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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Evol Dev. 2019 Nov 4;22(3):257–268. doi: 10.1111/ede.12326

Expression profiling of winged and wingless destined pea aphid embryos implicates insulin/insulin growth factor signaling in morph differences

Mary E Grantham 1,*, Alexander W Shingleton 2, Emma Dudley 1, Jennifer A Brisson 1,*
PMCID: PMC7196481  NIHMSID: NIHMS1054822  PMID: 31682317

Abstract

Developmental plasticity allows the matching of adult phenotypes to different environments. Although considerable effort has gone into understanding the evolution and ecology of plasticity, less is known about its developmental genetic basis. We focused on the pea aphid wing polyphenism, in which high- or low-density environments cause viviparous aphid mothers to produce winged or wingless offspring, respectively. Maternally provided ecdysone signals to embryos to be winged or wingless, but it is unknown how embryos respond to that signal. We used transcriptional profiling to investigate the gene expression state of winged-destined and wingless-destined embryos at two developmental stages. We found that embryos differed in a small number of genes, and that gene sets were enriched for the insulin-signaling portion of the FoxO pathway. To look for a global signature of insulin signaling, we examined the size and stage of winged- and wingless-destined embryos but found no differences. These data suggest the hypothesis that FoxO signaling is important for morph development in a tissue-specific manner. We posit that maternally supplied ecdysone affects embryonic FoXO signaling, which ultimately plays a role in alternative morph development. Our study is one of an increasing number that implicate insulin signaling in the generation of alternative environmentally-induced morphologies.

Keywords: phenotypic plasticity, gene expression, polyphenism, pea aphid, RNA-seq

Introduction

Adaptive phenotypic plasticity is an life-history strategy that allows organisms to match their phenotype to their environment (Bradshaw, 1965; West-Eberhard, 2003). With developmental plasticity, the alternative phenotypes are determined during development using environmental cues, but the matched phenotype is not realized until adulthood (Uller, 2008). In many cases, the cue can only be received during a critical window of development (Bateson, 1979). Developmental plasticity has fascinated researchers for decades and as a result the ecological causes and consequences of developmental plasticity are well known (Forsman, 2015; Nijhout, 1999; Scheiner, 1993; West-Eberhard, 2003). However, a major obstacle to the field of developmental plasticity has been the lack of knowledge about the specific molecular mechanisms controlling the switch between the alternative phenotypes.

Studies in a handful of taxa have begun elucidating the molecular mechanisms that underlie developmental plasticity. Many identified regulators have been of two types, neuroendocrine hormones (Rajakumar et al., 2012; Vellichirammal et al., 2017; Wolschin et al., 2011; Xu et al., 2015) and epigenetic factors (Ozawa et al., 2016; Simola et al., 2013; Spannhoff et al., 2011; Vellichirammal et al., 2016), which likely work in concert to achieve alternative morphologies (Grantham et al., 2015; Projecto-Garcia et al., 2017). Even in systems where the initial molecular mechanisms inducing alternative morphologies are known, how these cues ultimately lead to different adult phenotypes are not well understood.

The wing polyphenism (plasticity with discrete morphs) in the pea aphid, Acyrthosiphon pisum, has served as a model system for studying developmental plasticity (Brisson & Stern, 2006). It occurs during the summer months when females reproduce asexually and viviparously. Genetically identical females can be winged or wingless based on cues in the maternal environment (Blackman et al., 1987; Dixon, 1985). The adult female receives the cues and transmits them to the developing offspring in her ovarioles. High population density, predation, and insufficient resources all cause females to produce winged offspring in different aphid species, but the primary sensory cue is likely tactile stimulation in the pea aphid (Müller et al., 2001; Sutherland, 1969). As adults, winged females have wings, wing musculature, and expanded sensory systems, which are all absent in the wingless morph. Wingless females have shorter developmental time to adulthood and higher life-time fecundity compared to winged females (Braendle et al., 2006; Dixon, 1985).

Recent studies have begun to uncover the molecular mechanisms underlying wing morph determination in the pea aphid wing polyphenism. A transcriptomic approach initially identified ecdysone signaling as a candidate mechanism for how the mother transmits the environmental information to her offspring (Vellichirammal et al., 2016). Subsequent hormonal manipulations established more details about its role: increasing ecdysone signaling increases the production of wingless daughters, whereas reducing ecdysone signaling increases the production of winged daughters (Vellichirammal et al., 2017). Thus, while ecdysone is the hypothesized maternal signal in the wing polyphenism system, it is unclear how an embryo receives that signal and how signal reception instructs the developmental program to proceed down a wingless or winged course.

To explore the molecular basis of embryonic development of the winged and wingless phenotypes in pea aphids, we used transcriptomic profiling of two stages in winged and wingless destined embryos. These stages were carefully chosen for their relevance to the polyphenism. The critical embryonic induction period for wing plasticity is a late embryonic stage of development, stage 18 (Ishikawa & Miura, 2013). These embryos are either actively receiving the maternal signal to become winged or wingless, or may have recently received the signal (Ishikawa & Miura, 2013). By stage 20, embryos are determined into one morph or the other and cannot be reversed, thus early morph-specific development is likely to begin at this stage (Ishikawa & Miura, 2013) even though morphological differences are not seen until the second nymphal instar (Ogawa, Ishikawa, Kanbe, Akimoto, & Miura, 2012). We used mRNA-seq to profile winged and wingless-destined embryos at stage 18 and at stage 20. Our goal was to use an unbiased approach to identify aspects of how embryos respond to the maternal signal to become winged or wingless. We further hypothesized that at the earlier stage, embryos would express genes upstream in developmental pathways critical for achievement of either polyphenic morph, and that the older embryos would show corresponding downstream gene expression of the same pathway. Our results suggest the importance of the insulin signaling pathway in the aphid wing polyphenic response.

Methods and materials

Rearing Conditions.

We used a single pea aphid stain, BK10, that was collected from an alfalfa field in Berkley, MA, U.S.A., for all experiments. Asexual pea aphid females were reared at 18°C ±2°C on broadbean plants (Vicia fava) with a 16hr light, 8hr dark cycle at 40% ±5% humidity. Stock populations were maintained for at least three generations at low density (five per plant) before use to remove effects from formerly experienced higher density populations.

Embryo and nymph collection.

We used a combination of crowding and starvation for 24 hours to cue the production of winged offspring. We have shown previously (Grantham, et al., 2016; Vellichirammal et al., 2016) that 16–24 hours of crowding is sufficient to induce winged offspring production. Moreover, a single female will continue to produce winged offspring at the same percentage over multiple days (Grantham et al., 2016). Groups of ten adults from the stock population were crowded in a 35mm Petri dish (lined with moist filter paper to prevent desiccation) for 24 hours, females were moved to individual broadbean plants for 24 hours, and then embryos from relevant stages (see more below) were dissected from each female and placed in TRIzol® Reagent (Invitrogen) at −80°C. The nymphs produced on the plants after crowding were raised to adulthood and their phenotypes were counted. We will refer to this as the crowding treatment. For these crowded females, dissected embryos from females that produced either greater than 75% winged offspring (here called winged-destined embryos) or 0% winged offspring (wingless-destined embryos) during the 0–24 hours post-crowding were retained for use (S1 Fig). This resulted in crowding-treated winged and crowding-treated wingless embryo samples.

To obtain another group of wingless-destined offspring, we raised aphids on plants. Groups of ten females from the stock population were moved to a broadbean plant for 24 hours (same period as the crowding treatment on Petri dishes). Females were then moved individually to broadbean plants for an additional 24 hours, and then embryos were dissected from each female and placed in TRIzol®. The nymphs produced on the plants after crowding were raised to adulthood and their phenotypes were counted. We will refer to this as the plant treatment. For these plant-treated females, all females produced 0% winged offspring in the 24-hours post plant treatment and their dissected embryos were called wingless-destined embryos. The remaining samples were discarded.

RNA extraction.

We collected two stages of embryos for use: stage 18 and stage 20 (Miura et al., 2003). We combined 15 embryos (one each from 15 females) of each stage, from two treatments (crowding or plant treated). This resulted in crowding-treated winged and crowding-treated wingless, as well as plant-treated wingless samples. We had four replicates of each of the three samples, and two stages of each (stage 18 and stage 20), for a total of 24 samples. RNA was extracted via the TRIzol® protocol. The product was cleaned and DNA was digested using the Zymo RNA Clean and Concentrator Kit (Zymo Inc.) with the in-tube DNase treatment per manufacturer’s instructions. Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina). Single-end, 100 base pair libraries were sequenced on an Illumina HiSeq 2500 (University of Rochester Genomics Research Center). Stage 20 embryos (crowding and plant treated) were submitted to NCBI (Bioproject: PRJNA493136). Stage 18 embryos (crowding and plant treated) were submitted as Bioproject PRJNA445883. The crowding-treated stage 18 embryos were also the embryonic samples in Grantham & Brisson, 2018.

RNA-sequencing analysis.

All libraries were quality trimmed with trimmomatic version 0.32 (parameters: SE -phred33 SLIDINGWINDOW:4:20 TRAILING:13 LEADING:13 ILLUMINACLIPTruSeq3-SE.fa:2:30:10 MINLEN:15) (Bolger, Lohse, & Usadel, 2014). Reads were aligned to the pea aphid genome with STAR (version 2.5.2a) using a 99 base pair overhang (Dobin et al., 2013). Integer-based counts per gene were generated from the reverse stranded aligned reads using featureCounts in the Rsubread program version 1.26.1 (Liao et al., 2013) using the pea aphid official gene set version 2.1b (aphidbase.com).

We performed differential gene expression analyses with the packages DEseq2 (Love et al., 2014) and EdgeR (Robinson et al., 2010) in R version 3.4.1 (R Core Team, 2015). The programs were both used because they trade off a high positive detection rate (EdgeR) with a low false positive rate (DESeq2) (Soneson & Delorenzi, 2013). Genes were considered expressed if they had a count >= 0.1 reads per million in >= 4 libraries. A gene was called differentially expressed if the FDR < 0.1 for both EdgeR and DESeq2 analyses. We used this slightly relaxed FDR threshold because of the stringency we added by using both analysis pipelines. To test for main effects (developmental stage, treatment, and phenotype) we used generalized linear models from all 24 libraries. We used contrast comparisons from both EdgeR and DESeq2 to test for phenotypic differences in the stage 18, crowded only, embryo libraries and the stage 20, crowded only, embryo libraries, to mitigate the large effects of age and treatment on gene expression.

KEGG, INTERPRO, and GO enrichment analyses were conducted in DAVID version 6.8 (Huang, Sherman, & Lempicki, 2008, 2009). The background gene set consisted of all genes expressed by the embryonic samples in each respective test.

Principle components were calculated with prcomp in R and ggbiplot to plot (R Core Team, 2015). 95% CI ellipses were drawn using the following options: ellipse=true and ellipse.prob=0.95 in ggbiplot.

Nymphal Birth Weight.

Nymphs born on the first plant during collection (see Embryo and nymph collection) were weighed in groups of 15–30 nymphs on an AB54-Sscale (Mettler Toledo) reading to 0.1 mg and then returned to plants. The weighed aphids were raised to adulthood and phenotyped to find the proportion of winged nymphs per group. We measured the correlation coefficient (R2) and tested if there was significant correlation between the average nymphal mass and the proportion of winged nymphs with a Pearson’s correlation in R.

Tibia length.

After 24 hours of crowding or plant treatment, adult females were placed on a broadbean leaf to begin larvipositing. Immediately as nymphs were born, the right, hind leg was removed and sealed on a microscope slide. The nymphs were individually placed on a broadbean leaf that was inserted into agar in a 100mm Petri dish. The tibia segment of the leg was measured in imageJ and the nymph phenotype was recorded. A two-sample Wilcoxon rank sum test (equivalent to the Mann-Whitney U) was performed to test for tibia length differences between winged and wingless-destined embryos in R.

Sensilla presence analysis.

To identify the final embryonic molt in pea aphid embryos we used the absence (pre-molt) or presence (post-molt) of hair-like sensilla on the cuticle (Konopová et al., 2005). The pea aphid embryonic cuticle and sensilla are visible under fluorescent light in the green range. We observed embryos on a Leica SP5 confocal microscope with a 546 λm laser and detector range of 550–580λm at 10x magnification. An image was captured of the cuticle and of the body length for each embryo at ~stage 18 and older. Body length was used as a proxy for age and measured with Leica LAS AF. If sensilla could be observed on an embryo, it was counted as sensilla present and if not, it was counted as sensilla absent. To test for a correlation (Pearson’s product-moment correlation) between winged percentage and sensilla presence we compared the percentage of winged siblings from the same female and the presence of sensilla of offspring between the sizes of 800–1000μm in length.

Results

To identify gene expression differences between winged-destined (WD) and wingless-destined (WLD) embryos we used RNA sequencing at the two stages of development critical to the wing polyphenism mentioned above (stage 18 and stage 20). We used two treatments to produce the embryonic samples: a crowding treatment, from which we obtained WD and WLD embryos, and a low-density plant treatment, from which we obtained WLD embryos. Our rationale for collecting WD and WLD embryos from the same crowding treatment was that the samples would not have treatment differences. We were concerned, however, that if expression differences underlying the phenotypic switch worked as a threshold mechanism, this approach would potentially not reveal significant gene expression differences because they would be too subtle. We therefore performed the additional treatment of producing WLD embryos by growing them on plants.

For each stage (18 and 20), we performed four biological replicates for a total of 24 libraries (four WLD and four WD of both stages from the crowding treatment, four WLD of both stages from the plant treatment). Stranded transcriptome sequencing yielded an average of 26 million high quality reads per library mapped to reference genome v.2.1 (IAGC, 2010). We required ~2.6 reads for a gene to consider it expressed (EdgeR, counts per million > 0.1). Using this expression filter, we found 16,418 expressed genes that were used for downstream analyses.

Treatment and developmental stage heavily influence gene expression

To obtain an overview of how pea aphid embryonic gene expression is influenced by our three primary variables (phenotype, developmental stage, and treatment), we examined global patterns using a principle component analysis (PCA: Figure 1A). Principle component axis 1, which explained ~33% of the variance, indicated a large effect of treatment on the embryo samples. Principle component axis 2, with 26% of the variance, revealed that stage also strongly affected gene expression.

Figure 1.

Figure 1.

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Treatment and stage differences dominate expression differences among samples. A). A principle component analysis of gene expression levels across the two developmental stages (stage 18 and stage 20), the two treatments (crowding treated and plant treated), and two phenotypic outcomes (winged versus wingless). Dots are individual RNA-Seq libraries and ellipses are 95% confidence intervals. B) A Venn diagram showing the number of differentially expressed genes (DEG) between stages, treatment, and future phenotype (winged or wingless).

The numbers of differentially expressed genes mirrored these PCA results. To compile a conservative list of statistically differentially expressed genes, we used two methods for detection, DESeq2 and EdgeR. We chose these two methods because they trade-off a high true positive detection rate (EdgeR) and a low false positive detection rate (DESeq2) (Soneson & Delorenzi, 2013). Using the intersection of both methods (FDR < 0.1 for each), we found that of the 16,418 expressed genes, 8,473 genes were significantly different between developmental stages and 9,063 genes were differentially expressed between treatments (S1 Table). In other words, stage and treatment both had large effects on embryonic gene expression, with almost half of the expressed genes showing differences. In contrast, only 155 genes were differentially expressed between phenotypes (stages 18 and 20 winged samples, combined, compared to stages 18 and 20 wingless samples, combined) (Figure 1B).

Differential gene expression patterns between phenotypes at each stage implicates the involvement of insulin and FoxO signaling

Due to the overpowering influence of stage and treatment, we chose to proceed by restricting our gene expression analyses to the crowding-only treatment and to comparing the phenotypes within their respective developmental stages.

At the earlier developmental stage (stage 18), embryos are actively receiving the wing inducing cue or have just received the cue to be winged or wingless. We found 195 genes that were differentially expressed (FDR < 0.1, EdgeR: 225 genes, DESeq2: 226 genes; Figure 2A, S3 Table) between WD and WLD embryos. The majority of the differentially expressed genes had higher expression in the WLD embryos (143 of 195 genes). KEGG pathway analysis indicated an enrichment of FoxO signaling, lysosome, and metabolic pathways (Table 1). Additionally, when we performed other functional enrichment tests (GO and INTERPRO), we found an enrichment of insect cuticle-related and cytochrome p450 genes (S5 Table).

Figure 2.

Figure 2.

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Volcano plots of gene expression differences between wingless-destined and winged-destined embryos. Significantly differentially expressed genes (FDR < 0.1 in both EdgeR and DESeq2 analyses) are shown in red for A) stage 18 embryos and B) stage 20 embryos.

Table 1.

Enriched KEGG pathways of significantly differentially expressed genes between winged and wingless destined embryos.

Samples Signaling pathway (KEGG) Gene count % of differentially expressed genes EASE p-value*
Stage 18 significant genes (FDR<0.1) FoxO signaling pathway 4 3.1% 0.02
Lysosome 4 3.1% 0.06
Metabolic pathways 13 10.2% 0.07
Retinol metabolism 3 2.4% 0.09
Ascorbate and aldarate metabolism 3 2.4% 0.09
Stage 20 significant genes (FDR<0.1) Metabolic pathways 30 14.9% 0.00
Glycolysis / Gluconeogenesis 5 2.5% 0.01
Fructose and mannose metabolism 4 2.0% 0.01
Biosynthesis of antibiotics 9 4.5% 0.02
Galactose metabolism 4 2.0% 0.02
FoxO signaling pathway 5 2.5% 0.03
Biosynthesis of amino acids 5 2.5% 0.04
Valine, leucine and isoleucine biosynthesis 2 1.0% 0.05
Amino sugar and nucleotide sugar metabolism 4 2.0% 0.06
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*

DAVID 6.7, EASE: Modified Fishers Exact

At stage 20, when the embryos are committed to a single phenotype, 300 genes were differentially expressed (FDR < 0.1, EdgeR: 330 genes, DESeq2:450 genes; Figure 2B, S4 Table). Again, the majority showed higher expression in the WLD embryos: 257 genes, versus 43 genes with higher expression in WD embryos. We observed an increase in differentially expressed metabolic genes relative to stage 18 differences, particularly genes associated with glycolysis and gluconeogenesis (Table 1). We also observed similar overrepresented categories using GO and INTERPRO. We again found an enrichment of cytochrome p450 genes and FoxO signaling pathway genes (S5 Table).

Overall, only a small number of genes were differentially expressed between morphs and the number of differentially expressed genes increased with developmental time. Only 23 genes were differentially expressed between morphs at both stages (the overlap of the genes differentially expressed between morphs at stage 18 and the genes differentially expressed between morphs at stage 20); most of the differential expression between morphs is, therefore, stage-dependent.

Insulin-related genes are at higher levels in WLD embryos

Because we found FoxO signaling pathway enrichment between WD and WLD embryonic gene expression at both stages, we further examined FoxO-related gene expression between the phenotypes. We found that it was differential expression of genes in the insulin-signaling portion of the FoxO pathway that resulted in the pathway enrichment (Figure 3). KEGG pathway analysis identified four genes from this pathway at stage 18 and five at stage 20 (S6 Table). We also found four IIS-related genes that are not part of the KEGG FoxO pathway in our differentially expressed gene lists: target of brain insulin (aptobi, ACYPI001718, stage 18), Ecdysone-inducible gene-2 (apImpL2, ACYPI005323; stage 20), adipokinetic hormone (apAKH, ACYPI45399, stage 20), adipokinetic hormone receptor (apAKHr, ACYPI002471, stage 20). In fact, the most differentially expressed gene at stage 18 was aptobi with 3.5x higher expression in wingless-destined embryos. Pea aphids possess two insulin receptors (apInR1: ACYPI009339, apInR2: ACYPI010079). While both insulin receptors were expressed in our embryonic samples, only apInR1 was differentially expressed (at stage 18), with higher expression in wingless-destined embryos. The most obvious pattern across all IIS related genes was that they were expressed at higher levels in the WLD embryos.

Figure 3.

Figure 3.

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Differentially expressed genes are found in the insulin-signaling part of the FOXO signaling pathway. The KEGG pathway for FOXO signaling is shown, with genes highlighted in red if they were differentially expressed between winged- and wingless-destined embryos of either stage. Genes shown in blue are known to be present in the pea aphid genome.

Morphological traits differ by treatment, but not by phenotype

Our gene expression analysis implicated IIS as a candidate pathway for alternative phenotype development in the embryos. We therefore hypothesized that we would find size differences between WLD and WD embryos due to IIS differences. The observed differential expression of cuticle genes also led us to believe that we might observe stage differences between WD and WLD embryos, because cuticle genes are often expressed in a stage-specific manner during embryogenesis (Minelli et al., 2006; Riddiford et al., 2003; Zhang et al., 2014). Thus, we also hypothesized that we would find morphological evidence of stage differences between WLD and WD embryos.

We found no evidence for global size differences between WD and WLD embryos. We used two approaches, using weight and then length as measures of possible size differences. First, we examined the average nymphal weight of crowding-treated nymphs within 24 hours of birth and compared those measures to the group’s phenotype (winged or wingless) proportion. We observed no significant correlation between the two measures (Figure 4a; Pearson’s correlation, R2=−0.09, p = 0.61, wing percentage by nymphal weight), indicating that groups with more WD offspring were not significantly larger or smaller than groups with more WLD offspring. Second, we measured the length of the posterior, right tibia of newborn nymphs, using it as a proxy for body size. We then allowed those nymphs to grow up so that we could assay their phenotype (winged or wingless). We observed no differences (Figure 4b; Wilcox test, p = 0.503 winged vs. wingless by tibia length).

Figure 4.

Figure 4.

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Biometric assays do not indicate differences between winged and wingless embryos and 1st instar nymphs. A) Average weight of nymphs, measured in groups of 20–30 on the y-axis by their respective phenotype proportion per sibling group on the x-axis. B) Tibia length measured within one hour of birth does not correlate with phenotype and shows no effect by treatment (crowding-treated winged n=11, crowding-treated wingless n=15, and plant-treated wingless n=6). C) Sensilla indicates the molt from the 2nd to the final embryonic instar. There is no correlation between molt time and phenotype. However, this molt does appear to be different between treatments, where plant-treated embryos wait longer to molt. Dotted lines indicate the approximate window when crowding-treated embryos molt. Plant treated embryos only molt at the upper limit of the molting window (embryos measured from 30 crowding-treated adult females and 10 plant-treated females with ~11 embryos from each female).

We also found no evidence for stage differences between phenotypes. Stage 18 and stage 20 embryos are near the end of embryonic development, but major developmental changes occur between these stages. Between the two stages, embryos double in length, achieving their final embryonic size in stage 20, just prior to birth. Additionally, during stage 19, embryos undergo their final embryonic molt, which results in the development of the first nymphal instar cuticle. We examined if embryos had molted or not to assay their embryonic stage. As a measure of the molt, we visualized the absence (pre-molt) or presence (post-molt) of cuticle sensilla (Konopová et al., 2005), which are hair-like structures of the sensory system (Bromley et al., 1979). We again relied on sibling phenotype proportions to determinate probability of an embryo being WD or WLD. We saw no evidence of stage differences among the embryos (Figure 4c; Pearson’s product-moment correlation, R2=−0.06, p = 0.45, crowding-treated sensilla presence vs. winged offspring proportion). We did, however, see a large and interesting treatment effect. In plant-treated embryos, where almost all embryos are destined to be wingless, a critical size appears to be reached prior to molting (Figure 4c; nearly all embryos from plant-treatment reach 1000μm in length before exhibiting sensilla). In contrast, in crowding-treated embryos, they molt at an earlier, variable size (Figure 4c).

Discussion

Here we find evidence to suggest the involvement of IIS via FOXO in the developmental regulation of alternative morphs in pea aphids. Specifically, wingless morphs show an increase in the expression of genes that are transcriptionally-regulated by FOXO, the activity of which is negatively regulated by IIS. These data therefore suggest that the activity of IIS is reduced in embryos destined to be wingless versus winged adults.

The insulin/IGF-signaling pathway is canonically a regulator of growth and metabolism in response to environmental conditions, through the environmentally-regulated release of insulin-like peptides (Hietakangas & Cohen, 2009). These peptides bind to the insulin receptor of cells and activate the IIS pathway, which functions in part by repressing the transcriptional activity of FOXO (Neufeld, 2003) (Figure 3). Transcriptional targets of FOXO, the expression of which are upregulated when IIS activity is low, include negative growth regulators such as 4EBP (Jünger et al., 2003), genes involved in physiological stress response such as PEPCK (Jünger et al., 2003) and catalase (Sim & Denlinger, 2011), as well as genes in the insulin-signaling pathway itself, including InR (Puig & Tjian, 2005). We found many of these genes to be upregulated in the putatively wingless aphid morph (IIp, InR1, INSR [chico], PDK1, catalase, ATG8, BNIP3, PEPCK, atrogin-1, and imp-L2), indicating activation of FOXO and suppression of IIS. Because of its effects on the expression of these genes, the IIS pathway is thought of as a phenotypic plasticity pathway, generating bodies that match their environment with respect to size, shape and physiology. Correspondingly, multiple studies have implicated the IIS pathway in the regulation of a wide variety of plasticities (Emlen et al., 2012; Snell-Rood & Moczek, 2012; Tang et al., 2011; Wheeler et al., 2014; Wolschin et al., 2011; reviewed in Nijhout and McKenna, 2018), including late nymphal stage differences between winged and wingless morphs of two aphid species (Ding et al., 2017; Guo et al., 2016), and long and short-winged morphs of the brown planthopper, Nilaparvata lugens (Xu et al., 2015) and the soapberry bug (Fawcett et al., 2018). Interestingly, the latter two studies showed different functional requirements for the two insulin receptors, the paralogs InR1 and InR2. This may be the case in the aphid as well, since we only observed InR1 to be differentially expressed. In all of these studies, however, IIS signaling was manipulated or assayed during the generation of the phenotypically-plastic traits, be they morphological or physiology. In our study, the differences in IIS occurred well before any phenotypic differences between morphs can be detected (the morph differences are not discernible via histological sections until the second nymphal instar (Ogawa et al., 2012)), and before morph-specific development is likely to have begun.

While there is increasing evidence that insulin-signaling is involved in the generation of both discrete and continuous morphological variation across environmental conditions, there is a much older literature describing the effects of ecdysone and juvenile hormone (JH) in the regulation of polyphenisms. The latter include caste regulation by JH in bees, ants and termites by juvenile hormone (Simpson et al., 2011), and the regulation of seasonal polyphenisms by ecdysone in butterflies and moths (Nijhout, 1999). In the pea aphid, ecdysone-signaling during embryogenesis is involved in the wing polyphenism: aphids injected with ecdysone produce fewer winged offspring, and aphids with inhibited ecdysone signaling produce more winged offspring (Vellichirammal et al., 2017). JH and ecdysone have a well-established role in coordinating developmental transitions, particularly during molting and metamorphosis. Importantly, however, both JH and ecdysone are also regulators of insulin-signaling (Colombani et al., 2005; Mirth et al., 2014). The relationship between ecdysone and insulin-signaling has been well elucidated in Drosophila, where ecdysone synthesis by the prothoracic gland is positively regulated by insulin-signaling via FOXO (Koyama et al., 2014), while ecdysone-signaling in the fat body remotely suppresses the release of insulin-like-peptides from neurosecretory cells of the brain (Colombani et al., 2005). The activity of circulating insulin is further suppressed by the ecdysone-regulated synthesis of insulin-binding proteins impl2 (Honegger et al., 2008; Lee et al., 2018), which we found is at significantly higher levels in putatively wingless versus winged aphid embryos (Figure 2). Thus, it is possible that the observed suppression of insulin-signaling in embryos destined to be wingless is a consequence of elevated ecdysone signaling through maternally provided ecdysone (hypothesized relationship illustrated in Figure 5).

Figure 5.

Figure 5.

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Hypothesized model linking ecdysone and insulin signaling in the pea aphid wing polyphenism. In mothers that are at low densities, ecdysone signaling is inferred to be low (Vellichirammal et al., 2016; Vellichirammal et al., 2017). Evidence here suggests that, in these low density mothers, embryos have higher expression of FoXO-targeted genes and thus lower insulin-signaling activity, which ultimately results in their primarily wingless adult phenotype. The opposite is true in aphid mothers that experience high densities.

An open question, therefore, is whether differences in IIS activity between the winged and wingless morphs play a developmental role in generating the polyphenisms, or is IIS activity a pleiotropic consequence of differences in ecdysone level that acts independent of IIS to regulate the wing polyphenisms. Ecdysone has myriad developmental effects apart from its influence on IIS and this may be the primary purpose of maternally supplied ecdysone. Further, ecdysone also stimulates its own synthesis in Drosophila (Moeller et al., 2013), so early exposure to ecdysone may establish the synthesis of ecdysone later in development, when the polyphenism becomes apparent. Our observation that there is no difference in growth rate between wingless (high ecdysone/low IIS) and winged (low ecdysone/high IIS) morphs at a time when we can detect differences in the expression of genes regulated by IIS/FOXO-activity is, at first glance, consistent with the hypothesis that changes in IIS do not necessarily regulate the polyphenism.

The increased expression of FOXO-regulated genes in wingless-destined embryos may, however, reflect FOXO activation in specific tissues rather than in the body as a whole. We consider this to be the more likely scenario. In the brown planthopper Nilaparvata lugens, FOXO activity in the nymphal wing bud is thought to regulate the production of short-winged versus the long-winged adults (Xu et al., 2015). Knockdown of FOXO expression suppresses the short-wing phenotype, while FOXO activation (through knock-down of its inhibitor Akt) promotes the short-wing phenotype. The activation of FOXO in the short-winged phenotype appears to act on the growing wing buds alone and is not reported to correlate with any reduction in whole-body growth rate. Indeed, the production of winged forms – where FOXO activity is reduced – is associated with feeding on nutritionally-depleted hosts, conditions expected to increase FOXO activity and slow body growth. The production of winged pea aphids is similarly associated with reduced nutritional conditions, in the sense that crowding-treated aphids tend not to be able to feed normally. If the observed increases in the expression of FOXO-targeted genes in wingless aphids reflects tissue-specific rather than systemic activation of FOXO, this could explain why we did not observe differences in whole-body growth rate.

An obvious next step is to explore the functional consequences on wing development of manipulating insulin-signaling during aphid embryogenesis. This could be achieved by knockdown of FOXO or InR1 using dsRNA, or by manipulating insulin-signaling using pharmacological methods (Chowański et al., 2018). Specifically, if ecdysone injections generate wingless morphs via FOXO, this effect should be reduced in embryos where FOXO expression is also suppressed. Our increasing ability to manipulate gene expression using dsRNA make such experiments possible.

In conclusion, our study is one of an increasing number that implicate insulin-signaling in the generation of alternate environmentally-induced morphologies. While the role of insulin-like growth factors in regulating whole body growth in response to nutrition has long been understood, in this case the environmental-sensitivity of the IIS pathway may have been exploited to regulate the growth of individual traits in response to environmental conditions. Further, this environmental regulation appears to be at least partially indirect, via intermediaries such as ecdysone and juvenile hormone. These hormones have been implicated in the generation of polyphenisms in a wide diversity of insects. Whether they do so via insulin-signaling remains an open question.

Supplementary Material

Supp FigS1

S1 Figure. Collection and proportion of winged offspring from dissected females for RNA-seq. A) Females were placed in one of two conditions, crowding or plant treatment. For each treatment, 10 females were placed together for 24 hours (35mm petri dish for the crowding treatment, and fava bean seedling for the plant treatment). All females were then moved to fava bean seedlings and raised individually for 24 hours after which all embryos were dissected from adult females. Offspring born during the 24 hours while females were individually on plants were used to assess the winged offspring production of each female. If the proportion of winged offspring was greater than 0.75, we called that particular female's embryos winged-destined, and if it was 0, we called the embryos wingless-destined. B) Proportion of winged offspring produced from crowding-treated females (n=382 females with ~14 embryos each). Each circle represents a single female. C) Proportion of winged offspring from plant-treated females (n=69 females with ~8 embryo each).

Supp Tables

Research Highlights.

Transcriptional profiling of winged versus wingless destined pea aphid embryos implicated the importance of the insulin-signaling portion of the FoxO pathway.

No differences in size or stage were found in winged- versus wingless-destined embryos.

Together, these results raise the hypothesis that FoxO regulates morph development in a tissue-specific manner.

Acknowledgments

We thank Jennifer Keister for providing valuable technical assistance. This work was funded by NIGMS 5R01GM116867 to JAB.

Footnotes

Conflict of interest: The authors have no conflicts of interest to declare.

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Supplementary Materials

Supp FigS1

S1 Figure. Collection and proportion of winged offspring from dissected females for RNA-seq. A) Females were placed in one of two conditions, crowding or plant treatment. For each treatment, 10 females were placed together for 24 hours (35mm petri dish for the crowding treatment, and fava bean seedling for the plant treatment). All females were then moved to fava bean seedlings and raised individually for 24 hours after which all embryos were dissected from adult females. Offspring born during the 24 hours while females were individually on plants were used to assess the winged offspring production of each female. If the proportion of winged offspring was greater than 0.75, we called that particular female's embryos winged-destined, and if it was 0, we called the embryos wingless-destined. B) Proportion of winged offspring produced from crowding-treated females (n=382 females with ~14 embryos each). Each circle represents a single female. C) Proportion of winged offspring from plant-treated females (n=69 females with ~8 embryo each).

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Supp Tables
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