Makoto Kusakabe

Adaptive Divergence in the Thyroid Hormone Signaling Pathway in the Stickleback Radiation

During adaptive radiations, animals colonize diverse environments, which requires adaptation in multiple phenotypic traits. Because hormones mediate the dynamic regulation of suites of phenotypic traits, evolutionary changes in hormonal signaling pathways might contribute to adaptation to new environments. Here we report changes in the thyroid hormone signaling pathway in stream-resident ecotypes of threespine stickleback fish (Gasterosteus aculeatus), which have repeatedly evolved from ancestral marine ecotypes. Stream-resident fish exhibit a lower plasma concentration of thyroid hormone and a lower metabolic rate, which is likely adaptive for permanent residency in small streams. The thyroid-stimulating hormone-β2 (TSHβ2) gene exhibited significantly lower mRNA expression in pituitary glands of stream-resident sticklebacks relative to marine sticklebacks. Some of the difference in TSHβ2 transcript levels can be explained by cis-regulatory differences at the TSHβ2 gene locus. Consistent with these expression differences, a strong signature of divergent natural selection was found at the TSHβ2 genomic locus. By contrast, there were no differences between the marine and stream-resident ecotypes in mRNA levels or genomic sequence in the paralogous TSHβ1 gene. Our data indicate that evolutionary changes in hormonal signaling have played an important role in the postglacial adaptive radiation of sticklebacks.

A snapshot of a coral "holobiont": a transcriptome assembly of the scleractinian coral, porites, captures a wide variety of genes from both the host and symbiotic zooxanthellae.

Massive scleractinian corals of the genus Porites are important reef builders in the Indo-Pacific, and they are more resistant to thermal stress than other stony corals, such as the genus Acropora. Because coral health and survival largely depend on the interaction between a coral host and its symbionts, it is important to understand the molecular interactions of an entire “coral holobiont”. We simultaneously sequenced transcriptomes of Porites australiensis and its symbionts using the Illumina Hiseq2000 platform. We obtained 14.3 Gbp of sequencing data and assembled it into 74,997 contigs (average: 1,263 bp, N50 size: 2,037 bp). We successfully distinguished contigs originating from the host (Porites) and the symbiont (Symbiodinium) by aligning nucleotide sequences with the decoded Acropora digitifera and Symbiodinium minutum genomes. In contrast to previous coral transcriptome studies, at least 35% of the sequences were found to have originated from the symbionts, indicating that it is possible to analyze both host and symbiont transcriptomes simultaneously. Conserved protein domain and KEGG analyses showed that the dataset contains broad gene repertoires of both Porites and Symbiodinium. Effective utilization of sequence reads revealed that the polymorphism rate in P. australiensis is 1.0% and identified the major symbiotic Symbiodinium as Type C15. Analyses of amino acid biosynthetic pathways suggested that this Porites holobiont is probably able to synthesize most of the common amino acids and that Symbiodinium is potentially able to provide essential amino acids to its host. We believe this to be the first molecular evidence of complementarity in amino acid metabolism between coral hosts and their symbionts. We successfully assembled genes originating from both the host coral and the symbiotic Symbiodinium to create a snapshot of the coral holobiont transcriptome. This dataset will facilitate a deeper understanding of molecular mechanisms of coral symbioses and stress responses.

Differential suppressive effects of low physiological doses of estradiol-17β in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in previtellogenic ovarian follicles of rainbow trout.

Numerous recent reports have demonstrated effects of estrogenic chemicals on reproductive physiology of fish. However, there is little information available on the regulation of ovarian steroidogenesis by physiological levels of endogenous steroids in teleosts. Therefore, we analyzed the levels of mRNAs encoding steroidogenic proteins in ovaries of E2-treated rainbow trout Oncorhynchus mykiss). Previtellogenic (perinucleolar oocyte stage) trout received either blank or E2 implants (0.1 microg, 1 microg or 10 microg/g BW) for 7 days in order to achieve low, medium and high physiological levels of E2 in plasma. Plasma E2 levels were measured using radioimmunoassay. Levels of mRNAs encoding steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450 aromatase A (P450aromA) in the ovary were analyzed by real-time quantitative PCR. E2 levels in control animals were approximately 0.5 ng/ml. Levels in treated fish were approximately 1 ng/ml (0.1 microg implant), 2.6 ng/ml (1 microg implant) and 90 ng/ml (10 microg implant), within or just above the physiological range of immature and maturing female rainbow trout. StAR mRNA levels were significantly reduced by all E2 treatments. P450scc mRNA levels were not affected, but 3beta-HSD and P450arom mRNA levels were significantly decreased by the 1 and 10 microg E2/BW implants. These results indicate that E2, either directly or indirectly, downregulates expression of StAR and major steroidogenic enzyme genes in rainbow trout ovary. Furthermore, expression of the trout StAR gene seems particularly sensitive to E2.

Divergence in Sex Steroid Hormone Signaling between Sympatric Species of Japanese Threespine Stickleback

Sex steroids mediate the expression of sexually dimorphic or sex-specific traits that are important both for mate choice within species and for behavioral isolation between species. We investigated divergence in sex steroid signaling between two sympatric species of threespine stickleback (Gasterosteus aculeatus): the Japan Sea form and the Pacific Ocean form. These sympatric forms diverge in both male display traits and female mate choice behaviors, which together contribute to asymmetric behavioral isolation in sympatry. Here, we found that plasma levels of testosterone and 17β-estradiol differed between spawning females of the two sympatric forms. Transcript levels of follicle-stimulating hormone-β (FSHβ) gene were also higher in the pituitary gland of spawning Japan Sea females than in the pituitary gland of spawning Pacific Ocean females. By contrast, none of the sex steroids examined were significantly different between nesting males of the two forms. However, combining the plasma sex steroid data with testis transcriptome data suggested that the efficiency of the conversion of testosterone into 11-ketotestosterone has likely diverged between forms. Within forms, plasma testosterone levels in males were significantly correlated with male body size, a trait important for female mate choice in the two sympatric species. These results demonstrate that substantial divergence in sex steroid signaling can occur between incipient sympatric species. We suggest that investigation of the genetic and ecological mechanisms underlying divergence in hormonal signaling between incipient sympatric species will provide a better understanding of the mechanisms of speciation in animals.

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.

Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

Unilateral ovariectomy increases egg size and reduces follicular atresia in the semelparous coho salmon, Oncorhynchus kisutch.

Unilateral ovariectomy (ULO, removal of one ovary) is a powerful technique for studying aspects of reproductive physiology, including follicular recruitment and growth. To examine effects of ULO for the first time in a semelparous species, coho salmon (Oncorhynchus kisutch) were unilaterally ovariectomized during mid-vitellogenesis approximately 3 months before spawning. At termination of the study (79 days post-surgery), single ovaries of ULO fish were gravimetrically equivalent to paired ovaries of sham surgery, control fish. There was no evidence of recruitment of new vitellogenic follicles. Instead, the dramatic increase in ovary mass was attributable to hypertrophy of existing vitellogenic follicles (33% increase in volume) and increased fecundity achieved through a greater than two-fold reduction in follicular atresia. The composition of whole ovaries on a dry weight basis from ULO fish was greater in protein, but lower in lipid than that of control fish. Expressing the data on a per follicle basis, however, showed that follicles of ULO fish contained more protein, ash, water, and lipid. The results indicate that ULO of coho salmon induces compensatory hypertrophy of existing vitellogenic follicles, while maximizing fecundity through reduction of atresia. Thus, 3 months before spawning, coho salmon exhibit the ability to adjust final egg size and number when faced with significant depletion of ovarian follicles. This in vivo system provides a platform for further study of physiological mechanisms regulating follicular growth and atresia, and the trade-off between egg size and egg number.

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.

Different contributions of local‐ and distant‐regulatory changes to transcriptome divergence between stickleback ecotypes

Differential gene expression can play an important role in phenotypic evolution and divergent adaptation. Although differential gene expression can be caused by both local‐ and distant‐regulatory changes, we know little about their relative contribution to transcriptome evolution in natural populations. Here, we conducted expression quantitative trait loci (eQTL) analysis to investigate the genetic architecture underlying transcriptome divergence between marine and stream ecotypes of threespine sticklebacks (Gasterosteus aculeatus). We identified both local and distant eQTLs, some of which constitute hotspots, regions with a disproportionate number of significant eQTLs relative to the genomic background. The majority of local eQTLs including those in the hotspots caused expression changes consistent with the direction of transcriptomic divergence between ecotypes. Genome scan analysis showed that many local eQTLs overlapped with genomic regions of high differentiation. In contrast, nearly half of the distant eQTLs including those in the hotspots caused opposite expression changes, and few overlapped with regions of high differentiation, indicating that distant eQTLs may act as a constraint of transcriptome evolution. Finally, a comparison between two salinity conditions revealed that nearly half of eQTL hotspots were environment specific, suggesting that analysis of genetic architecture in multiple conditions is essential for predicting response to selection.

Morphological and functional characterization of a novel Na + /K + -ATPase-immunoreactive, follicle-like structure on the gill septum of Japanese banded houndshark, Triakis scyllium

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na+/K+-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na+/H+ exchanger 3 and Ca2+ transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.