The Role of the Insulin Signaling Pathway in Wing Polyphenism

Meghan M. FawcettDepartment of Biology, Colby College, Waterville, ME

Background Polyphenism is an adaptive trait that confers two stable adult endpoints from the same genotype. In Jadera haematoloma, this polyphenism is manifested in wing morph—either long winged or short winged. Polyphenism has developed in a number of different species as an adaptation to variable environments. Case precedent suggests that polyphenism in J. haematoloma is determined by the interactions between both genes and environment. Many assumptions in my study are based off of an aphid case study wherein genotypically identical females, with no wings, produce offspring with wings if conditions indicate a dangerous or unfit environment. Generally, when predation risk is high, food availability low, or crowding too great, females produce only long winged offspring (Purandare, 2014). It is assumed, that in times of pressure, individuals grow longer wings to fly away from the poor environment in favor of a better one. It is further postulated that individuals who fail to grow wings instead reallocate their resources towards fecundity, as they do not need to escape inhospitable environments. It is possible that the wing polyphenism of the soapberry bug also functions on this model.

similarly to those in Jadera. In N. lujens, the first insulin receptor (InR1) promotes long winged growth if active whereas InR2, promotes short winged morphs when active and long winged morphs (Xu, 2015). The third unit Chico, is an adaptor protein attached to both insulin receptors, that when downregulated promotes smaller body size and in some cases increased life span (Ismail, 2015). Finally Akt is the target protein of insulin receptor signals from PK13 and Chico, whose deactivation also promotes smaller body size and increased longevity (Jevtov, 2015). I hypothesized individuals in low food conditions, heavy crowding, and small space should produce more long-winged individuals. Additionally, the results of the InR1 knockdown experiments should show smaller individuals with short wings, and InR2 knockdown should result in more long winged individuals. Finally, the knockdown of both Akt and Chico should result in smaller, predominantly short winged individuals.

MethodsEnvironmental Manipulation

Bugs were placed in high and low food conditions, one dish of food was provided to them at the beginning of the study and never replaced or refreshed. For individuals in low food conditions one small dish was filled (about 20 balloon vine seeds), and for high food one larger dish was filled (about 80 seeds). For both conditions tank size remained constant. In order to induce crowding conditions, high crowding environments typically contained about four times as many bugs as the low crowding environments.

RNAi

First double stranded RNA was made using template DNA from Jadera, polymerase, dNTPs, and T7 primers to produce an amplified sequence. Next, in-vitro transcription amplifies sequences from the previous reaction and anneals the dsRNA. The dsRNA is purified using ammonium acetate and ethanol to precipate the dsRNA. dsRNA is purified, resuspended, and diluted with buffer and McCormick green food dye. Only 4th instar bugs collected for injection. Each bug is injected in the abdomen at 10psi and pulse duration of 6. Visual confirmation of injection is verified by a green spot in the abdomen of the bug.

Figure 2. Number of short winged morphs in relation to condition in which the bugs

were placed. In each situation, the amount of food per individual increased, as did the

number of short winged individuals. Control bugs only show a slight trend toward short

winged morphs whereas all tip box individuals were shortwinged. Tank size and relative food availability significantly affects

wing morph ratio (p<.0001 )

The Akt and Chico experiments yielded similar results that were not supported by my hypothesis. In both experiments, the data suggests that both Chico and Akt result in delayed molting, or potentially the inability to molt altogether. This is most likely due to the fact that chico is the only adaptor protein in the insulin signaling pathway, and so manipulation of the gene causes large and very visible effects. Similarly, Akt is linked to decreased sensitivity to insulin, which might also result in the observed phenotypes. Furthermore upon dissection of the Chico females, excess fat storage and water retion was observed (Fig. 4.h)

AcknowledgmentsI would like to thank my advisor Dr. Angelini for providing me with guidance, advice, materials, and discussion throughout my experiment. I would also like to thank our Lab Technician, Beth Richards, for taking care of our bugs and experimental cages as well as performing other vital tasks in the lab. Finally, I would like to thank Stacey Hou for her help during the summer months on the preliminary portions of this experiment.

Works Cited Ismail, M.Z., Hodges, M.D., Boylan, M., Achall, R., Shirras, A. and Broughton, S.J. (2015). The Drosophila insulin receptor independently

modulates lifespan and locomotor senescence. PLoS One 10: e0125312Jevtov, I., et al. (2015). TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules. J Cell Sci

[Epub ahead of print]. PubMed ID: 26054799Ogawa, K., & Miura, T. (2013). Two developmental switch points for the wing polymorphisms in the pea aphid Acyrthosiphon

pisum. EvoDevo, 4, 30. Purandare, S. R., Tenhumberg, B., & Brisson, J. A. (2014). Comparison of the wing polyphenic response of pea aphids (Acyrthosiphon

pisum) to crowding and predator cues. Ecological Entomology, 39(2), 263–266. Slade, J. D. and Staveley, B. E. (2015). Compensatory growth in novel Drosophila Akt1 mutants. BMC Res Notes 8: 77.Wheeler D.E., Buck N.A., & Evans J.D. (2013). Expression of insulin/insulin-like signaling and TOR pathway genes in honeybee caste

determination. Insect Mol. Bio. 23(1):113-21.Xu HJ, Xue J, Lu B, Zhang XC, Zhuo JC, He SF et. al. (2015) Two insulin receptors determine alternative wing morphs in planthoppers. Nature, 519(7544):464-7

Environmental ManipulationsThe results of the first experiment

exhibit that environmental conditions have a significant affect on wing morphs. The main contributing factor is most likely the accessibility of nutrients and resources. Short winged morphs are more common in resource rich environments and might allocate resources away from wing growth in favor of fecundity. In situations where food is not as abundant, the trend is towards more long winged morphs, this trend can be explained by the need to fly in order to relocate in search of more favorable environments. Finally, these results allowed us to more accurately test gene manipulations by controlling for high and low food situations.

InR1/InR2 RNAi

InR 1 InR2 High InR2 Low GFP High GFP Low0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Long Winged Short Winged

Treatment Type

Perc

enta

ge in

d. o

f eac

h m

orph

Figure 3. Percentage of short and long winged morphs in relation to condition in which the bugs were placed. GFP control

treatments showed only a slight trend toward long winged morphs, which was not

significantly different to InR2 low treatment (p=0.16). InR1 knockdown had a marginal affect (p=0.041). Finally, InR2 high treatment was significantly different

(p=.0057)

InR1 and 2 knockdown therapies presented interesting results that were only slightly in keeping with my original hypothesis. Only InR2 high food treatment and InR1 treatment provided any significant difference to the control. This might suggest that the insulin signaling pathway is more robust than previously anticipated, and contains a “back up system” in case of damage to one insulin receptor. A double knock out trial is now beginning to examine the effects of simultaneous down regulation. Furthermore, qPCR samples of exemplary bugs from each morph as well as cDNA have been obtained.

The insulin signaling pathway is integral to the development of wing in J. haematoloma, therefore I chose to study the effects of four specific units within the signaling pathway: InR1, InR2, Chico and Akt. Both of the insulin receptors are on the cell surface and respond to signals from insulin binding. Similarly to J. haematoloma, the migratory brown planthopper, Nilaparvata lugens, also has two insulin receptors that may function

Figure 1. A vast oversimplification of the key elements of the insulin

signaling pathway

Figure 4. (a) GFP, high-food long winged female (b) GFP, low-food short winged male (c) InR1, high-food long winged female (d) InR2, ventral low-food short winged male (e) dorsal view of (d)

Akt/Chico RNAi

Akt High Akt Low Chico High Chico Low GFP High GFP Low 0

2

4

6

8

10

12

14

16

18

20

22

Unmolted Long WingedShort Winged

Treatment type

Num

ber o

f Ind

. of e

ach

mor

ph

Figure 5. A graph showing the number of live individuals one month after injection and their wing morphs. Akt has a majority of unmolted individuals, and the only adult morph is short

winged. Chico treated bugs also have a majority unmolted individuals, with only short winged adults. GFP treatment bugs (same as above)

show only a slight tendency towards long winged morphs, with all live individuals having

reached adulthood. All four treatments (Akt/ Chico, High/Low) showed a statistical

significance in comparison to the control (p<0.0001).

Future/Ongoing StudiesNow that qualitative data have been recorded and analyzed, quantitative data

is needed to asses gene expression of each one of the genes manipulated in the experiments. qPCR will be particularly helpful in analysis of the InR1 and 2 experiments, as it will either support or refute my hypothesis of robustness—if I am correct, there will be an upregulation of the one insulin receptor in the absence of the other.

Figure 6. (a) Chico, L5 high-food female, dorsal (b) ventral view of (a)(c) Dissection of Chico female abdomen, arrows indicate egg sacs & ovaries respectively (d) Akt, L5 high-food, dorsal (e) ventral view of (d)

(a) (b) (c)

(d) (e)

(a) (b) (c)

(d) (e)