Photoperiod controls wing polyphenism in a water strider independently of insulin receptor signalling

Keywords: water strider, polyphenism, insulin receptor, RNA interference, developmental plasticity, FOXO

(a) . Gerrid cultures and rearing

The G. buenoi population was originally collected from a pond in Toronto, Ontario, Canada, during 2012 and was replenished in 2015 with 30 individuals from a population in the same area. The photoperiods for the stock population were from 2012 to May 2019 16 h light : 8 h dark (16 L : 8 D) and since then 22 L : 2 D. From May 2019 until present, the generation of adults in the breeding stock was primarily from individuals that had been reared during their nymphal stages in either 12 L : 12 D or 18 L : 6 D, constituting a mix of short-winged (henceforth micropterous) and long-winged (henceforth macropterous) morphs. A smaller proportion of breeders came from nymphs reared at high densities in 22 L : 2 D which produces a small frequency of macropterous individuals.

Gerrids in the stock population were fed five times a week with frozen crickets (instar 2–3 Acheta domestica) and kept at room temperature. Pieces of Styrofoam were provided as a substrate for egg laying and resting.

(b) . Determination of environmental factors governing wing morph determination in Gerris buenoi

(c) . RNA interference

dsRNA was synthesized from PCR products generated from genomic DNA or cDNA using the primers in the electronic supplementary material, table S2. The primers have the T7 RNA polymerase promotor sequence as overhangs (bold letters in sequences electronic supplementary material, table S2) to facilitate generation of dsRNA through in vitro transcription. Each DNA template was verified with Sanger sequencing (Eurofins Genomics Mix2Seq) before in vitro transcription overnight with T7 RNA polymerase (Thermo Fisher Scientific). The dsRNA was treated with DNase I (Thermo Fisher Scientific) and then purified using phenol : chloroform salt extraction or GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fisher Scientific). Elution or resuspension was done in Spradling injection buffer [29]. Microinjections of dsRNA were performed with Eppendorf Celltram Vario with attached 1 mm outer diameter glass capillary needles (Narishige).

For injections, nymphs of instar 3, 4 or 5 which were hatched and reared in either 12 L : 12 D or 18 L : 6 D were sedated with CO₂ and gently attached on double-sided adhesive tape to hold them still. Injections were made in between the 6th and 5th abdominal segment ventrally delivering dsRNA solution of 0.2–0.3 µl for instar 3, 0.3–0.4 µl for instar 4 and 0.4–0.6 µl for instar 5, with the concentrations specified in the electronic supplementary material, table S3. After injection, replicate groups of 7–8 injected nymphs were held in either photoperiod 19 × 26 cm boxes of (1.6 individuals per 100 cm²) and fed with crickets (instar 2–3) five times a week until being phenotyped as adults (final sample size indicated in figure 3a–c). To confirm knockdown, we extracted total RNA using Trizol (Qiagen) from seven whole individuals (males) per dsRNA treatment 2 days after moulting into instar 4 (about 5 days after injection) and performed quantitative reverse transcriptase PCR (qRT-PCR), see electronic supplementary material, table S2 for primers. For confirmation of the effect of FOXO RNAi in wing buds, striders were injected in instar 3 and sampled 2 days after moulting into the 5th instar. Furthermore, to confirm the functionality of our RNAi protocol, we knocked down the expression of Distalless (Dll) with RNAi, where we injected Dll dsRNA in instar 3 nymphs hatched and reared in 12 L : 12 D and evaluated developmental effects expected from manipulating the expression of Dll (e.g. abnormal leg shape and shorter than normal wing buds). The RNAi knockdown treatment for each gene is referred to as dsGFP, dsINR1, dsINR2, dsINR1-like, dsFOXO, INR-cocktail or dsDLL below. The negative control in the RNAi experiments is the dsGFP treatment, where dsRNA with the sequence of green fluorescent protein (GFP) is injected into individuals but should not interfere with gene expression, thus controlling for potential effects of the injection procedure in itself on wing morph determination.

(d) . Body size measurements and data statistical analysis

Individuals used for measurements were stored in 70% ethanol at 4°C until photographed on millimetre paper using a Nikon SMZ800 stereo microscope with Nikon DS Fi1 camera attachment. Measurements on these photographed specimens were made with ImageJ (v. 1.53 k, see electronic supplementary material, figure S2 for definition of measurements). All statistical analysis was performed in R (v. 4.0.3). ANOVAs, t-tests, Pearson's χ² and generalized linear models were performed within the R set of core functions (stats 4.0.3). All code and data are available in the electronic supplementary material. The estimation of pairwise sequence identity of the inr genes coding sequences was done with Muscle (v. 3.8.425 (alignment in Geneious (v. 2021.1.1) using standard parameters.

(a) . Wing morph in Gerris buenoi is determined by photoperiod and is independent of parental phenotype

All nymphs became macropterous in the 12 L : 12 D and 14 L : 10 D photoperiod treatments, whereas all nymphs in 18 L : 6 D became micropterous (figure 1c). Intermediate frequencies of adult wing morphs resulted from the 15 L : 9 D and 16 L : 8 D photoperiod treatments (figure 1c). To assess whether the state of wing morph in parents influences the determination of wing morph in the offspring, we tested whether the frequencies of wing morphs in 15 L : 9 D differed between offspring generated from different controlled crosses but no significant difference was found (electronic supplementary material, table S4, χ² = 4.66, d.f. = 6, p = 0.58). We detected a significant sex-difference in wing morph frequencies in 15 L : 9 D (electronic supplementary material, table S4, χ² = 11.82, d.f. = 2, p = 0.002) where females were less likely to become macropterous. Together, these results show that constant photoperiod strongly influences wing morph determination in G. buenoi and that it does so independently of the parental wing morph phenotype.

(b) . Exposure to 18 L : 6 D as early as instar 3 biases development toward the micropterous morph

Adult individuals differed significantly in wing morph depending on the direction of shift in photoperiod experienced as nymphs in instars 3–5 (figure 1d,e). For individuals experiencing the 18 L : 6 D to 12 L : 12 D shift, a tendency to develop to micropterous adults was seen at instar 3 (approx. 50%) and no macropterous individuals were found when the shift occurred after early instar 4 (i.e. these individuals were committed to micropterous development when the shift in photoperiod occurred). By contrast, individuals experiencing the 12 L : 12 D to 18 L : 6 D shift in photoperiod were seemingly uncommitted with regard to developing a particular wing morph until as late as day 2 of instar 5. The effects of instar, direction of photoperiod shift and their interaction were highly significant (χ² = 63,27, d.f. = 5, p = 2.56 × 10⁻¹², χ² = 125,87, d.f. = 1, p < 2.2 × 10⁻¹⁶, d.f. = 5, χ² = 14,18, p = 0.015, respectively). Thus, the onset of commitment to a particular wing morph differed markedly during development in either 12 L : 12 D or 18 L : 6 D. We found no evidence of sex-differences in morph determination (electronic supplementary material, figure S3, χ² = 1.39, d.f. = 1, p = 0.24 and χ² = 0.36, d.f. = 1, p = 0.55 for increase or decrease shifts respectively).

(c) . Wing polyphenism in Gerris buenoi is sensitive to nymphal density but not nutrient availability

In addition to photoperiod, we also explored nymphal density and nutrition as environmental factors for wing morph determination in G. buenoi (figure 2). Almost all nymphs reared in 12 L : 12 D across all density regimes became macropterous (three out of 574 in total became micropterous), whereas in 18 L : 6 D most individuals became micropterous across all densities (figure 2a). The frequency of the macropterous morph in 18 L : 6 D significantly increased with density (χ² = 24,39, d.f. = 3, p = 2 × 10⁻⁵). Interestingly, the frequency of macropterous individuals in 18 L : 6 D in the extreme density was higher among females than males (χ² = 18,23, d.f. = 6, p = 0.006).

When exploring the effect of nutrient availability, the ratio of wing morphs remained unchanged in 12 L : 12 D (100% macropterous individuals), and in 18 L : 6 D only two macropterous individuals appeared of 129 in total across all dietary regimes. While we found a significant difference in the frequency of mesopterous adults among individuals treated with different nutrient regimes from instar 1 (figure 2b, χ² = 17,08, d.f. = 2, p = 0.0002), sample size was very low in the low diet and thus this result should be treated with caution. Among the individuals reared in the nutrient regimes from instar 4 group (figure 2c), we found that the morph frequencies did not differ significantly between low- and high-nutrient regimes (χ² = 2,00, d.f. = 2, p = 0.37), although they differed profoundly between photoperiods. We assessed how the nutrient regimes affected mortality, developmental duration and adult body size, in order to test if the restricted diet regimes were stressful. We found a clear effect of nutritional regime on mortality and developmental duration and, furthermore, adults that had been reared in restricted diets were significantly smaller than adults reared in the highest diet (electronic supplementary material, figure S4A–G). These results show that the dietary regimes that we tested were stressful for the gerrids and that wing morph determination is robust to restricted nutritional availability.

(d) . Depletion of genes in the insulin/insulin-like receptor signalling pathway does not alter wing morph

To test whether the IIS pathway has a role in controlling wing polyphenism in G. buenoi, we injected male and female nymphs grown in 12 L : 12 D and 18 L : 6 D during the 3rd, 4th or 5th instar with dsRNA targeting INR1, INR2, INR1-like or FOXO, or a cocktail of all three insulin receptor genes. GFP dsRNA was used as a negative control. We found no differences compared to control treatment in frequencies of the wing morphs for any of the genes subjected to gene expression knockdown in the RNAi treatments (figure 3a–c). A proportion (16%) of individuals in the instar 4 injection group expressed an aberrant wing phenotype (electronic supplementary material, figure S5) where one or both of the wings had abnormal morphology. This was, however, shown to be a result of handling during injections and not from knock down of any specific gene as it was present in non-injected control individuals as well in the dsGFP negative control treatment.

We used reverse transcription-quantitative PCR (RT-qPCR) to confirm knockdown of each mRNA. All had reduced levels as compared to the dsGFP treatment (figure 3d–h). Additionally, a strong reduction in FOXO mRNA in wing buds was found in dsFOXO-treated instar 5 nymphs, showing that the injected dsRNA is able to knock down gene expression in the wing bud tissue and that the effect lasts from the time point of injection, in this case from instar 3, until instar 5, which is the stage when adult wing development occurs. As an additional control to validate our RNAi protocol, we injected dsRNA against the developmentally important gene distalless (dll, electronic supplementary material, figure S6). Wing bud phenotypes in dll RNAi-treated individuals were observed before in a different water strider species (Limnoporus dissortis, [30]), in which the hind wing bud protrudes from under the abnormally short forewing bud. We found a very similar phenotype in the dll RNAi-treated G. buenoi individuals (electronic supplementary material, figure S6). In addition to shortened forewing buds, these individuals also had abnormal appendages and experienced problems during moulting. Overall, our results show that, contrary to findings in several other insect species, knockdown of components in the main IIS pathway does not alter wing morph frequencies in G. buenoi.

(e) . Forkhead box O RNAi causes an increase in body size but not wing size

As we did not detect differences in wing morph determination in dsGFP-treated individuals and those with an altered IIS pathway, we explored whether manipulated individuals showed differences in body and wing size within wing morph categories. Body size differences are expected in IIS-manipulated animals, given the amount of evidence of size regulation orchestrated by the IIS pathway [31]. We found that dsFOXO individuals in both wing morphs and sexes were significantly larger than dsGFP individuals (4.2% and 4.3% longer for females and males, respectively, electronic supplementary material, figure S7). Thus, FOXO appears to act as a general negative regulator of body size in G. buenoi, but interestingly, the increased body size in dsFOXO-treated males and females were not matched by an effect on wing length in either micropterous or macropterous individuals (electronic supplementary material, figure S7).

We also found significant differences in body and wing length between dsINR1-like females and dsGFP females, and between dsINR1 males and dsGFP males, within morph categories (electronic supplementary material, figure S7). However, it should be noted that the reduced body size in dsINR1 and dsINR1-like individuals cannot reliably be attributed to the effect of RNAi against these respective genes, as there is significant cross-knockdown of all three InRs in these treatments (figure 3d–f).

(a) . Photoperiod and nymphal rearing density but not nutrition control wing polyphenism in Gerris buenoi

By exposing nymphs throughout development to a range of photoperiods, we have demonstrated a strong relationship between (constant) photoperiod and wing morph frequency in G. buenoi. Previous work on the role of environmental induction of wing morph in G. buenoi was performed in field enclosures and focused on patterns of wing morph frequencies in relation to nymphal stage at the summer solstice [19]. Spence [19] found that no individuals which reached the adult stage before the summer solstice became macropterous, but after the solstice, there was a step-wise increase in the frequency of macropters among individuals in increasingly younger stages at solstice. All individuals that were either instar 1 or 2 at the solstice developed to the macropterous morph [19]. These results suggest that the direction of change in photoperiod is an important cue for wing morph determination in G. buenoi, as it appears to be also in the closely related Gerris odontogaster [20].

Nonetheless, this interpretation is not entirely satisfactory because the Alberta population of G. buenoi produced 100% macropters in the laboratory under 16 L : 8 D constant photoperiod [19], but a mix of macropterous and micropterous morphs at 19 L : 5 D [24]. Although it is tempting to suspect that direction of change in day length is important, changes in photoperiod around the solstice are very small in relation to the developmental duration of late instars of G. buenoi in field conditions (roughly 10 days on average for instar 4 and 5 combined, [19]). Under these circumstances, there is little opportunity for a switch to operate on decreasing photoperiod to generate the macropterous individuals seen in fig. 4 in [19]. Thus, the exact contribution of either absolute photoperiod or changing photoperiod on induction during natural conditions remains to be thoroughly understood. Nonetheless, it is clear that at least in laboratory conditions, G. buenoi can use absolute photoperiod as a cue for the wing morph switch.

One particularly interesting finding from our work is that females were less likely to become macropterous than males when held at 15 L : 9 D. A similar tendency has been shown before under field conditions [19], where G. buenoi females consistently showed lower frequency of macropters than males. We also found a female bias in morph induction in response to high density, where females kept at high density had a higher frequency of macropterous individuals than males (figure 2a). Interestingly, Han [25] recently showed that wing morph induction in the gerrid Tenagogerris euphrosyne is sex-biased, with females equally likely to become macropters as males at low density but that females were less likely to become macropterous than males at high density. Taken together, these results suggest that the threshold for induction of wing morphs can be sexually dimorphic in water striders and that the direction of the response can differ between species. For a detailed discussion on the adaptive significance of sex-specific wing morph determination see [25].

In line with several other studies on gerromorphans [23,25,38], we found that higher juvenile density led to increased frequency of macropterous individuals (figure 2). This is in contrast with results from Harada and Spence [24] who found that high density increased the frequency of micropterous G. buenoi individuals. It is not immediately clear why, although it is possible that differences in photoperiods between our study and that of Harada and Spence [24] played a role. Increased macropter frequencies in response to high nymphal density is likely an adaptive response to enable dispersal when conditions might become detrimental in the future, e.g. due to competition for food. However, the induction of micropters in response to crowding may emphasize that the morph induction can be rather complex; see [24] for a more comprehensive discussion on life-history strategies in response to crowding conditions in water striders.

When we exposed individuals to restricted nutrition regimes of different magnitudes, we found that food availability did not directly influence wing morph determination, at least not in the photoperiods we used (figure 2b,c). The low food regime was associated with increased mortality, longer developmental duration and smaller body size. Although no direct effects of nutrition were found on wing morph induction in laboratory conditions, indirect effects may be evident in natural conditions. For example, a longer developmental time may push the development of individuals across solstice and into conditions that favour the development of macropterous morphs.

From the experiments with constant photoperiod, we show that exposure to a long photoperiod of at least 15 h throughout development resulted in some micropterous development, whereas exposure to shorter photoperiods with either 12 or 14 h of daylight resulted strictly in macropterous development. To explore the sensitive stages of wing morph determination in G. buenoi, we exposed nymphs to a shift in photoperiod at different developmental stages, similar to an experimental design reported by Inoue & Harada [28] in a study of A. paludum. For our population, exposure to a long photoperiod (18 L : 6 D) was highly inductive of the micropterous morph and the sensitive window for this induction lasts from at least instar 3 until latest day 2 in instar 5. Furthermore, our results show that the growth of the adult wings, which occurs in instar 5, can by some mechanism be stunted by photoperiod cues that are received as early as instar 3. It should be noted that these results may be specific for the tested conditions, especially considering that 12 L : 12 D and 18 L : 6 D and the 6 h shifts in photoperiod is extreme and not experienced by individuals in natural conditions. Nevertheless, these experiments provide valuable information on the potential limits of induction, including the responsiveness of the mechanism that relay information of photoperiod to the growing adult wing tissue. We suggest that the pattern of commitment to wing morph we observe is in line with a view that the default developmental trajectory is towards the macropterous morph, but that this development can be arrested by an unknown mechanism induced by a long photoperiod during a rather long sensitive window, and lead to micropterous development. Interestingly, there is variation in wing morphs within some experimental groups (e.g. I5E and I5M in the 12 L : 12 D to 18 L : 6 D shift, figure 1e), which suggests genetic variation in the sensitive stage for induction.

(b) . Insulin/insulin-like receptor signalling pathway is not involved in wing polyphenism in Gerris buenoi

Multiple studies have implicated the IIS pathway as an important driver in wing length polyphenism in several insect species [14,15,37]. Our data, however, suggest that IIS does not play a role in wing polyphenism in G. buenoi. This may be because our RNAi knockdown did not reduce protein expression enough to see an effect, or because pathways other than IIS regulate the switch between wing morphs in G. buenoi. We argue that the latter alternative is the most-likely explanation based on the following observations. (i) There is no clear link between wing morph determination and nutritional availability in G. buenoi. (ii) As expected from the conserved role of IIS in growth regulation, manipulation of IIS components resulted in phenotypic effects similar to the effect of restricted nutrition (e.g. smaller body size), demonstrating that our RNAi treatments induced phenotypic alterations. (iii) The expression of all genes we tested was significantly reduced in individuals that were treated with the corresponding dsRNA, and the knockdown levels were of similar or higher effect sizes compared to similar studies that have reported positive results (e.g. [5,10,12]).

We note, however, that there was significant cross-knockdown among the insulin receptors, so that each InR dsRNA produced a significant knockdown of all InR genes. This is not surprising as the three expressed mRNAs share a considerable amount of sequence identity (approx. 41%, electronic supplementary material, table S5). In principle, cross-knockdown is not desirable, but since we do not see any change in wing morph frequencies in any InR dsRNA treatment, we argue that the observed cross-knockdown should not alter our conclusion.

It is possible that no wing morph switches were induced because the InRs are redundant, meaning that they all can serve as growth regulators of wing development. However, we detected a strong reduction in FOXO mRNA expression in wing buds of dsFOXO-treated individuals. Not only does the FOXO KD show that RNAi works efficiently in wing buds but also that normal levels of FOXO are not required for switching between wing morphs. As FOXO is the main IIS transcriptional output component [31], we interpret the robust reduction of FOXO mRNA in both wing buds and body in our RNAi experiment as our strongest evidence to suggest that the role of the nutrient-sensing IIS pathway is not involved in G. buenoi and thus that the IIS is not ubiquitous in regulating insect wing polyphenisms.

Our results therefore suggest that G. buenoi use a different genetic pathway to regulate wing morph differentiation than at least two other hemipterans with wing polyphenisms, the brown planthopper [14] and the soapberry bug [15] in which the IIS pathway controls wing morph determination. We speculate that a potential explanation could be higher environmental stochasticity faced by G. buenoi compared to the other species in terms of nutrient availability. The environmental cues for morph determination in the brown planthopper and soapberry bug are associated with the quality of their respective host plants, sugar content [12] and seeds available [15], respectively. The degree of signalling of the nutrient-sensitive IIS pathway thus provides a direct link between environmental cue and growth/induction. G. buenoi and other gerrids opportunistically consume a variety of arthropods that fall onto the water surface and thus are subjected to more day to day stochasticity in nutrient availability than species that follow the phenology of a host plant. This view is in line with previous discussions suggesting that nutrition is not a likely factor for wing morph determination in gerrids [33]. The stochastic nature of nutrient availability may thus have favoured other genetic pathways than IIS to be coupled to the wing regulatory network in the evolution of wing polyphenism in G. buenoi, and possibly also in other gerrids.

It is not surprising to find that environmental induction can occur with different mechanisms in different species, given that many different genes and pathways can be involved in wing shape and size regulation in insects [39]. In G. buenoi, it is clear that the genetic pathway responsible for control of wing length has evolved to be under the influence of photoperiod and will be very interesting to study further.

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Data Citations

1. Gudmunds E, Narayanan S, Lachivier E, Duchemin M, Khila A, Husby A. 2022. Data from: Photoperiod controls wing polyphenism in a water strider independently of insulin receptor signalling. Dryad Digital Repository. ( 10.5061/dryad.zs7h44jbz) [DOI] [PMC free article] [PubMed]
2. Gudmunds E, Narayanan S, Lachivier E, Duchemin M, Khila A, Husby A. 2022. Photoperiod controls wing polyphenism in a water strider independently of insulin receptor signalling. FigShare. ( 10.6084/m9.figshare.c.5926244) [DOI] [PMC free article] [PubMed]

Supplementary Materials

Data Availability Statement

The data used in this manuscript can be found in the file named ‘Data_file_Gudmunds_etal_20211221.xslx’. To analyse these data, the R-script ‘R_code_Gudmunds_etal_20211221.R’ can be used. Available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.zs7h44jbz [40].

The data are provided in the electronic supplementary material [41].