Insect Biochemistry and Molecular Biology 145 (2022) 103773

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier.com/locate/ibmb

Quantity versus quality: Effects of diet protein-carbohydrate ratios and

amounts on insect herbivore gene expression
Carrie A. Deans a, b, *, Gregory A. Sword a, Heiko Vogel c, Spencer T. Behmer a
a
Department of Entomology, Texas A&M University, TAMU 2475, College Station, TX, 77843, USA
b
Department of Entomology, University of Minnesota, 219 Hodson Hall, St. Paul, MN, 55108, USA
c
Department of Entomology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, Jena, GER, 07745, USA

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

Keywords: Dietary protein and digestible carbohydrates are two key macronutrients for insect herbivores, but the amounts
Transcriptome and ratios of these two macronutrients in plant vegetative tissues can be highly variable. Typically, insect her­
Geometric framework bivores regulate their protein-carbohydrate intake by feeding selectively on nutritionally complementary plant
Nutrient regulation
tissues, but this may not always be possible. Interestingly, lab experiments consistently demonstrate that per­
Physiology
Helicoverpa zea
formance – especially growth and survival – does not vary greatly when caterpillars and nymphal grasshoppers
Digestive enzymes are reared on diets that differ in their protein-carbohydrate content. This suggests insect herbivores employ post-
ingestive physiological mechanisms to compensate for variation in diet protein-carbohydrate profile. However,
the molecular mechanisms that underlie this compensation are not well understood. Here we explore, for the first
time in an insect herbivore, the transcriptional effects of two dietary factors: protein-to-carbohydrate ratio (p:c)
and total macronutrient (p + c) content. Specifically, we reared Helicoverpa zea caterpillars on three diets that
varied in diet p:c ratio and one diet that varied in total p + c concentration, all within an ecologically-relevant
range. We observed two key findings. Caterpillars reared on diets with elevated total p + c content showed large
differences in gene expression. In contrast, only small differences in gene expression were observed when cat­
erpillars were reared on diets with different p:c ratios (spanning from protein-biased to carbohydrate-biased).
The invariable expression of many metabolic genes across these variable diets suggests that H. zea caterpillars
employ a strategy of constitutive expression to deal with protein-carbohydrate imbalances rather than diet-
specific changes. This is further supported by two findings. First, few genes were uniquely associated with
feeding on a protein- and carbohydrate-biased diet. Second, many differentially-expressed genes were shared
across protein-biased, carbohydrate-biased, and concentrated diet treatments. Our study provides insights into
the post-ingestive physiological mechanisms insect herbivores employ to regulate protein-carbohydrate intake.
Most notably, it suggests that H. zea, and perhaps other generalist species, use similar post-ingestive mechanisms
to deal with protein-carbohydrate imbalances – regardless of the direction of the imbalance.

1. Introduction
Insect herbivores, like all animals, require multiple nutrients to fuel
growth and reproduction (Scriber and Slansky, 1981; Bernays and
Chapman, 1994; Simpson et al., 2004). Protein and digestible carbo­
hydrates are especially important for insect herbivores (Behmer 2009),
but the concentrations and ratios of these two key macronutrients in
plant vegetative tissue are often highly variable. This variation occurs
spatially both within and between individual plants, and temporally as
plants grow and as seasons change. For example, leaf protein content
from a range of different plants varies between 1 and 40% dry mass
(Bernays and Chapman, 1994; Schoonhoven et al., 2005), and generally
the protein content of plant reproductive tissues exceeds that of foliar
tissues (Scriber and Slansky, 1981; Bernays and Chapman, 1994; Deans
et al., 2016a,b, 2018). Plant protein content also tends to decrease as
tissues age throughout the growing season and can be significantly
impacted by fertilization, herbivory, pathogen infection, and other
environmental effects (Scriber and Slansky, 1981; Bernays and
Chapman, 1994; Lenhart et al., 2015; Deans et al., 2016a,b). Likewise,
plant digestible carbohydrate content is highly variable. This occurs in
response to a number of factors, including seasonal changes (e.g., light,
temperature, and precipitation), plant ontogeny, and plant

* Corresponding author. Entomology Department University of Minnesota St. Paul, MN, 55108, USA.
E-mail address: dean0179@umn.edu (C.A. Deans).

https://doi.org/10.1016/j.ibmb.2022.103773
Received 22 November 2021; Received in revised form 8 March 2022; Accepted 1 April 2022
Available online 9 April 2022
0965-1748/© 2022 Elsevier Ltd. All rights reserved.
C.A. Deans et al. Insect Biochemistry and Molecular Biology 145 (2022) 103773

photosynthetic pathways (Bernays and Chapman, 1994; Lenhart et al., Table 1

2015; Deans et al., 2016a,b, 2018). The four diet treatments used for rearing H. zea caterpillars. The first three diets
As a mechanism to manage nutritional heterogeneity, virtually all had a total macronutrient content (protein + digestible carbohydrates) of 42%:
insect herbivores actively regulate their intake of dietary protein and (1) carb-biased (12% protein and 30% carbohydrate; p12:c30), (2) balanced
digestible carbohydrates when provided the opportunity (Behmer (p24:c18), and (3) protein-biased (p30:c12). The balanced diet represents the p:
c ratio H. zea caterpillars self-select they were allowed to choose between two
2009). This self-selected ratio – termed an intake target – is often
nutritionally sub-optimal, but complementary diets. The fourth diet had the
species-specific, and insects reared on diets that closely match their
same p:c ratio as the balanced diet, but 26% more total macronutrients (p39:
intake targets perform best and maximize their fitness (Simpson et al., c29). Each of these treatments corresponds to a particular host plant tissue eaten
2004a,b; Lee et al., 2006; Behmer and Joern 2008; Jensen et al., 2012; by H. zea caterpillars (Deans et al., 2015, 2016a, 2018).
Roeder and Behmer 2014). When insect herbivores cannot reach their
Treatment P:C Ratio Total P + C Reference
intake target, due to various biological constraints (e.g., suboptimal host Content
plants, risks from predation), they can experience reductions in perfor­
carb-biased p12:c30 42% sweet corn kernels
mance and fitness. But this is not always the case. Many insect herbi­
(0.4)
vores, particularly dietary generalists, exhibit impressive nutritional Balanced p24:c18 42% cotton squares (pre-
plasticity and are able to maintain performance when feeding on diets (1.3) flowers), self-selected IT
that diverge considerably from their intake target. For example, when protein- p30:c12 42% cotton leaves
Deans et al. (2015) reared Helicoverpa zea larvae on four different arti­ biased (2.4)
ficial diets that mimicked the range of protein-carbohydrate ratios (p:c) balanced+ p39:c29 68% developing cotton seed,
(1.3) self-selected IT
and total macronutrient concentrations (p + c) found in cotton (a
common resource for H. zea larvae), few differences in developmental
time, pupal mass, and growth rate were observed. A similar result was
seen for pupation success, developmental time, and pupal mass (Deans
et al. (2017)). Although several other studies have documented signifi­
cant effects of diet p:c on insect performance, most of these effects were
only observed on the most extreme and ecologically unrealistic diets.
Performance across diet treatments that correspond to more
field-relevant p:c ratios, like those tested by Deans et al. (2015, 2017),
showed few significant differences and/or effects with limited biological
significance (Lee et al., 2004, 2006; Thompson et al., 2005; Despland
and Noseworthy, 2006; Merkx-Jacques et al., 2008).
There is a wealth of data connecting dietary protein and carbohy­
drates to insect herbivore fitness, but the mechanisms that mediate these
effects are still largely ambiguous. Transcriptomic analyses offer the
means to explore diet-mediated effects on gene expression and the op­
portunity to identify molecular and physiological mechanisms associ­
ated with nutritional plasticity. Additionally, the affordability of next-
generation sequencing and advancements in de novo techniques have
made gene expression studies more feasible, including for non-model
organisms. However, of the handful of studies that have measured
gene expression or gene products across insect diets, there has been little
consistency among the dietary factors tested. Several studies have
focused on gene expression across different host plants or undefined
artificial diets (Chougule et al., 2005; Osborn, 2011; Gog et al., 2014;
Celorio-Mancera et al., 2012; Vogel et al., 2014) without documenting
the macronutrient differences between them. Instead, the focus of these Fig. 1. The four diets used in this study. Each is shown relative to the others,
studies has largely been on the effects of plant secondary compounds positioned in soluble protein (x-asis) and digestible carbohydrate (y-axis)
(Govind et al., 2010; Oppert et al., 2010; Celorio-Mancera et al., 2012; nutrient space. The legend shows how the protein-carbohydrate content of each
Alon et al., 2012; Gog et al., 2014; Vogel et al., 2014) or diet corresponds to plant tissues – corn, developing cotton seed, cotton leaves,
and cotton squares (preflowers) – that are regularly consumed by H. zea (based
protease-inhibitors (Moon et al., 2004; Chougule et al., 2005; Erlandson
on Deans et al., 2016a, 2018). Iso-caloric lines (dotted) are shown for the three
et al., 2010). Our study is the first to explicitly measure transcriptional
diets with 42% total macronutrient content, and for the one diet with 68% total
effects in response to quantified variation in diet protein-carbohydrate macronutrient content.
ratios (p:c) and total amounts (p + c) in an insect herbivore.
In the current study we utilized RNA-seq to measure whole-body
Three of these diets had a total p + c concentration of 42%; this is a
gene expression in H. zea (commonly known as cotton bollworm and/
moderate macronutrient concentration for agricultural crops (Deans
or corn earworm) caterpillars fed on artificial diets that varied in both p:
et al., 2016a,b, 2018). The fourth diet contained a higher total p + c
c ratio and total p + c content. This allowed us to identify genes and
concentration of 68%, which is more indicative of reproductive tissues
pathways that respond to both the relative amounts of protein and
like cotton seed that are also consumed by H. zea larvae (Deans et al.,
carbohydrates (p:c) and the total amount of diet comprised of protein
2016a,b). These four diets also directly correspond to the control
and carbohydrates by dry mass (p + c). In total, we created four
treatments that were used to measure larval performance in Deans et al.
ecologically-relevant artificial diet treatments that match the p:c ratio
(2017), which allows us to make comparisons between diet-specific gene
and p + c content of different plant tissues in two common hosts of H. zea
expression profiles, diet consumption, and larval performance.
– cotton (Gossypium hirsutum) and sweet corn (Zea mays L.) (Deans et al.,
Given that H. zea larvae demonstrate an ability to maintain larval
2016a,b, 2018). Two of the diet treatments also had p:c ratios that
performance across diets that vary in p:c ratio and total protein-
approximated the self-selected protein-carbohydrate intake target re­
carbohydrate concentration (Deans et al., 2015, 2017), most likely as
ported for H. zea (Deans et al., 2015). These four diets, and the plant
a function of post-ingestive nutrient regulation (Behmer 2009), we
tissues they correspond to, are listed in Table 1 and shown in Fig. 1.

2
C.A. Deans et al.
hypothesized that considerable transcriptional alterations would be
required to mitigate the physiological effects of the nutritional differ­
ences between the diets. In general, we expected to see a high level of
differential gene expression across the different protein-carbohydrate
diets. In particular, we expected to see compensation in the expression
of digestive enzymes. For example, genes encoding proteases were ex­
pected to be up-regulated in caterpillars fed protein-poor diets. In
contrast, we expected genes encoding glycosidases to be up-regulated in
caterpillars reared on the carbohydrate-poor food. On the diet contain­
ing high protein-carbohydrate content (68%), we anticipated that genes
involved in energy metabolism would be up-regulated. Collectively our
results provide unique insights into the post-ingestive mechanism insect
herbivores use to regulate their protein-carbohydrate intake.
2. Methods
2.1. Insects
Caterpillar eggs (Helicoverpa zea) were obtained from Benzon
Research (Carlisle, PA). Neonates were hatched in the laboratory and
transferred to individual cells in plastic trays using a fine-tipped paint
brush. Larvae typically go through 5 instars in the lab. Throughout the
experiment larvae were housed in a growth chamber Model I-66VL;
Percival Scientific, Perry, IA, USA) set to 25 ◦ C with a 14:10 L:D cycle.
2.2. Diet treatments
Neonates (n = 10) were assigned to one of four diet treatments. We
used the modified artificial diet of Jing et al. (2013) to create four diets
with specific macronutrient profiles. These diets were selected because
they represent a range of field-relevant p:c ratios and total p + c con­
centrations, in that they mimic the macronutrient profiles of two com­
mon hosts for H. zea – cotton and sweet corn (Deans et al., 2016a,b,
2018). Table 1 shows the name of each diet treatment, its p:c ratio, total
p + c concentration, and the plant tissue it corresponds to, while Fig. 1
shows the location of each treatment in nutrient space. Treatments
consisted of three diets with different p:c ratios but the same total p + c
concentration of 42%, which is indicative of plant vegetative tissues.
This included a diet that approximates the reported intake target for
H. zea and matches the nutrient content of immature cotton flowers, or
squares (balanced) (Deans et al., 2015), a diet that reflects the nutrient
content of mature leaves (protein-biased), and a diet that mimics sweet
corn kernels (carb-biased). The balanced diet was also tested at a higher
total p + c concentration of 68% (balanced+), which is indicative of
plant reproductive tissues such as developing cotton seeds, a preferred
resource for H. zea larvae. Larvae were reared on their respective diets
during their entire larval development until sampled and were fed ad
libitum. Fresh diet was provided at a minimum of every four days.
2.3. Protocol
Twelve hours after molting to the 4th instar, larvae were dipped in
liquid nitrogen and stored individually at − 80 ◦ C. We sampled 4th in­
stars, which allowed ample time for the larvae to feed on the experi­
mental diets, and we waited 12 h after molting to prevent molting-
related gene transcripts from dominating our RNA samples. Develop­
mental rates were comparable across diet treatments and all samples
were collected over two days. Frozen larvae were crushed with a
disposable pestle in a 1.5 mL microcentrifuge tube and mixed with 1 mL
of TRIzol reagent. Due to the exploratory nature of this study, whole-
body samples were used; however, midgut tissue likely accounted for
the majority of larval biomass. Each sample consisted of one individual
larva. RNA extractions were then done on a 50% dilution of this mixture
(0.5 mL of mixture added to 0.5 mL of TRIzol reagent). Extractions were
done using Direct-zol™ RNA MiniPrep columns (Zymo Research) with
10 μL elutions. Extractions of four replicates from each treatment (16
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Insect Biochemistry and Molecular Biology 145 (2022) 103773
samples total) were sent to the Texas A&M University’s Genomics and
Bioinformatic Service for quality analysis and sequencing. Sixteen
strand-specific libraries were prepared using Illumina TruSeq library
prep and 150bp paired-end reads were sequenced using an Illumina
HiSeq2500. Library fragment size selection was kept small (75bp) to
reduce the exclusion of short-sequence immunity genes.
2.4. Bioinformatics
Tophat (Trapnell et al., 2013) was used to align processed reads to an
early version of the H. zea genome (provided by Tom Walsh, CSIRO).
The complete genome is now available on the NCBI website (accession
#: GCA_002150865.1) (Pearce et al., 2017). The R program FactoMineR
was used to create a PCA plot to show individual variation. A differential
gene expression analysis was done using edgeR within BLAST2GO, using
a GLM likelihood ratio test with an FDR threshold of 0.05 and a log
fold-change threshold of 1 (Götz et al., 2008; Robinson et al., 2010).
Contrasts were made between the intake target diet and the other three
diets, resulting in three different comparisons: (1) balanced vs bal­
anced+, (2) balanced vs protein-biased, and (3) balanced vs carb-biased.
The balanced vs balanced + contrast identified genes related to changes
in total p + c content; the balanced vs protein-biased and balanced vs
carb-biased contrasts identified genes related to changes in dietary p:c
ratio. Differentially-expressed (DE) gene sequences were
BLAST-searched (blastp-fast) against the non-redundant (nr) ncbi data­
base and were cross-referenced against the InterPro database using the
public EMBL-EMI InterPro web-service (May 2016). The BLAST results
were then mapped using data from the Gene Ontology Association and
UniProt database. All these analyses were performed using BLAST2GO
(Götz et al., 2008).
For each diet contrast, up- and down-regulated DE genes were an­
notated with gene ontology (GO) terms using BLAST2GO. Gene ontology
was compared across contrasts by documenting the terms that were
uniquely up- or down-regulated within each contrast (only present in the
up- or down-regulated DE gene list), as well as terms that had a signif­
icantly higher number of genes up- or down-regulated, as determined by
a Chi-square test, for each contrast. Differentially-expressed genes sets
were also manually annotated based on gene name and GO terms to
identify genes related to specific digestive functions: (1) protein diges­
tion (trypsin, chymotrypsin, aminopeptidase, and carboxypeptidase),
(2) carbohydrate digestion (amylase, alpha-N- acetylgalactosaminidase,
trehalase, lactase, and maltase), (3) lipid digestion (lipase), and (4)
energy metabolism (respiration).
A Gene Set Enrichment Analysis (GSEA) was also performed using
GSEA (GSEA 3.0, Broad Institute) to determine whether the above cat­
egories of digestive function genes were correlated with specific diet
treatments. Gene sets were manually curated by searching for genes
within the H. zea genome that contained the relevant names and/or GO
terminology. In GSEA, the expression of each gene in a gene set is
compared across contrasts to determine if the gene set in question is
significantly correlated with a specific treatment. Gene sets are
comprised of genes with specific putative gene functions, whether they
have been shown to be DE or not. In this way, GSEA looks at cumulative
expression across genes within specific gene categories to determine if
that function is associated with a particular treatment.
Finally, to determine the connection between the diet treatments and
the expression of genes related to specific metabolic pathways, we uti­
lized consumption data from Deans et al. (2017), shown in SI Table 1, to
calculate the mass-specific protein and carbohydrate consumption
across diets. We then used this consumption data to adjust the total
expression of protease (trypsin, chymotrypsin, aminopeptidase, and
carboxypeptidase) and glycosidase (amylase, alpha-N- acetylgalactosa­
minidase, trehalase, lactase, and maltase) genes to account for the
amount of protein and carbohydrates consumed in each diet treatment.
Specifically, we estimated the total expression (in
transcripts-per-million, or TPM) of protease and glycosidase genes per
C.A. Deans et al.
mean mg of protein or carbohydrate consumed on a mass-specific basis
(mg p or c/mg insect/day). This allowed us to account for protein and
carbohydrate intake when assessing differences in the transcriptional
response of specific digestive enzymes. Because the expression of pro­
tease and glycosidase genes were measured simultaneously in the same
samples, we used a MANOVA (SPSS Statistics 24 Inc., Chicago, IL, USA)
to detect significant diet effects on the multivariate expression across
diets for total and consumption-specific protease and glycosidase
expression. Univariate analyses were subsequently used to determine
the contribution of protease and glycosidase genes to any differences in
the multivariate response. Discriminant analyses were used post-hoc to
determine specific differences between diets.
3. Results
3.1. Summary statistics
Across all samples we obtained 320,277,088 total reads, with an
average of 20,017,318 reads per sample. About 90% of our reads suc­
cessfully mapped to our reference genome, and over 80% of our read
pairs showed concordant alignments between pairs. We were able to
obtain BLAST matches for almost all of the differentially-expressed (DE)
sequences (98%) and obtained gene ontology information for about 70%
(SI Fig. 1). Fig. 2 shows the individual variation between replicates
within each diet treatment. Clustering was apparent, with the protein-
biased treatment exhibiting the tightest clustering among replicates
and the balanced and balanced + having a greater spread.
3.2. Diet effects
To determine the transcriptional effects of diet p:c ratio and total p +
c concentration, contrasts were made relative to the balanced diet (also
the IT for H. zea). First, we compared the balanced + diet (68% total p +
c) to the balanced diet (42% total p + c). This allowed us to assess gene
expression as a function of variation in total p + c concentration. Next,
we compared the protein-biased and the carb-biased to the balanced
diet. This allowed us to assess gene expression as a function of variation
in dietary p:c ratio. The major takeaway from these comparisons was
that feeding on a diet with a high total p + c content had a greater impact
on overall transcription compared to feeding on diets that had imbal­
anced p:c ratios.
3.2.1. Balanced vs Balanced+
Increasing the protein-carbohydrate content of the balanced diet
from 42% to 68% resulted in the differential expression of 1,870 genes;
roughly half were up-regulated (Table 2). The up-regulated DE genes
corresponded to 248 GO terms, while the down-regulated genes
Fig. 2. PCA plot of gene expression for all individual replicates. This figure
shows the extent to which the replicates of the four diets cluster together, while
also showing how the different diets are separated from one another. The four
diets are: (1) carb-biased (p12:c30; green), (2) balanced (p24:c18; orange), (3)
protein-biased (p30:c12; blue), and (4) balanced + (p39:c29; red).
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Insect Biochemistry and Molecular Biology 145 (2022) 103773
corresponded to 198 GO terms. Although many of these terms were
found among both up- and down-regulated DE genes, those related to
binding, biological regulation, response to stimulus, and signaling were
more strongly associated with the up-regulated genes (Fig. 3). Catalytic
activity, structural molecule activity, and general cellular and metabolic
processes were significantly associated with the down-regulated genes
(Fig. 4).
With respect to digestive enzymes, the balanced + diet had a higher
number of regulated genes related to protein, lipid, and energy meta­
bolism compared to the other diets. The number of up- and down-
regulated genes for protein and lipid metabolism were comparable,
although there were far more up-regulated genes related to energy
metabolism (Fig. 5). The GSEA results show that the overall expression
of protease and energy metabolism genes, but not lipases, were signifi­
cantly correlated with feeding on the balanced + diet (Table 3).
3.2.2. Balanced vs protein-biased
Feeding on the protein-biased diet impacted the regulation of 107
genes, with about 80% of DE genes being down-regulated and 20%
being up-regulated (Table 2). The up-regulated and down-regulated DE
genes corresponded to 55 and 87 GO terms respectively. For the protein-
biased diet, biological process of localization was the only term signif­
icantly up-regulated among DE genes (Fig. 3). In contrast, genes related
to the extracellular region and cellular processes were the only terms
significantly associated with down-regulated DE genes (Fig. 4).
Only 11 genes related to digestive enzymes were regulated in the
protein-biased treatment and most were down-regulated. Fig. 5 shows
that in comparison to the other diet treatments, protein and lipid
metabolism were most impacted in the protein-biased treatment.
Despite finding a limited number of DE genes encoding digestive en­
zymes, the GSEA showed that expression levels across proteases, gly­
cosidases, and energy metabolism genes were significantly correlated
with the protein-biased treatment (Table 3).
3.2.3. Balanced vs carb-biased
Feeding on the carbohydrate-biased diet resulted in the differential
regulation of 281 genes (Table 2). The majority of these genes, about
85%, were down-regulated while 15% were up-regulated. The up-
regulated DE genes corresponded to 39 GO terms, but only genes
related to membrane components and localization were significantly up-
regulated (Fig. 3). Several other cellular components, including the cell,
extracellular region, and organelle, were uniquely down-regulated,
along with structural molecule activity and molecular function regula­
tors. In addition, the biological process terms signaling, response to
stimulus, and biological regulation were significantly down-regulated
(Fig. 4).
Twenty-one genes related to digestive enzymes were regulated in the
carb-biased treatment. Most of these genes were related to protein
metabolism and were down-regulated (Fig. 5). The GSEA results showed
that only aminopeptidase and energy metabolism expression was
significantly correlated with feeding on a carb-biased diet.
3.3. Contrast similarities
In order to further understand how deviations away from a balanced
diet impact global gene expression, it is useful to explore the overlap
between the differentially-expressed genes in each contrast. The DE
genes that are unique to each contrast show the specific response to how
that diet diverges from the balanced diet, while those genes that are
shared across contrasts indicate a generalized response that is broadly
associated with feeding on an unbalanced diet.
Interestingly, Table 2 shows that very few DE genes associated with
the protein- and carb-biased treatments were unique, at only 7% and
11% respectively. In contrast, 86% of the DE genes in the balanced +
treatment were unique. The carb-biased treatment shared a greater
number of DE genes with the balanced + treatment than the protein-
C.A. Deans et al. Insect Biochemistry and Molecular Biology 145 (2022) 103773

Table 2
The number of differentially-expressed (DE) up- and down-regulated genes across the three different diet contrasts. The first three rows compare up-regulated genes,
while the last three rows compare down-regulated genes. The columns show the number of genes unique to each contrast, as well as the number shared across
contrasts.
Number of DE genes

Regulation Contrasts Total Unique Shared (protein-biased) Shared (carb-biased) Shared (all)

Up-regulated balanced vs balanced+ 962 916 6 28 12

balanced vs protein-biased 22 4 – 0
balanced vs 43 3 0 –
carb-biased
Down-regulated balanced vs balanced+ 908 702 2 129 75
balanced vs protein-biased 85 3 – 5
balanced vs 238 29 5 –
carb-biased

Fig. 3. Gene ontology for up-regulated genes. Pie charts show the proportion of DE genes associated with each significantly up-regulated GO term. They are
organized into three columns (cellular components, molecular function, and biological processes) and three rows (diet contrasts). GO terms marked with dots identify
those that have a significantly higher number of up-regulated genes than down-regulated genes as indicated by a Chi-square test (P ≤ 0.05). Pie slices with diagonal
lines show terms that were uniquely up-regulated (no down-regulated gene were listed).

biased diet (157 compared to 8). The protein- and carb-biased treat­
ments only shared a total of 5 genes; however, a substantial number of
DE genes, 81% of protein-biased and 31% of carb-biased, were shared
across all three treatments (Table 2).
In terms of the function of these shared genes, SI Tables 2 and 3 list
manually-assigned putative functional terms for these shared DE genes,
based gene name, and gene ontology. The up-regulated genes shared
between the carb-biased and balanced + contrast relate mostly to energy
metabolism, sugar transport, and various forms of binding (SI Table 2).
The down-regulated genes related most strongly to energy, chitin, and
lipid metabolism, protease genes, and protein binding (SI Table 3). The
up-regulated genes shared across all three contrasts include those
related to amino acid and lipid metabolism, transport, and calcium
binding (SI Table 2). The down-regulated genes across all three contrasts
represented a diverse array of gene functions. However, those related to
lipid and chitin metabolism, uncharacterized membrane proteins, pro­
teases, and cuticle proteins were the most numerous (SI Table 3).
3.4. Consumption-specific transcriptional effects
To account for the effect of differences in protein and carbohydrate
consumption on the transcription of digestive enzymes across treat­
ments, we used consumption data from Deans et al. (2017) to calculate
the expression of protease and glycosidase genes adjusted for

5
C.A. Deans et al.
Fig. 4. Gene ontology for down-regulated genes. Pie charts show the proportion of DE genes associated with each significantly down-regulated GO term. They are
organized into three columns (cellular components, molecular function, and biological processes) and three rows (diet contrasts). GO terms marked with dots identify
those that have a significantly higher number of down-regulated genes than up-regulated genes as indicated by a Chi-square test (P ≤ 0.05). Pie slices with diagonal
lines show terms that were uniquely down-regulated (no up-regulated gene were listed).
Fig. 5. Differential-expression of genes encoding digestive enzymes. Bar graphs
show, for each diet contrast, the number of up- and down-regulated DE genes of
digestive enzymes associated with protein, carbohydrate, lipid, and energy
metabolism (respiration). Genes were manually annotated based on gene name
and GO terminology.
mass-specific protein and carbohydrate consumption (grams consumed
per gram of insect per day). Deans et al. (2017) reported that diet
treatment had no significant effect on total consumption or mass-specific
consumption for larvae on the same diets tested in this study. However,
mass-specific protein and carbohydrate consumption of these same diets
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Insect Biochemistry and Molecular Biology 145 (2022) 103773
were statistically different due to differences in diet p:c ratio (SI
Table 1). Mass-specific protein consumption differed significantly be­
tween each diet, scaling with the proportion of protein in the diet.
Mass-specific carbohydrate consumption also scaled significantly with
dietary carbohydrate content (SI Table 1). The diet matching our
protein-biased treatment showed the lowest mass-specific carbohydrate
consumption, followed by the balanced diet. The carbohydrate-biased
diet and balanced + diets showed statistically similar carbohydrate
consumption, likely because they had a similar percentage of dietary
carbohydrates (~30%).
With respect to transcription in the current study, Table 4 shows that
diet treatment had a significant effect on the multivariate gene expres­
sion of proteases (trypsin, chymotrypsin, aminopeptidase, and
carboxypeptidase) and glycosidases (amylase, alpha-N-
acetylgalactosaminidase, trehalase, lactase, and maltase). The univari­
ate results show that the differences in total expression were largely
driven by differences in glycosidase genes, as protease levels were
higher and less variable across diets (Fig. 6a). Fig. 6a shows that the
carb-biased, balanced, and balanced + treatments had statistically
similar total expression levels, but the protein-biased treatment had
significantly higher protease and glycosidase expression. When protein
and carbohydrate consumption were accounted for, this pattern
changed. Consumption-specific expression in the balanced and
balanced + diets remained statistically similar, while the protein-biased
treatment had higher multivariate expression and the carb-biased
treatment had the highest expression (Fig. 6b). The univariate results
show that these differences were due to significant changes in both
protease and glycosidase expression (Table 4). Accounting for
C.A. Deans et al. Insect Biochemistry and Molecular Biology 145 (2022) 103773

Table 3
Gene set enrichment analysis (GSEA) results for genes related to digestive function. The FDR values, with the absolute value of the normalized enrichment
scores (NES) in parentheses, for each gene set. Bold values indicate gene sets that were significantly enriched (FDR ≤ 0.05).
Gene Set balanced vs balanced+ balanced vs protein-biased balanced vs carb-biased

balanced balanced+ balanced protein-biased balanced carb-biased

Protein
all proteases <0.001 (2.0) 0.242 (1.1)
trypsin 0.001(1.9) 0.001 (1.7) 0.090 (1.3)
chymotrypsin 0.002 (1.8) <0.001 (2.1) 0.223 (1.2)
aminopeptidase 0.999 (0.52) 0.014 (1.5) 0.016 (1.7)
carboxypeptidase 0.015 (1.5) 0.031 (1.4) 0.205 (1.2)
Carbohydrates
glycosidases 0.610 (1.0) 0.002 (1.8) 0.130 (1.2)
Lipids
lipases 0.684 (0.90) 0.124 (1.2) 0.176 (1.3)
Energy <0.001 (2.3) 0.001 (1.7) <0.001 (2.0)

Table 4
MANOVA results and univariate statistics for diet treatment effects on the
expression of protease and glycosidase genes. The statistics for the diet effects on
total gene expression (TPM) and consumption-specific gene expression (TPM/
mass-specific consumption (mg/mg/day)) are reported. Bold values indicate
statistical significance (P ≤ 0.05).
Effect Pillai’s df F P-value
trace statistic
Total Expression (MANOVA) 0.796 6,24 2.646
protease expression (univariate) 3,12 2.873
glycosidase expression (univariate) 3,12 5.575
Consumption-Specific Expression 1.609 6,24 16.436
(MANOVA)
protease expression (univariate) 3,12 22.414
glycosidase expression (univariate) 3,12 12.315
consumption did not impact the relative expression of protease and
glycosidase genes in the balanced treatment, but did affect the other
treatments in different ways. While the total expression of protease
genes was higher than glycosidase genes for all diets, expression of both
enzymes were equal in the balanced + treatment when adjusted for
consumption. Consumption-specific glycosidase expression exceeded
that of protease genes in the protein-biased treatment and consumption-
specific expression of protease genes increased while glycosidase genes
decreased in the carb-biased treatment (Fig. 6b). This resulted in larvae
exhibiting greater consumption-specific expression for glycosidases in
the protein-biased treatment and for proteases in the carb-biased
treatment.
We also looked at the total and consumption-specific expression of
different types of proteases and glycosidases across diets. Chymotrypsin
was the only protease to show significant differences in total expression
across diets (SI Fig. 2a), but when adjustments were made for protein
consumption, all proteases varied significantly across diets (Table 5).
Despite significant diet effects on consumption-specific expression, SI
Fig. 2b shows that the expressional patterns for each diet were quite
similar across gene families, with the carb-biased treatment having
consistently high expression across all proteases, followed by the
balanced and protein-biased treatments, which were statistically
similar, and the balanced + treatment, which had the lowest expression
across proteases (SI Fig. 2b).
The total expression of α-amylase, α-N-acetylgalactosaminidase,
lactase, and maltase were significantly different across diets (SI Fig. 3a),
but after accounting for carbohydrate consumption all gene families
except α-glucosidase showed significant differences across diets
(Table 5). As with proteases, the pattern of glycosidase expression was
consistent for each treatment across gene families, with the protein-
biased treatment showing the highest expression across genes and the
other three diets being comparable (SI Fig. 3b).
4. Discussion
This study is the first to explore how ecologically-relevant variation
in dietary p:c ratio and total p + c content impact gene expression in an
insect herbivore. Comparing the expression profiles of larvae fed a
protein- or a carb-biased diet to those fed on a balanced diet (that ap­
proximates the insects’ self-selected intake target) allowed us to
examine the effect of p:c ratio on transcription. Additionally, comparing
gene expression in larvae fed a balanced diet to those fed on a more
0.041
0.080 concentrated balanced + diet with the same p:c ratio, allowed us to
0.012 examine the impact of total macronutrient content on transcription.
<0.0001 Deans et al. (2017) observed few differences in H. zea larval perfor­
mance across the same diets tested in the current study, thus we ex­
pected that the homeostatic regulation required to maintain
<0.0001
0.001
performance across diets would require significant changes at the
transcriptional level. We hypothesized that the expression of metabolic
genes would be impacted the most, particularly those related to diges­
tive enzymes and transport, as they may be used to offset macronutrient
imbalance. While some of our hypotheses were confirmed, we observed
a rather limited transcriptional response to large changes in dietary p:c
ratio. We also found that the expressional signatures of the treatment
diets, which diverged from the balanced diet in different ways (pro­
tein-biased, carb-biased, concentrated macronutrients), were not as
unique as expected, with many differentially-expressed genes being
shared across treatment contrasts.
Diet total p + c content had a strong impact on gene expression,
resulting in the regulation of between 6.6 and 17.5 times more genes
than the p:c ratio contrasts. These DE genes corresponded to a diversity
of different GO terms, including many higher order processes. The
strong transcriptional response to an increased total p + c content of
68% was somewhat unexpected, given that a total macronutrient con­
tent of 42% is already at the higher end of the range found in plant
tissues (Deans et al., 2016a,b, 2018; Lenhart et al., 2015) and not likely
limiting, particularly considering that artificial diets are more easily
digested than plant tissues (Lee et al., 2004; Clissold, 2007). Such a
strong transcriptional response suggests that many physiological pro­
cesses are routinely constrained by total p + c content in nature and that
larvae have evolved mechanisms to take advantage of high calorie foods
when they are available. This is further supported by the fact that Deans
et al. (2017) did not observe a reduction in consumption for larvae fed
on the p39:c29 diet (comparable to our balanced + diet), despite it being
more concentrated.
In contrast to the effect of total macronutrients, the numbers of DE
genes observed across p:c ratio contrasts were relatively small. Relative
to the balanced diet, only 281 genes were differentially-regulated in the
carb-biased treatment and even fewer, 107, in the protein-biased
treatment (though this diet was more similar to the balanced diet than
the carb-biased diet). On the other hand, the impact of total p + c
content on gene regulation was substantial, as 1870 genes were

7
C.A. Deans et al. Insect Biochemistry and Molecular Biology 145 (2022) 103773

Fig. 6. Total and consumption-adjusted expression of

protease and glycosidase genes across diets. Panel (a)
shows the mean (± SEM) total number of protease
and glycosidase expression in transcripts per million
(TPM) in larvae from each diet treatment. Panel (b)
shows the mean (± SEM) protease and glycosidase
expression adjusted for protein and carbohydrate
consumption. Different letters indicate significant
differences in the multivariate expression of proteases
and glycosidases genes, as determined by a MANOVA
and subsequent discriminant analyses (P ≤ 0.05).

Table 5
ANOVA statistics for diet effects on protease and glycosidase gene expression. The top half of the table shows results for the effect of diet on the total expression and
consumption-specific expression of specific protease classes (trypsin, chymotrypsin, aminopeptidase, and carboxypeptidase). The bottom half of the table shows results
for the effect of diet on specific glycosidase classes (amylase, alpha-N-acetylgalactosaminidase, trehalase, lactase, and maltase genes). Bold values indicate statistical
significance (P ≤ 0.05).
Genes Total expression Consumption-specific expression

df F statistic P-value df F statistic P-value

Proteases
trypsin 3,12 2.42 0.117 3,12 9.41 0.002
chymotrypsin 3,12 5.61 0.012 3,12 22.56 <0.0001
aminopeptidase 3,12 1.70 0.219 3,12 4.96 0.018
carboxypeptidase 3,12 1.86 0.190 3,12 19.60 <0.0001
Glycosidases
α-amylase 3,12 8.83 0.002 3,12 15.57 <0.0001
α-trehalase 3,12 2.15 0.147 3,12 9.19 0.002
α-glucosidase 3,12 0.35 0.789 3,12 3.03 0.071
α-N-acetylgalactosaminidase 3,12 3.67 0.044 3,12 7.32 0.005
lactase 3,12 8.53 0.003 3,12 9.27 0.002
maltase 3,12 5.79 0.011 3,12 7.88 0.004

8
C.A. Deans et al.
regulated in the balanced + treatment. Of these DE genes, a much larger
proportion were down-versus up-regulated in the p:c ratio contrasts,
while a comparable proportion were both up- and down-regulated in the
balanced + treatment. This pattern suggests that feeding on an imbal­
anced diet, regardless of whether it is protein- or carb-biased, has a
suppressive effect on gene expression. This strategy may function to
limit resource allocation to physiological functions, including general
transcription, when dietary resources do not match physiological re­
quirements. When considering the proportion of genes that were
uniquely regulated in each p:c contrast, the numbers of DE genes were
further reduced. Only 32 genes, or 11% of the total DE genes, were
uniquely expressed in the carb-biased treatment and only 7, or 6.5% of
DE genes, were unique the protein-biased treatment. In comparison, 85
DE genes were shared by all three diet contrasts, representing 30% and
75% of the total DE genes in the protein- and carb-biased contrasts,
respectively. This result supports the notion that H. zea larvae primarily
respond to general rather than nutrient-specific deviations away from
their balanced diet and that similar mechanisms may be utilized to deal
with nutritional imbalances, regardless of where the imbalance occurs.
We initially expected to see a high degree of transcriptional activity
across metabolic genes in larvae fed on imbalanced diets due to the
regulation of physiological trade-offs related to macronutrient limita­
tions and excesses, but instead, we observed a strategy that relies on
constitutive expression (i.e., lack of plasticity). The consumption data
from Deans et al. (2017) showed that when confronted with imbalanced
diets H. zea do not change their eating patterns. Larvae consume similar
amounts of balanced and imbalanced foods, which leads to differences
in protein and carbohydrate intake across diets. Our transcriptional data
show that the total expression levels of protease and glycosidase genes
do not vary substantially in larvae fed different diets, indicating patterns
of constitutive expression when faced with nutritional variation. A
similar result was documented for trypsin activity in H. zea by Broadway
and Duffey (1986). After accounting for differences in protein and car­
bohydrate consumption, however, our consumption-specific expression
data clearly show that a strategy of fixed consumption coupled with
constitutive enzyme expression automatically leads to compensation for
the most limiting macronutrient. This is exemplified by the fact that (1)
the carb-biased diet showed the highest consumption-specific protease
expression and the lowest glycosidase expression, (2) the protein-biased
treatment had higher consumption-specific glycosidase expression than
protease expression, and (3) the balanced and balanced + treatments
had significantly lower and similar consumption-specific expression of
both enzymes. These patterns demonstrate an effort to increase the
digestion and uptake of limiting nutrients while feeding on a balanced
diet requires few expressional changes.
It is unclear whether a strategy of fixed consumption and constitutive
expression of digestive enzymes is common among other insect herbi­
vores. Clissold (2007) found that locusts fed protein-biased diets showed
significant decreases in α-chymotrypsin activity compared to those fed
on a 1:1 or carb-biased diet. Similarly, α-amylase-like enzyme activity
was also significantly reduced in locusts fed high-carbohydrate on diets.
This compensatory response makes sense for locusts, as they display
much stricter pre-ingestive nutrient regulation. They are also grass
specialists and are more mobile than H. zea caterpillars. Despite seeing
no differences in the total expression of digestive enzyme encoding
genes in this study, we did observe a similar pattern of compensation at
the transcriptional level after accounting for nutrient intake. Of course,
it is possible that post-transcriptional and post-translational regulation
may produce similar patterns in enzyme activity in H. zea.
There is a wealth of data available on the relationship between host
plant use and digestive enzyme activity in generalist lepidopterans
(Patankar et al., 2001, Kotkar et al., 2009; Chougule et al., 2005), as well
as gene expression across host plant consumption (Celorio-Mancera
et al., 2012; Roy et al., 2016). However, most of these studies focus on
the effects of plant defensive compounds, including
proteinase-inhibitors (Brioschi et al., 2007; Chikate et al., 2013; Kuwar
9
Insect Biochemistry and Molecular Biology 145 (2022) 103773
et al., 2015) and allelochemicals (Li et al., 2002; Pauchet et al., 2010;
Celorio-Mancera et al., 2012; Vogel et al., 2014) rather than nutrients. In
fact, because differences in defensive compounds and nutrients between
host plants are rarely quantified and reported, it is often impossible to
tease out the nutritional contribution to transcriptional changes. There
is no doubt that plant allelochemicals play a significant, if not primary,
role in host plant adaptation, as few, if any, insect herbivores feed on
resources devoid of allelochemicals. Because metabolic responses to
nutrients have evolved in this context, it is possible that
nutrient-mediated changes in transcription may be dependent on the
presence of these other compounds and that the observed expression in
their absence does not reflect field-relevant responses to nutritional
variability. Research suggests that the expression of detoxification en­
zymes, such as cytochrome P450s, esterases, glutathione S-transferases,
UDP-glucuronosyltransferases, ATP-binding cassette transporters, etc.,
are responsive to differences in host plants (Li et al., 2002; Pauchet et al.,
2010; Celorio-Mancera et al., 2012; Koenig et al., 2015; Vogel et al.,
2014), but interactions between nutritional state and responses to alle­
lochemicals are still not well understood (Deans et al., 2016a,b, 2017).
Ultimately, the interplay between meeting nutritional requirements,
minimizing consumption of allelochemicals, and effectively allocating
resources to multiple physiological processes must be more thoroughly
studied. The results presented in this study contribute to a better un­
derstanding of the nutritional perspective of this complex process and
highlight the importance of nutrient regulation, particularly the role
that nutritional optimum (i.e., intake target) play in transcriptional re­
sponses and plasticity (Illius et al., 2002; Simpson et al., 2004a,b;
Simpson and Raubenheimer, 2012).
Funding sources
This work was supported by a USDA Biotechnology and Risk
Assessment Grant [2015-33522-24099] (awarded to GAS and STB), as
well as an AgriLife Genomics Grant from the College of Agriculture and
Life Sciences at Texas A&M University.
Acknowledgements
We would like to thank all those who provided edits and suggestions
on the manuscript, including Dr. Hojun Song, Dr. Tyler Raszick, and Bert
Foquet.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ibmb.2022.103773.
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