Asano Ishikawa

Gene up-regulation in response to predator kairomones in the water flea, Daphnia pulex

BackgroundNumerous cases of predator-induced polyphenisms, in which alternate phenotypes are produced in response to extrinsic stimuli, have been reported in aquatic taxa to date. The genus Daphnia (Branchiopoda, Cladocera) provides a model experimental system for the study of the developmental mechanisms and evolutionary processes associated with predator-induced polyphenisms. In D. pulex, juveniles form neckteeth in response to predatory kairomones released by Chaoborus larvae (Insecta, Diptera).ResultsPrevious studies suggest that the timing of the sensitivity to kairomones in D. pulex can generally be divided into the embryonic and postembryonic developmental periods. We therefore examined which of the genes in the embryonic and first-instar juvenile stages exhibit different expression levels in the presence or absence of predator kairomones. Employing a candidate gene approach and identifying differentially-expressed genes revealed that the morphogenetic factors, Hox3, extradenticle and escargot, were up-regulated by kairomones in the postembryonic stage and may potentially be responsible for defense morph formation. In addition, the juvenile hormone pathway genes, JHAMT and Met, and the insulin signaling pathway genes, InR and IRS-1, were up-regulated in the first-instar stage. It is well known that these hormonal pathways are involved in physiological regulation following morphogenesis in many insect species. During the embryonic stage when morphotypes were determined, one of the novel genes identified by differential display was up-regulated, suggesting that this gene may be related to morphotype determination. Biological functions of the up-regulated genes are discussed in the context of defense morph formation.ConclusionsIt is suggested that, following the reception of kairomone signals, the identified genes are involved in a series of defensive phenotypic alterations and the production of a defensive phenotype.

Developmental Link between Sex and Nutrition; doublesex Regulates Sex-Specific Mandible Growth via Juvenile Hormone Signaling in Stag Beetles

Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.

Wing development genes of the pea aphid and differential gene expression between winged and unwinged morphs.

Little is known about when, how or even if the wing development gene network elucidated in Drosophila is deployed in direct‐developing insects. Here we identify the wing development genes (as determined in Drosophila) of the pea aphid (Acyrthosiphon pisum), which produces winged or unwinged adults in response to environmental cues. We find that the principal wing development genes studied in Drosophila are present in the aphid genome and that apterous and decapentaplegic exhibit duplications. We followed expression levels of 11 of these developmental genes at embryogenesis and across the nymphal instars. Six showed significant stage‐specific expression level effects and apterous1 exhibited significantly different expression levels between winged and unwinged morphs, suggesting this gene acts proximately to realize polyphenic development.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive‐mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive‐mode change. Using liquid chromatography‐mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short‐day conditions than in aphids producing parthenogenetic morphs under long‐day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real‐time quantitative reverse‐transcription‐PCR across the reproductive‐mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short‐day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short‐days, leading to the production of sexual morphs.

Morphological and histological examination of polyphenic wing formation in the pea aphid Acyrthosiphon pisum (Hemiptera, Hexapoda)

Aphids display divergent adult phenotypes, depending on environmental conditions experienced during their embyonic and nymphal stages in their complex life cycles. The plastic developmental mode is an extreme case of phenotypic plasticity, so-called “polyphenism”, in which discrete multiple phenotypes are produced based on a single genome. For example, winged and wingless adult females are derived from a single genotype. However, the developmental mechanisms producing these polyphenic traits according to the extrinsic stimuli, such as density conditions, still remain unknown. In this study, to analyze the developmental processes underlying the wing polyphenism, we extensively observed and compared wing development in the winged and wingless individuals in parthenogenetic generations of the aphid Acyrthosiphon pisum (Harris), using scanning electron microscopy and histological sectioning. At the first-instar stage, the wing primordia were observed both in the future winged (W) and wingless (WL) nymphs. Developmental differences can be seen from the second-instar stage, when wing primordia degenerate in the WL nymphs, while they develop and become more thickened in the W nymphs, suggesting that the developmental programs should be launched prior to this stage. Furthermore, during the third- to fifth-instar stages, wing buds and flight muscles were well developed in the W nymphs, while wing primordia completely disappeared in the WL ones. In addition, the observation on the detailed developmental process of wing primordia during the third-instar W nymphs showed that the wing buds become swollen especially at the basal part, even during the intermolt period. This was caused by the development of wing epithelia under the cuticle of this instar nymph. Actually on the surface of the cuticle of wing-bud bases, there were numerous furrows, which gradually expand during the intermolt period. The similar situation was also observed at the forth-instar nymphs, in which the wings are formed in the complicated manner inside the wing pads. Furthermore, the developmental process of flight muscles was also described in detail. These dynamic developmental differences between the wing morphs should be regulated under the gene expression cascades that switch according to environmental stimuli.

Physiological and genetic basis for variation in migratory behavior in the three-spined stickleback, Gasterosteus aculeatus

Closely related species of fish often exhibit different migration patterns. Even within species, anadromous and resident populations can be found in a diverse number of taxa. Although several environmental factors that regulate behavioral and physiological changes associated with fish migration have been identified, the genetic mechanisms underlying the variation in the ability to respond to these environmental cues in fishes that show different migratory behaviors are not well known. The three-spined stickleback Gasterosteus aculeatus (Linnaeus 1758) is a good model system for elucidation of the genetic basis for variation in migratory behaviors and other physiological changes associated with migration. First, the three-spined stickleback exhibits great inter-population variation in migration patterns. Second, genetic and genomic tools are now available for studying this species. In the present study, variation in the migration patterns among G. aculeatus populations and the recent progress in our understanding of the genetic and physiological basis for variation in traits important for G. aculeatus migration are reviewed.

Genetic basis for variation in salinity tolerance between stickleback ecotypes.

Adaptation to different salinities can drive and maintain divergence between populations of aquatic organisms. Anadromous and stream ecotypes of threespine stickleback (Gasterosteus aculeatus) are an excellent model to explore the genetic mechanisms underlying osmoregulation divergence. Using a parapatric pair of anadromous and stream stickleback ecotypes, we employed an integrated genomic approach to identify candidate genes important for adaptation to different salinity environments. Quantitative trait loci (QTL) mapping of plasma sodium concentrations under a seawater challenge experiment identified a significant QTL on chromosome 16. To identify candidate genes within this QTL, we first conducted RNA‐seq and microarray analysis on gill tissue to find ecotypic differences in gene expression that were associated with plasma Na+ levels. This resulted in the identification of ten candidate genes. Quantitative PCR analysis on gill tissue of additional Japanese stickleback populations revealed that the majority of the candidate genes showed parallel divergence in expression levels. Second, we conducted whole‐genome sequencing and found five genes that are predicted to have functionally important amino acid substitutions. Finally, we conducted genome scan analysis and found that eight of these candidate genes were located in genomic islands of high differentiation, suggesting that they may be under divergent selection. The candidate genes included those involved in ATP synthesis and hormonal signalling, whose expression or amino acid changes may underlie the variation in salinity tolerance. Further functional molecular analysis of these genes will reveal the causative genetic and genomic changes underlying divergent adaptation.

Differential regulations of wing and ovarian development and heterochronic changes of embryogenesis between morphs in wing polyphenism of the vetch aphid.

SUMMARY In wing polyphenisms that produced alternative wing morphs depending on environmental conditions, the developmental regulations to balance between flight and reproductive abilities should be important. Many species of aphids exhibit wing polyphenisms, and the development of wing and flight muscles is thought to incur costs of reproductive ability. To evaluate the relationship between flight and reproduction, the fecundity and the wing‐ and ovarian development in the parthenogenetic generations were compared between winged and wingless aphids in the vetch aphid Megoura crassicauda. Although no differences in offspring number and size were detected, the onset of larviposition after imaginal molt was delayed in winged adults. The comparison of growth in flight apparatus revealed that, after the second‐instar nymphs, the flight‐apparatus primordia of presumptive wingless aphids were degenerated while those of winged nymphs rapidly developed. In the ovaries of winged line, the embryo size was smaller and the embryonic stages were delayed from third to fifth instars, although these differences had disappeared by the time of larviposition. It is therefore likely that the delay in larviposition in winged aphids is due to the slower embryonic development. The correlation between embryo size and developmental stage suggests that the embryos of winged aphids are better developed than similarly sized embryos in wingless aphids. These heterochronic shifts would facilitate the rapid onset of larviposition after the dispersal flight. This developmental regulation of embryogenesis in the aphid wing polyphenism is suggested to be an adaptation that compensates the delay of reproduction caused by the wing development.

Speciation in ninespine stickleback: reproductive isolation and phenotypic divergence among cryptic species of Japanese ninespine stickleback

Although similar patterns of phenotypic diversification are often observed in phylogenetically independent lineages, differences in the magnitude and direction of phenotypic divergence have been also observed among independent lineages, even when exposed to the same ecological gradients. The stickleback family is a good model with which to explore the ecological and genetic basis of parallel and nonparallel patterns of phenotypic evolution, because there are a variety of populations and species that are locally adapted to divergent environments. Although the patterns of phenotypic divergence as well as the genetic and ecological mechanisms have been well characterized in threespine sticklebacks, Gasterosteus aculeatus, we know little about the patterns of phenotypic diversification in other stickleback lineages. In eastern Hokkaido, Japan, there are three species of ninespine sticklebacks, Pungitius tymensis and the freshwater type and the brackish‐water type of the P. pungitius–P. sinensis species complex. They utilize divergent habitats along coast–stream gradients of rivers. Here, we investigated genetic, ecological and phenotypic divergence among three species of Japanese ninespine sticklebacks. Divergence in trophic morphology and salinity tolerance occurred in the direction predicted by the patterns observed in threespine sticklebacks. However, the patterns of divergence in armour plate were different from those previously found in threespine sticklebacks. Furthermore, the genetic basis of plate variation may differ from that in threespine sticklebacks. Because threespine sticklebacks are well‐established model for evolutionary research, the sympatric trio of ninespine sticklebacks will be an invaluable resource for ecological and genetic studies on both common and lineage‐specific patterns of phenotypic diversification.

Gene expression changes during caste-specific neuronal development in the damp-wood termite Hodotermopsis sjostedti

BackgroundOne of the key characters of social insects is the division of labor, in which different tasks are allocated to various castes. In termites, one of the representative groups of social insects, morphological differences as well as behavioral differences can be recognized among castes. However, very little is known about the neuronal and molecular bases of caste differentiation and caste-specific behavior. In almost all termite species, soldiers play defensive roles in their colonies, and their morphology and behavior are largely different from workers (or pseudergates). Therefore, we predicted that some genes linked to defensive behavior and/or those required for neuronal changes are differentially expressed between workers and soldiers, or during the soldier differentiation, respectively.ResultsUsing the brain and suboesophageal ganglion (SOG) of the damp-wood termite Hodotermopsis sjostedti, we first screened genes specifically expressed in soldiers or during soldier differentiation by the differential display method, followed by quantitative real-time polymerase chain reaction. No distinctive differences in expression patterns were detected between pseudergates and soldiers. In the course of soldier differentiation, however, five genes were found to be up-regulated in brain and/or SOG: 14-3-3epsilon, fibrillin2, beta-tubulin, ciboulot, and a hypothetical protein containing a SAP motif. Some of these genes are thought to be associated with cytoskeletal structure or motor-associated proteins in neuronal tissues.ConclusionThe identified five genes could be involved in soldier-specific neuronal modifications, resulting in defensive behaviors in termite soldiers. The temporal expression patterns of these genes were consistent with the neuronal changes during soldier differentiation, suggesting that molecular machineries, in which the identified factors would participate, play important roles in behavioral differentiation of termite soldiers.