Transcriptome profiling of ontogeny in the acridid grasshopper Chorthippus biguttulus.

Acridid grasshoppers (Orthoptera:Acrididae) are widely used model organisms for developmental, evolutionary, and neurobiological research. Although there has been recent influx of orthopteran transcriptomic resources, many use pooled ontogenetic stages obscuring information about changes in gene expression during development. Here we developed a de novo transcriptome spanning 7 stages in the life cycle of the acridid grasshopper Chorthippus biguttulus. Samples from different stages encompassing embryonic development through adults were used for transcriptomic profiling, revealing patterns of differential gene expression that highlight processes in the different life stages. These patterns were validated with semi-quantitative RT-PCR. Embryonic development showed a strongly differentiated expression pattern compared to all of the other stages and genes upregulated in this stage were involved in signaling, cellular differentiation, and organ development. Our study is one of the first to examine gene expression during post-embryonic development in a hemimetabolous insect and we found that only the fourth and fifth instars had clusters of genes upregulated during these stages. These genes are involved in various processes ranging from synthesis of biogenic amines to chitin binding. These observations indicate that post-embryonic ontogeny is not a continuous process and that some instars are differentiated. Finally, genes upregulated in the imago were generally involved in aging and immunity. Our study highlights the importance of looking at ontogeny as a whole and indicates promising directions for future research in orthopteran development.

Exploring the hidden landscape of female preferences for complex signals.

A major challenge in evolutionary biology is explaining the origins of complex phenotypic diversity. In animal communication, complex signals may evolve from simpler signals because novel signal elements exploit preexisting biases in receivers' sensory systems. Investigating the shape of female preference functions for novel signal characteristics is a powerful, but underutilized, method to describe the adaptive landscape potentially guiding complex signal evolution. We measured female preference functions for characteristics of acoustic appendages added to male calling songs in the grasshopper Chorthippus biguttulus, which naturally produces only simple songs. We discovered both hidden preferences for and biases against novel complex songs, and identified rules governing song attractiveness based on multiple characteristics of both the base song and appendage. The appendage's temporal position and duration were especially important: long appendages preceding the song often made songs less attractive, while following appendages were neutral or weakly attractive. Appendages had stronger effects on songs of shorter duration, but did not restore the attractiveness of very unattractive songs. We conclude that sensory biases favor, within predictable limits, the evolution of complex songs in grasshoppers. The function-valued approach is an important tool in determining the generality of these limits in other taxa and signaling modalities.

Divergence of cuticular hydrocarbons in two sympatric grasshopper species and the evolution of fatty acid synthases and elongases across insects.

Cuticular hydrocarbons (CHCs) play a major role in the evolution of reproductive isolation between insect species. The CHC profiles of two closely related sympatric grasshopper species, Chorthippus biguttulus and C. mollis, differ mainly in the position of the first methyl group in major methyl-branched CHCs. The position of methyl branches is determined either by a fatty acid synthase (FAS) or by elongases. Both protein families showed an expansion in insects. Interestingly, the FAS family showed several lineage-specific expansions, especially in insect orders with highly diverse methyl-branched CHC profiles. We found five putative FASs and 12 putative elongases in the reference transcriptomes for both species. A dN/dS test showed no evidence for positive selection acting on FASs and elongases in these grasshoppers. However, one candidate FAS showed species-specific transcriptional differences and may contribute to the shift of the methyl-branch position between the species. In addition, transcript levels of four elongases were expressed differentially between the sexes. Our study indicates that complex methyl-branched CHC profiles are linked to an expansion of FASs genes, but that species differences can also mediated at the transcriptional level.

Chemical cues from females trigger male courtship behaviour in grasshoppers.

Gomphocerine grasshoppers use species-specific calling songs for sex recognition and mate attraction. In two closely related species, Chorthippus biguttulus and C. mollis, acoustic communication is the only experimentally characterized communication channel that elicits male courtship behaviour. However, courtship in these species involves extensive close-range interactions that are likely to be mediated by other signalling modalities, in particular chemical cues. We developed a bioassay to determine if female cuticular hydrocarbons (CHCs) act as chemical cues that induce courtship behaviour, and if males assess variation in CHCs to determine whether or not to court a female. The results of this approach provide evidence that grasshopper males use species- and sex-specific information from CHC signals and respond with a courtship song to the CHC profile of conspecific females but not to the CHC profile of heterospecific females and conspecific males. We conclude that males of C. biguttulus and C. mollis use multimodal channels for mating decisions, based on both acoustic and olfactory cues. We discuss various factors that might favour the evolution of male choosiness in grasshoppers.