Author post: An amino acid polymorphism in the Drosophila insulin receptor demonstrates pleiotropic and adaptive function in life history trait

This next guest post is by Annalise Paaby on her paper: Paaby et al. “An amino acid polymorphism in the Drosophila insulin receptor demonstrates pleiotropic and adaptive function in life history traits” bioRxived here.

Find the alleles!
Organisms vary, even within populations, in ways that appear adaptive. We would very much like to identify the genetic elements that encode these phenotypic differences—but this is a challenging task. For polygenic traits, the tiny contributions of single loci can be near-impossible to detect in an experimental setting. In contrast, natural selection operates on a grand scale, with power to discriminate between alleles. We took advantage of the fact that Drosophila melanogaster are distributed across an extreme environmental gradient in order to identify a specific polymorphism that contributes to adaptive variation.D. melanogaster live along the east coasts of North America and Australia. On both continents, flies in low-latitude, warm environments develop faster and are more fecund, while flies in high-latitude, cold environments live longer and are more resistant to most stresses.Knocking out insulin signaling genes extends lifespan, increases stress tolerance, and reduces reproduction. Given these phenotypes, we wondered whether insulin signaling genes might vary in natural populations and influence life history. In a paper published a few years ago, we showed that alleles of a polymorphism in the Insulin-like Receptor (InR) showed clines in frequency in both North America and Australia. Since the populations were founded at different times from different source populations, the replicated pattern on separate continents is good evidence that the polymorphism is a target of selection.

What is this polymorphism?
The polymorphism we discovered is a complex indel that disrupts a region of glutamines and histidines in the first exon of InR. In our original survey, we found many segregating alleles, all differing in length by multiples of three nucleotides.H owever, two alleles comprise the majority. An allele we call InRshort is common at high latitudes, and InRlong, which is six nucleotides longer, is common at low latitudes. The alleles differ in four amino acids across a span of 16 residues.

The alleles affect signaling
In our current study, we show that InRshort and InRlong affect levels of insulin signaling. We took InRshort and InRlong flies from a single population in New York, replaced the X and second chromosomes, and randomized the genetic backgrounds of the third chromosome, on which InR resides. We measured levels of insulin signaling in test lines by performing qPCR on seven transcriptional targets in the pathway, all downstream of the receptor.We found that for five of the seven targets (four of which were significant), signaling was highest in InRlong, lowest in InRshort, and intermediate in the heterozygote—suggesting that InRshort and InRlong act additively on signaling levels. The directionality of these results makes sense: reduction of insulin signaling is known to extend lifespan, increase stress tolerance and reduce reproductive success, and these are the phenotypes we see at high latitudes where InRshort is common.

Fluctuations over time
In our new study, we returned to the North American populations we evaluated five years prior. However, this time around we mapped 100-bp paired-end reads from pooled population samples. (These data relate to Alan Bergland’s larger exploration of spatial and temporal variation in D. melanogaster, described here on arXiv.) We called each of the discrete polymorphisms within the complex indel polymorphism—SNPs or small indels—individually. Some of those discrete polymorphisms distinguish between the InRshort and InRlong alleles, and they confirm that the clines persist in North America.We reasoned that alleles prevalent in high-latitude, cold climates might be selected for in the winter, and alleles prevalent in low-latitude, warm climates might be selected for in the summer. We examined a Pennsylvania population at multiple timepoints over three years and saw dramatic fluctuations in allele frequency (changes of approximately 20%) for discrete polymorphisms associated with InRshort and InRlong. As predicted, the “winter” and “summer” alleles were those common at high and low latitudes, respectively.However, the polymorphisms that showed the most dramatic fluctuations over seasonal time were not necessarily those with the strongest clines in frequency across geographical space. We suggest that aspects of demography and selection probably vary between seasonal and geographical environments, even in the face of apparently similar climatic pressures.

A question of pleiotropy
A longstanding question in the field of life history evolution is whether single alleles affect multiple traits at once (pleiotropy) or affect traits individually but reside near each other (linkage). The question itself arises from the observation, made many times over, that life history traits are typically correlated. For example, long-lived individuals often show reduced reproductive fitness. Longevity is also often positively correlated with the ability to tolerate stress. Do the same genetic variants encode multiple trait phenotypes?We assayed our InRshort and InRlong test lines for multiple phenotypes: fecundity, development time, body size and allometry, body weight and lipid content, tolerance for multiple stresses, and lifespan. We used the test lines described above, a replicate set of InRshort and InRlong lines derived from a second population, and lines in which we measured the effects of InRshort and InRlong in an InRhypomorph mutant background.Our full report can be found in the manuscript, but the take-home message is that InRshort and InRlong are significantly associated with all of the tested traits, in directions predicted by a selection regime favoring fast development time, rapid egg-laying, and high heat tolerance in warm climates, and resistance to cold and starvation stresses in cold climates. The InRshort allele was also associated with increased lifespan in males, though we do not necessarily expect that lifespan itself is associated with fitness.In conclusion, our results implicate insulin signaling as a major mediator of life history adaptation in D. melanogaster, and suggest that tradeoffs can be explained by extensive pleiotropy at a single locus.

Some other things I would like to mention
I value this study for its functional tests—phenotypic effects of candidate polymorphisms are often missing from evolutionary studies. However, and this is a major caveat: the InRshort and InRlong alleles were embedded in genotypic backgrounds that extended well beyond the locus in the test lines. On their own, I do not consider the functional tests definitive. But D. melanogaster have low linkage disequilibrium, which we know decays rapidly just outside our candidate polymorphism. In my opinion, the segregation of InRshort and InRlong in large, recombining wild populations pinpoints the functional alleles, while the experimental assays confirm our hypotheses about the selection regime.When we first measured fecundity, we counted every single egg laid by every single female over every single one of their lives. And the InRlong females, which we knew were more fecund—their culture bottles grew like gangbusters—laid only five more eggs on average than InRshort females! Highly non-significant. But, it looked like the InRlong flies laid eggs faster. We set up a different assay to measure eggs laid in the first day, and InRlong was six times more fecund. I think this provides an important lesson. We can easily imagine big fitness consequences for egg laying rate, but we might not think to measure it in the lab. Many studies, especially those from a molecular genetics point of view, have been keen to emphasize decoupling of lifespan and reproduction for so-called longevity genes. For conclusions drawn about natural genetic variants (which are the ones of utmost relevance, in my opinion), the question of tradeoffs must consider those fitness axes that are relevant to the wild organism. And these are often unknowable.We found that InRshort and InRlong were associated with smaller and larger body sizes, respectively. This makes sense in terms of levels of insulin signaling, but not in terms of body sizes in wild populations. High latitude flies are typically larger, not smaller. So, if InRshort and InRlong alleles affect body size, they either do so epistatically with other body size loci or they suffer antagonistic selection pressures along multiple fitness axes. Interesting!

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