Sex-determining Gene Research Articles

Gonadal transcriptomic analysis and differentially expressed genes between the testes and ovaries in Trachinotus ovatus

The Trachinotus ovatus is a popular aquaculture species in China. There are no obvious morphological differences between male and female fish, even during maturity, prompting research studies on sex-related features of this fish. To examine sex determination- and gonadal development-related genes, we conducted transcriptome analysis of the ovaries and testes of T. ovatus. A total of 345,972,132 high-quality clean reads were obtained from 12 libraries. In addition, 28,137 gonad-expressed unigenes were obtained by mapping the clean reads to the T. ovatus reference genome. A total of 8,237 differentially expressed genes (DEGs) were identified between stage I ovaries and testes, including 3,235 testicular upregulated and 5,002 ovarian upregulated genes. Furthermore, 13,448 DEGs were obtained between stage III ovarian and testicular libraries, including 7,576 testicular upregulated and 5,872 ovarian upregulated genes. The DEGs included some sex-determining genes such as sry, dmrt1, and amh. DEGs between ovarian and testicular libraries were significantly enriched with KEGG pathways that are involved in gonadal development, sex determination, and gonadal function. Then, 10 DEGs (seven testicular upregulated and three ovarian upregulated unigenes) were selected for quantitative real-time PCR analysis. Four genes (zinc-binding protein A33-like, pro-opiomelanocortin-like, leucine-rich repeat and transmembrane domain-containing protein 2-like, and forkhead boxC1) showed a specific testicular expression pattern. The gonadally expressed as well as testicular and ovarian DEGs provide useful information for further research on the reproductive biology of T. ovatus.

Autosomal sdY Pseudogenes Explain Discordances Between Phenotypic Sex and DNA Marker for Sex Identification in Atlantic Salmon

Despite the key role that sex-determination plays in evolutionary processes, it is still poorly understood in many species. In salmonids, which are among the best studied fishes, the master sex-determining gene sexually dimorphic on the Y-chromosome (sdY) has been identified. However, sdY displays unexplained discordance to the phenotypic sex, with a variable frequency of phenotypic females being reported as genetic males. Multiple sex determining loci in Atlantic salmon have also been reported, possibly as a result of recent transposition events in this species. We hypothesized the existence of an autosomal copy of sdY, causing apparent discordance between phenotypic and genetic sex, that is transmitted in accordance with autosomal inheritance. To test this, we developed a qPCR methodology to detect the total number of sdY copies present in the genome. Based on the observed phenotype/genotype frequencies and linkage analysis among 2,025 offspring from 64 pedigree-controlled families of accurately phenotyped Atlantic salmon, we identified both males and females carrying one or two autosomal copies of sdY in addition to the Y-specific copy present in males. Patterns across families were highly consistent with autosomal inheritance. These autosomal sdY copies appear to have lost the ability to function as a sex determining gene and were only occasionally assigned to the actual sex chromosome in any of the affected families.

Genomes of the Banyan Tree and Pollinator Wasp Provide Insights into Fig-Wasp Coevolution

Banyan trees are distinguished by their extraordinary aerial roots. The Ficus genus includes species that have evolved a species-specific mutualism system with wasp pollinators. We sequenced genomes of the Chinese banyan tree, F.microcarpa, and a species lacking aerial roots, F.hispida, and one wasp genome coevolving with F.microcarpa, Eupristina verticillata. Comparative analysis of the two Ficus genomes revealed dynamic karyotype variation associated with adaptive evolution. Copy number expansion of auxin-related genes from duplications and elevated auxin production are associated with aerial root development in F.microcarpa. A male-specific AGAMOUS paralog, FhAG2, was identified as a candidate gene for sex determination in F.hispida. Population genomic analyses of Ficus species revealed genomic signatures of morphological and physiological coadaptation with their pollinators involving terpenoid- and benzenoid-derived compounds. These three genomes offer insights into and genomic resources for investigating the geneses of aerial roots, monoecy and dioecy, and codiversification in a symbiotic system.

Two rather than one

Sex Determination For several decades, it has been believed that the mammalian sex-determining gene Sry contains a single exon. Miyawaki et al. have now identified a cryptic second exon of mouse Sry . Loss- and gain-of-function analyses revealed that the two-exon SRY (SRY-T), not the canonical single exon–encoded SRY (SRY-S), is the bona fide testis-determining factor. Sry exon2 is composed of retrotransposon-derived sequences. The SRY-S carboxyl terminus contains a degradation sequence (degron), whereas the SRY-T carboxyl terminus encoded in the Sry exon2 is degron free, thereby conferring protein stability on SRY-T. Science , this issue p. [121][1] [1]: /lookup/doi/10.1126/science.abb6430

Comparative proteomic study on fem-1b in female and male gonads in Hyriopsis cumingii

Sex regulation of bivalves is a complex process, and there has also been little research on their sex-determining genes. To better understand the sex determination/differentiation of bivalves, we analyzed the gonadal proteomics of Hyriopsis cumingii. We identified 673 peptides and 76 proteins. The 35 differentially expressed proteins identified included 19 upregulated and 16 downregulated proteins. FEM-1B protein associated with sex determination showed female-specific expression; the function of fem-1b gene was explored. The full-length cDNA of fem-1b cloned by RACE was 2105 bp. Quantitative PCR (qPCR) was performed on male and female tissues and early developmental stages; the results showed that fem-1b was expressed in both male and female tissues, and its expression level in gonads was significantly higher in females than in males (P < 0.01). In early embryonic development, the fem-1b mRNA expression increased significantly on the third day after fertilization (cleavage stage). Results of in situ hybridization showed a strong signal on oocyte membranes and no signal in male gonads. Our results indicate that fem-1b plays a certain role in sex determination and oocyte development of H. cumingii. This study provides valuable resources for understanding the mechanism and pathway of sex determination/differentiation of H. cumingii.

The genomes of a monogenic fly: views of primitive sex chromosomes

The production of male and female offspring is often determined by the presence of specific sex chromosomes which control sex-specific expression, and sex chromosomes evolve through reduced recombination and specialized gene content. Here we present the genomes of Chrysomya rufifacies, a monogenic blow fly (females produce female or male offspring, exclusively) by separately sequencing and assembling each type of female and the male. The genomes (> 25X coverage) do not appear to have any sex-linked Muller F elements (typical for many Diptera) and exhibit little differentiation between groups supporting the morphological assessments of C. rufifacies homomorphic chromosomes. Males in this species are associated with a unimodal coverage distribution while females exhibit bimodal coverage distributions, suggesting a potential difference in genomic architecture. The presence of the individual-sex draft genomes herein provides new clues regarding the origination and evolution of the diverse sex-determining mechanisms observed within Diptera. Additional genomic analysis of sex chromosomes and sex-determining genes of other blow flies will allow a refined evolutionary understanding of how flies with a typical X/Y heterogametic amphogeny (male and female offspring in similar ratios) sex determination systems evolved into one with a dominant factor that results in single sex progeny in a chromosomally monomorphic system.

Donor Cell Fate in Particulated Juvenile Allograft Cartilage for the Repair of Articular Cartilage Defects

Background: Particulated juvenile allograft cartilage (PJAC) has demonstrated good clinical efficacy in repairing articular cartilage defects, but the related repair mechanism after transplant and the biological characteristics of the transplanted cells are still unclear. Purpose: To study the efficacy of PJAC in repairing full-thickness cartilage defects and the specific fate of donor cells to provide experimental evidence for its clinical application. Study Design: Controlled laboratory study. Methods: Twenty female Guizhou minipigs were randomly divided into an experimental group and a control group. An 8-mm cylindrical full-thickness cartilage defect was created in the femoral trochlea of 1 knee in all minipigs. The experimental group received transplant of PJAC from 5 male juvenile Guizhou minipigs (PJAC group; n = 10) and the control group received autologous cartilage chips (ACC group; n = 10). Follow-up assessments were conducted at 1 month and 3 months to track the transplanted cells by the male-specific sex-determining region Y-linked (SRY) gene; tissue sections were hybridized in situ, and O’Driscoll histological scoring was performed according to hematoxylin and eosin staining, safranin O and fast green staining, and toluidine blue O staining, as well as immunohistochemical evaluation of aggrecan and Sry-type HMG-box 9 (SOX9). Results: All 20 Guizhou minipigs were followed; no infection or incision healing disorder occurred after the operation. By SRY in situ hybridization, the SRY signal of the transplanted cells was positive in the repaired tissue of the defect, and the SRY positive signal could still be detected in repaired tissue at 3 months postoperatively. The average number of positive cells was 68.6 ± 11.91 at 1 month and 32.6 ± 3.03 at 3 months (confocal microscope: ×400), and the difference was statistically significant. The O’Driscoll histological scores were 14 ± 0.71 in the ACC group and 9.8 ± 0.84 in the PJAC group at 1 month, and 18 ± 1.20 in the ACC group and 17.4 ± 1.14 in the PJAC group at 3 months. The scores were statistically significant between the ACC group and PJAC group at 1 month. The positive rates of SOX9 in the PJAC and ACC groups at 1 month were 67.6% ± 3.78% and 63.4% ± 5.30%, respectively, and the difference was not statistically significant (P > .05). The positive rates of SOX9 in the PJAC and ACC groups at 3 months were 68.8% ± 2.69% and 17.1% ± 1.26%, respectively, and the difference was statistically significant (P < .05). The positive rates of aggrecan in the PJAC and ACC groups at 1 month were 40.5% ± 2.78% and 42.4% ± 0.54% respectively, and the difference was not statistically significant (P > .05). The positive rates of aggrecan in the PJAC and ACC groups at 3 months were 40.8% ± 1.50% and 30.1% ± 2.44%, respectively, and the difference was not statistically significant (P > .05). Conclusion: An animal model was established with Guizhou minipigs, and the cartilage defect was repaired with PJAC from male minipigs. The SRY gene positive signal could be detected from the repaired tissue by in situ hybridization, indicating that the transplanted cells survived at least 3 months. The key genes of cartilage formation, SOX9 and aggrecan, were expressed at 1 month and 3 months, and SOX9 expression was stronger in the PJAC group than the ACC group at 3 months. Clinical Relevance: This study suggests that it is feasible to study the biological characteristics of transplanted cells in the cartilage region by the sex-determining gene.

Construction of high-density genetic maps defined sex determination region of the Y chromosome in spinach

Spinach (Spinacia olracea L.) is a dioecious leafy vegetable with a highly repetitive genome of around 990Mb, which is challenging for de-novo genome assembly. In our study, a segregating F1 (double pseudo-testcross) population from 'Viroflay' × 'Cornell-NO. 9' was used for genetic mapping by resequencing genotyping. In the paternal 'Cornell-NO. 9' map, 212,414 SNPs were mapped, and the total linkage distance was 476.83cM; the maternal 'Viroflay' map included 29,282 SNPs with 401.28cM total genetic distance. Both paternal and maternal maps have the expected number of six linkage groups (LGs). A non-recombining region with 5678 SNPs (39 bin markers) co-segregates with sex type which located at 45.2cM of LG1 in the 'Cornell-NO. 9' map while indicates the sex determination region (SDR). Integration of two maps into a consensus map guided us to anchor additional 1242 contigs to six pseudomolecules from the published reference genome, which improved additional 233Mb (23.4%) assembly based on spinach estimated genome size. Particularly, the X counterpart of SDR in our assembly is estimated around 18.4Mb which locates at the largest chromosome, as consensus with sex-biased FISH signals from previous cytogenetics studies. The region is featured by reduced gene density, higher percentage of repetitive sequences, and no recombination. Our linkage maps provide the resource for improving spinach genome de-novo assembly and identification of sex-determining genes in spinach.

Reconstruction of the birth of a male sex chromosome present in Atlantic herring

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an ∼500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination.

Transcriptomic analysis reveals ethylene signal transduction genes involved in pistil development of pumpkin.

Development of female flowers is an important process that directly affects the yield of Cucubits. Little information is available on the sex determination and development of female flowers in pumpkin, a typical monoecious plant. In the present study, we used aborted and normal pistils of pumpkin for RNA-Seq analysis and determined the differentially expressed genes (DEGs) to gain insights into the molecular mechanism underlying pistil development in pumpkin. A total of 3,817 DEGs were identified, among which 1,341 were upregulated and 2,476 were downregulated. The results of transcriptome analysis were confirmed by real-time quantitative RT-PCR. KEGG enrichment analysis showed that the DEGs were significantly enriched in plant hormone signal transduction and phenylpropanoid biosynthesis pathway. Eighty-four DEGs were enriched in the plant hormone signal transduction pathway, which accounted for 12.54% of the significant DEGs, and most of them were annotated as predicted ethylene responsive or insensitive transcription factor genes. Furthermore, the expression levels of four ethylene signal transduction genes in different flower structures (female calyx, pistil, male calyx, stamen, leaf, and ovary) were investigated. The ethyleneresponsive DNA binding factor, ERDBF3, and ethylene responsive transcription factor, ERTF10, showed the highest expression in pistils and the lowest expression in stamens, and their expression levels were 78- and 162-times more than that in stamens, respectively. These results suggest that plant hormone signal transduction genes, especially ethylene signal transduction genes, play an important role in the development of pistils in pumpkin. Our study provides a theoretical basis for further understanding of the mechanism of regulation of ethylene signal transduction genes in pistil development and sex determination in pumpkin.

The role of mab-3 in spermatogenesis and ontogenesis of pinewood nematode, Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is one of the most destructive invasive species, causing extensive economic losses worldwide. The sex ratio of female to male of B. xylophilus plays an important role in the nematode infestation. However, little is known about the processes of its sex determination. The double sex/mab-3-related family of transcription factors are highly conserved in animals, playing crucial roles in sex determination, spermatogenesis and ontogenesis. We therefore investigated its orthologue, Bxy-mab-3, in B. xylophilus. Bxy-mab-3 has two typical conserved DNA-binding domains. It was observed in J2 (the second-stage of juveniles), J3, J4 and male adults (specifically on the spicules), but not in eggs or female adults via mRNA in situ hybridization. RNA-Seq indicated significantly higher expression in males. RNAi showed that the body size and sperm size of male adults were markedly smaller than those of the controls. Meanwhile, almost all the RNAi-treated males failed to mate with the normal females, even 26.34% of interfered males did not produce sperm. However, RNAi of Bxy-mab-3 had no effect on the sex ratio of B. xylophilus. Bxy-mab-3 is indispensable for spermatogenesis, ontogenesis and mating behavior. It is a typical sex-determination gene with differential expression in males and females. However, knocking down Bxy-mab-3 expression could not alter the sex ratio as seen in other species. Our findings contribute towards a better understanding of the molecular events of Bxy-mab-3 in B. xylophilus, which provides promising hints for control of pine wilt disease by blocking ontogenesis and decreasing nematode fecundity.

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs.

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.

An Alternative, High Throughput Method to Identify Csd Alleles of the Honey Bee.

Applying instrumental insemination in closely related honey bee colonies often leads to frequent lethality of offspring causing colony collapse. This is due to the peculiarities of honey bee reproductive biology, where the complementary sex determination (csd) gene drives sex determination within a haplodiploid system. Diploid drones containing homozygous genotypes are lethal. Tracking of csd alleles using molecular markers prevents this unwanted event in closed breeding programs. Our approach described here is based on high throughput sequencing (HTS) that provides more data than traditional molecular techniques and is capable of analysing sources containing multiple alleles, including diploid individuals as the bee queen. The approach combines HTS technique and clipping wings as a minimally invasive method to detect the complementary sex determiner (csd) alleles directly from honey bee queens. Furthermore, it might also be suitable for screening alleles of honey harvested from hives of a closed breeding facility. Data on alleles of the csd gene from different honey bee subspecies are provided. It might contribute to future databases that could potentially be used to track the origin of honey. With the help of tracking csd alleles, more focused crossings will be possible, which could in turn accelerate honey bee breeding programmes targeting increase tolerance against varroosis as well.

Default Sex and Single Gene Sex Determination in Dioecious Plants.

A well-established hypothesis for the evolution of dioecy involves two genes linked at a sex-determining region (SDR). Recently there has been increased interest in possible single gene sex determination. Work in Populus has finally provided direct experimental evidence for single gene sex determination in plants using CRISPR-Cas9 to knock out a single gene and convert individuals from female to male. In poplar, the feminizing factor popARR17 acts as a “master regulator”, analogous to the mammalian masculinizing factor SRY. The production of fully functional males from females by a simple single gene knockout is experimental evidence that an antagonistic male-determining factor does not exist in Populus. Mammals have a “default sex” (female), as do poplar trees (Populus), although the default sex in poplars is male. The occurrence of single gene sex determination with a default sex may be much commoner in plants than hitherto expected, especially when dioecy evolved via monoecy. The master regulator does not even need to be at the SDR (although it may be). In most poplars the feminizing factor popARR17 is not at the SDR, but instead a negative regulator of it. So far there is little information on how high-level regulators are connected to floral phenotype. A model is presented of how sex-determining genes could lead to different floral morphologies via MADS-box floral developmental genes.

Two androecious mutations reveal the crucial role of ethylene receptors in the initiation of female flower development in Cucurbita pepo.

Ethylene is the key regulator of sex determination in monoecious species of the family Cucurbitaceae. This hormone determines which individual floral meristems develop as female or male flowers and the female flowering transition. The sex determination genes discovered so far code for ethylene biosynthesis enzymes, but little is known about the importance of ethylene signaling components. In this paper we characterize two novel ethylene-insensitive mutations (etr1a-1 and etr1b) which block the female flowering transition of Cucurbita pepo; this makes plants produce male flowers indefinitely (androecy). Two missense mutations in the ethylene-binding domain of the ethylene receptors CpETR1A or CpETR1B were identified as the causal mutations of these phenotypes by using whole-genome resequencing. The distinctive phenotypes of single and double mutants for four etr mutations have demonstrated that the final level of ethylene insensitivity depends upon the strength and dosage of mutant alleles for at least three cooperating ETR genes, and that the level of ethylene insensitivity determines the final sex phenotype of the plant. The sex phenotype ranges from monoecy in ethylene-sensitive wild-type plants to androecy in the strongest ethylene-insensitive ones, via andromonoecy in partially ethylene-insensitive plants. The induction of female flowering transition was found to be associated with upregulation of CpACS11, CpACO2 and CpACS27, three ethylene biosynthesis genes required for female flower development. A model is proposed herein, integrating both ethylene biosynthesis and receptor genes into the genetic network which regulates sex determination in C.pepo.

Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution

BackgroundHeteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome.ResultsWe combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy).ConclusionsTogether, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.

The identification of a major sex QTL in the white-leg shrimp, Litopenaeus vannamei

The white-leg shrimp, Litopenaeus vannamei, is the most widely farmed shrimp species globally. In recent years, interest in developing selective breeding programs and identifying the mechanisms controlling sex determination in L. vannamei has increased in the hope that improvements can be made on productivity efficiency. L. vannamei exhibits sexual dimorphism whereby females grow faster and larger than males. The discovery of novel genes or regions involved in sex-determination within L. vannamei would allow farmers to exploit this natural sexual dimorphism to enhance productivity. This study utilised a revised genetic linkage map for L. vannamei (whereby linkage groups 42 and 44 were merged as LG42.44) to conduct QTL and GWAS analyses from 20 full-sib families to identify one major QTL across 20 mapping families. The QTL was identified on LG42.44 and had a 95% confidence interval of 0–12.7 cM across all families (428 progeny). This region explained from 79.4–100% of the phenotypic variation. Independent genome wide association studies (GWAS) across 1952 individuals identified 11 significant SNPs (in high linkage disequilibrium) located on LG42.44 (0–12.7 cM) with one SNP 5790_511 with an extreme -log10 (P-value) of 294. For SNP 5790_511, 93% of homozygous genotypes were males and 97.6% of heterozygous were females indicating relatively close proximity of this SNP to the sex-determination locus. The SNP heritability for the sex phenotype was 0.84 +-0.007 suggesting a strong genetic basis of sex determination. A previously described feminisation gene, Pvfem-1, characterised as being involved in male and female gonad differentiation, was annotated to LG16; however no significant associations or QTL for sex were identified at this region in this study. Even though no known sex-determination genes could be identified under the QTL region of LG42.44, the large proportion of phenotypic variance explained by the significantly associated markers underlying the QTL region is promising and highlights this region for further fine mapping and sex-determination gene discovery efforts.

The Potential Sex Determination Genes, Sox9a and Cyp19a, in Walleye Pollock (Gadus Chalcogrammus) Are Influenced by Water Temperature

Our aim was to study the relationship between the sex-determining genes, sox9a and cyp19a, and water temperature in Gadus chalcogrammus. We assessed the sex ratio based on the expression levels of sox9a and cyp19a at different water temperatures (5, 8, 11, and 14 °C) and at different stages of walleye pollock development (embryos, larvae, and juveniles). Next, we used immature walleye pollock to assess sox9a expression in males and cyp19a and vitellogenin (VTG) expression in females at different water temperatures. Males expressed sox9a in the gonadal tissues, while females expressed cyp19a in the gonadal tissues and VTG in the blood plasma. In the first experiment, cyp19a expression was higher at 5 °C and 8 °C, and sox9a expression was higher at 11 and 14 °C. In the second experiment, sox9a expression remained relatively stable, but cyp19a expression decreased with increasing temperature, decreasing significantly after 14 °C. Similar patterns were also observed for VTG expression. These results indicate that lower water temperatures increase cyp19a expression, which increases the female ratio. Higher water temperatures increase sox9a expression, which increases the male ratio. Therefore, this study highlights the potential of the sex-determining genes and the influence of water temperature.

Epigenetic regulation of gonadal and brain aromatase expression in a cichlid fish with environmental sex determination

A fit animal must develop testes or ovaries, with brain and physiology to match. In species with alternative male morphs this coordination of development across tissues operates within sexes as well as between. For Pelvicachromis pulcher, an African cichlid in which early pH exposure influences both sex and alternative male morph, we sequence both copies of aromatase (cyp19a1), a key gene for sex determination. We analyze gene expression and epigenetic state, comparing gonad and brain tissue from females, alternative male morphs, and fry. Relative to brain, we find elevated expression of the A-copy in the ovaries but not testes. Methylation analysis suggests strong epigenetic regulation, with one region specifying sex and another specifying tissue. We find elevated brain expression of the B-copy with no sex or male morph differences. B-copy methylation follows that of the A-copy rather than corresponding to B-copy expression. In 30-day old fry, we see elevated B-copy expression in the head, but we do not see the expected elevated A-copy expression in the trunk that would reflect ovarian development. Interestingly, the A-copy epialleles that distinguish ovaries from testes are among the most explanatory patterns for variation among fry, suggesting epigenetic marking of sex prior to differentiation and thus laying the groundwork for mechanistic studies of epigenetic regulation of sex and morph differentiation.

Transcriptome Analysis of the Oriental Fruit Fly Bactrocera dorsalis Early Embryos.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most devastating and highly invasive agricultural pests world-wide, resulting in severe economic loss. Thus, it is of great interest to understand the transcriptional changes that occur during the activation of its zygotic genome at the early stages of embryonic development, especially the expression of genes involved in sex determination and the cellularization processes. In this study, we applied Illumina sequencing to identify B. dorsalis sex determination genes and early zygotic genes by analyzing transcripts from three early embryonic stages at 0–1, 2–4, and 5–8 h post-oviposition, which include the initiation of sex determination and cellularization. These tests generated 13,489 unigenes with an average length of 2185 bp. In total, 1683, 3201 and 3134 unigenes had significant changes in expression levels at times after oviposition including at 2–4 h versus 0–1 h, 5–8 h versus 0–1 h, and 5–8 h versus 2–4 h, respectively. Clusters of gene orthology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed throughout embryonic development to better understand the functions of differentially expressed unigenes. We observed that the RNA binding and spliceosome pathways were highly enriched and overrepresented during the early stage of embryogenesis. Additionally, transcripts for 21 sex-determination and three cellularization genes were identified, and expression pattern analysis revealed that the majority of these genes were highly expressed during embryogenesis. This study is the first assembly performed for B. dorsalis based on Illumina next-generation sequencing technology during embryogenesis. Our data should contribute significantly to the fundamental understanding of sex determination and early embryogenesis in tephritid fruit flies, and provide gene promoter and effector gene candidates for transgenic pest-management strategies for these economically important species.