myzus
are more closely connected to other nodes than expected at persicae; U.amb, Uroleucon ambrosiae; C.san, Cycloneda sanguinea;
E.con, Eriopis connexa; H.axy, Harmonia axyridis; Hi.con, Hippoda-
random, and nodes tend to interact primarily with a small mia convergens 
cluster of nodes. In a small world topology, interactions tend
to be clustered or compartmentalized, but there are enough
connections among clusters that it takes only a few con- exhibit high clustering (Paula et al., unpublished), although
nections for clusters to influence each other. For example, few have been examined to know if these results are general.
Milgram (1967) suggested that every person in the world is A recently identified network property is a network
only “six degrees of separation” from every other human, motif (SI Glossary). A network motif is a substructure
which means, for example, a disease can transmit through- of the network and is a recurring pattern of interconnec-
out the world via transmission to only six people. Watts and tions among a small number of nodes that occurs more
Strogatz (1998) formalized tests to determine if a network commonly than random (Fig. 4, Milo et al. 2002). There
exhibits “small world” topologies. The first is evaluated by are several possible motifs in food webs involving three
the characteristic path length (e.g., six people), which in a populations. The tri-trophic interaction was more common
food web is the average of the smallest number of linkages than random in nearly all empirical food webs, which cor-
connecting a prey to a predator (SI Glossary). The second is responds to the naive view that communities are organized
evaluated by the clustering coefficient (SI Glossary), which into food chains (Stouffer et al. 2007). Food webs divided
in a sink food web is the fraction of intraguild predation into two motif types. The larger group of food webs, which
linkages among the potential intraguild linkages for the includes most of the terrestrial food webs, had an over-
predators feeding on the same prey. A short characteristic representation of omnivory/intraguild predation and an
path length may result in a rapid spread of a perturbation underrepresentation of shared predation and apparent com-
(e.g., species invasion or extirpation) through the food web petition (Fig. 4). The smaller group of food webs had low
(Watts and Strogatz 1998; Williams et al. 2002) but a high omnivory/intraguild predation and high shared predation
clustering coefficient (e.g., close to 1) may be associated and apparent competition. The reason for the difference in
with high connectance (SI Glossary) and greater resilience these two groups of food webs is not known. As intragu-
within clusters to perturbation (Dunne et al. 2002). Molecu- ild predation has been commonly found in molecular sink
lar sink food webs have short critical path lengths but do not food webs (Paula et al. 2016; Andow and Paula, unpub-
lished), elaboration of molecular sink webs will probably
increase the number of the first group of food webs.

13

Fig. 4  Common three-species motifs in empirical food webs, showing
the motif in black and white and all examples of the motif embedded
in a food web with red linkages. A motif must contain all indicated
Future Avenues
Biological control research can benefit tremendously of the
advances in DNA-based methods, such as HTS, not only for
detection of species and their interactions, but also to assess
the strength of such interactions. The capacity of detecting
multiple and previously unknown interactions provided by
metabarcoding and Lazaro, is enabling an unpreceded abil-
ity to construct complex multitrophic food webs at various
spatial and temporal resolutions, which otherwise would be
impractical to obtain from natural systems. These have direct
implications for the understanding of community dynamics
and improvement of predictions of the biological control
potential to regulate pests and not have adverse impacts on
non-targets. However, there remains considerable room for
fundamental research to control for false-positive and -nega-
tive identifications and to produce relative read abundances
that provide accurate quantitative estimates of predation
rates.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13744-0​ 22-0​ 1011-3.
Author Contribution  Conception of the work: D. P. P.
Literature search: D. P. P. and D. A. A.
Data analysis: D. A. A.
Draft version: D. P. P. and D. A. A.
13
D. P. Paula, D. A. Andow
linkages and only those indicated linkages. a Tri-trophic interaction;
b omnivory/intraguild predation; c shared predation; d apparent com-
petition
Critical revision: D. P. P. and D. A. A.
Declarations 
Conflict of Interest  The authors declare no competing interests.
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