Gap-free genome assembly of Salangid icefish Neosalanx taihuensis.

Neosalanx taihuensis is widely distributed in freshwater and brackish water areas in China. Due to its high commercial value, it has been artificially introduced into many lakes and reservoirs, showing strong ecological adaptability. Here, a gap-free chromosome-level reference genome was constructed by combining short reads, PacBio HiFi long reads, Nanopore ultralong reads and Hi-C data. The reference genome of N. taihuensis was 397.29 Mb with a contig N50 of 15.61 Mb. The assembled sequences were anchored to 28 chromosomes. Furthermore, 20,024 protein-coding genes and 98.16% of the predicted genes were annotated in publicly available biological databases. This high-quality gap-free assembled genome will provide an essential reference for studying the evolution and ecological adaptability of N. taihuensis.

Assessment of Genetic Diversity of the Salangid, Neosalanx taihuensis, Based on the Mitochondrial COI Gene in Different Chinese River Basins.

The salangid Neosalanx taihuensis (Salangidae) is a commercially important economical fish endemic to China and restricted to large freshwater systems with a wide-ranging distribution. This fish species has continuous distribution ranges and a long-introduced aquaculture history in Chinese basins. However, the research on its population genetic differentiation within and between basins is very limited. In this regard, 197 individuals were sampled from 11 populations in the Nenjiang River Basin (A1-A4), Songhua River Basin (B1), Yellow River Basin (C1-C2), Yangtze River Basin (D1), Lanchang River Basin (E1-E2) and Huaihe River Basin (F1). Based on the COI sequence, the N.taihuensis population's genetic difference within and between river basins was investigated. The haplotypes and their frequency distributions were strongly skewed, with most haplotypes (n = 13) represented only in single samples each and thus restricted to a single population. The most common haplotype (H4, 67/197) was found in all individuals. The analysis of molecular variance (AMOVA) revealed a random pattern in the distribution of genetic diversity, which is inconsistent with contemporary hydrological structure. The mismatch between the distribution and neutrality tests supported the evidence of a population expansion, which occurred during the late Pleistocene (0.041-0.051 million years ago). Significant levels of genetic subdivision were detected among populations within basins rather than between the six basins. Population history dynamics showed that N. taihuensis experienced an expansion during the glacial period in the late Pleistocene. Therefore, different populations should be considered as different management units to achieve effective conservation and management purposes. These results have great significance for the evaluation and exploitation of the germplasm resources of N. taihuensis.

Genetic Diversity Evaluation and Conservation of Topmouth Culter (Culter alburnus) Germplasm in Five River Basins in China.

To study the genetic diversity of Culter alburnus (C. alburnus) populations, we analyzed the genetic diversity of five C. alburnus populations from Songhua Lake (SH), Huaihe River (HH), Changjiang River (CJ), Taihu Lake (TH), and Gehu Lake (GH) based on mitochondrial COI gene sequences. The results showed that the average contents of bases T, C, A, and G in the 526 bp COI gene sequence were 25.3%, 18.1%, 28.1%, and 28.6%, respectively, which showed AT bias. A total of 115 polymorphic sites were detected in the five populations, and 11 haplotypes (Hap) were defined. The nucleotide diversity (Pi) of the five populations ranged from 0.00053 to 0.01834, and the haplotype diversity (Hd) ranged from 0.280 to 0.746, with the highest genetic diversity in the TH population, followed by the SH population, with lower genetic diversity in the HH, CJ and GH populations. The analysis of the fixation index (Fst) and the genetic distance between populations showed that there was significant genetic differentiation between the SH population and the other populations, and the genetic distances between all of them were far; the genetic diversity within populations was higher than that between populations. Neutral tests, mismatch distributions, and Bayesian skyline plot (BSP) analyses showed that the C. alburnus populations have not experienced population expansion and are relatively stable in historical dynamics.

Transcriptome changes of Takifugu obscurus liver after acute exposure to phenanthrene.

Phenanthrene (Phe) is a model compound in polycyclic aromatic hydrocarbon (PAH) research. Reportedly, Phe treatment induced oxidative stress and histological disorders to Takifugu obscurus liver. In this study, to further explore the molecular responses of T. obscurus liver to Phe exposure, transcriptome sequencing was applied to compare mRNA transcription profiles between Phe treatment and the control. Compared with the control, 1,581 and 1,428 genes were significantly upregulated and downregulated in Phe treatment, respectively. Further analysis revealed that Phe treatment mainly upregulated genes in Ras-MAPK and PI3K-akt signaling pathways, which represented insulin resistance and further activated the FOXO signaling pathway. The triacylglycerol biosynthesis was promoted but the gluconeogenesis process was inhibited in response to Phe treatment, demonstrating that Phe exposure disturbed the sugar and lipid metabolism. Moreover, Phe treatment upregulated the Apelin-APJ and ErbB signaling pathways, promoting angiogenesis in T. obscurus liver. Insulin resistance, promoted triacylglycerol biosynthesis, and angiogenesis might explain the molecular mechanisms underlying carcinogenic toxicity of Phe. Overall, this study provides new insights to understand the environmental risk of Phe to fishes.

Transcriptome changes of Takifugu obscurus liver after acute exposure to the oxygenated-PAH 9,10-phenanthrenequione.

Contamination with polycyclic aromatic hydrocarbons (PAHs) causes noticeable ecological problems in aquatic ecosystems. 9,10-Phenanthrenequione (9,10-PQ) is an oxidized PAH and is highly toxic to aquatic animals. However, the effects of 9,10-PQ on the molecular metabolism of fish remain largely unknown. In this study, Takifugu obscurus juveniles were acutely exposed to 44.30 µg/L 9,10-PQ for 3 days. The transcriptome profile changes in their livers were compared between the 9,10-PQ treatment group and the control using T. rubripes as the reference genome. The results identified 22,414 genes in our transcriptome. Among them, 767 genes were differentially expressed after exposure to 9,10-PQ, which enriched 16 KEGG pathways. Among them, the glycolysis, phagosome, and FOXO signaling pathways were significantly activated in 9,10-PQ treatment compared with the control. These data indicate that 9,10-PQ increased the glycolysis capacity to produce more energy for resistance and harmed immune function. Moreover, several genes related to tumorigenesis were significantly upregulated in response to 9,10-PQ, displaying the carcinogenic toxicity of 9,10-PQ to T. obscurus. Genes in steroid biosynthesis pathways were downregulated in the 9,10-PQ treatment group, suggesting interference with the endocrine system. Overall, these findings provide information to help evaluate the environmental risks that oxygenated-PAHs present to T. obscurus.

Transcriptome analysis of Takifugu obscurus liver in response to acute retene exposure.

Retene (1-methyl-7-isopropyl-phenanthrene, RET) is an alkyl polycyclic aromatic hydrocarbon (PAH) with environmental risk to aquatic animals. Takifugu obscurus is a migratory fish species with high economic and ecological value. To assess the toxic effects of RET on molecular metabolism, juvenile T. obscurus in this study were acutely exposed to 44.30 µg/L of RET for four days. The transcriptome profiles of livers were compared between RET treatment group and the control, and the results revealed that 1,897 genes were significantly differentially expressed (DEGs) after exposure to RET, which enriched 17 KEGG pathways. Among these, glycerolipid metabolism, glycerophospholipid metabolism, insulin signaling pathway, and FOXO signaling pathways were significantly activated. Further exploration indicated that RET exposure disrupted glucose metabolism, stimulated insulin metabolism, and activated cell proliferation genes. Overall, these findings help explain the molecular mechanisms underlying RET toxicity, and may offer evidence to support T. obscurus protection.

Discovery of the indicator role of period 2 in yellow catfish (Pelteobagrus fulvidraco) food intake during early life development stages.

The early development stages of fish are a highly ordered and tightly regulated, involving many circadian rhythm-related gene and protein processes. Nonetheless, there are few reports on the effects of circadian clock genes on the early development stages of fish. We studied Pelteobagrus fulvidraco Period 2 (Pf-Per 2) gene structures and expression patterns during the early life stages of development, including the fertilized embryo, yolk absorption, preliminary food, rotifer breeding, and mixed food stages. cDNA of Pf-Per 2 is 4593 bp in length, with 357 bp 5'-untranslated region (5'UTR), 216 bp 3'UTR. The 4020 bp open reading frame consists of 1339 encoded amino acids. By multiple sequence alignment and phylogenetic analysis, the sequence was found to demonstrate high similarity to humans, rodents, microorganisms, and other fish species. Expression patterns of mRNA transcripts showed existence of rhythmic oscillations in the yellow catfish during the early development phase. The higher expression level of Per 2 is obviously present in the early embryonic development stage; the continuous downward trend of Per 2 was observed in the embryonic development and yolk nutrition absorption stages; additionally, the expression of Per 2 mRNA was significantly increased during individual development, rotifer breeding, and mixed food stages. Moreover, immunohistochemistry studies revealed strongest immune-labeled positive signals of Per 2 proteins mainly located in the cytoplasm of the olfactory bulb cell. Our findings reveal Pf-Per 2 serves important functions and may be useful as an indicator of P. fulvidraco early life development and initial food intake process stages.

Histological, oxidative and immune changes in response to 9,10-phenanthrenequione, retene and phenanthrene in Takifugu obscurus liver.

Polycyclic aromatic hydrocarbons (PAHs) are typical pollutants and may be alkylated and oxygenated to form alkyl-PAHs and oxygenated-PAHs (oxy-PAHs), respectively. Takifugu obscurus is an important anadromous fish species and displays a high risk of being exposed to PAHs-contaminated areas. In the present study, the effects of acute exposure to 44.29 µg L-1 9,10-phenanthrenequione (9,10-PQ), retene and phenanthrene (Phe) on T. obscurus liver histology, antioxidant enzymes and immune indices were compared. After exposure to these three compounds, histological sections showed damages of hepatocyte, and the activities of alanine and aspartate aminotransferase increased in plasma, indicating direct hepatic toxicity. Hepatic malondialdehyde (MDA) content increased, but superoxide dismutase (SOD) and catalase (CAT) activities decreased in response to treatments with Phe, retene and 9,10-PQ. These results revealed peroxidative effects on T. obscurus hepatocytes. In addition, total immunoglobulin content and lysozyme activity in plasma increased in treatments with Phe, retene and 9,10-PQ, which might be resulted from the damaged liver cells and the subsequently hepatic inflammation. Besides, the changes were more severe in treatment with 9,10-PQ than those with Phe and retene, demonstrating higher toxicity of 9,10-PQ than the other two compounds. Overall, the present study posed a high environmental risk of PAH derivatives to aquatic ecosystems.

Toll-like receptors (TLRs) respond to tributyltin chloride (TBT-Cl) exposure in the river pufferfish (Takifugu obscurus): Evidences for its toxic injury function.

Tributyltin chloride (TBT-Cl) residual in water body had become a noticeable ecological problem for aquatic ecosystems. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting nonself antigens and immune system activation. In this study, we explored the effect of TBT-Cl exposure on four TLRs expression in river pufferfish, Takifugu obscurus. The four T. obscurus Toll-like receptors (To-TLRs) contained different types of domains such as leucine-rich repeats (LRRs), leucine-rich repeats, typical subfamily (LRR_TYP) and other special domains. The To-TLRs mRNA transcripts expressed in all tissues, also To-TLR2 was investigated with higher level in kidney, as well as To-TLR3 in kidney, while To-TLR18 in liver and To-TLR22 in intestine. After the acute and chronic exposure of TBT-Cl, To-TLR2 and To-TLR3 mRNA transcripts were significantly down-regulated in gill. However, To-TLR18 and To-TLR22 were significantly up-regulated in gill and liver. Moreover, the histology and immunohistochemistry (IHC) results showed the different injury degrees of TBT-Cl in liver and gill and implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-TLRs to TBT-Cl exposure. All the results indicated that To-TLRs might involve in sensing and mediating innate immune responses caused by TBT-Cl for keeping detoxification homeostasis.

FoxL2 combined with Cyp19a1a regulate the spawning upstream migration in Coilia nasus.

FoxL2 is a member of the forkhead/HNF-3-related family of transcription factors which provides tissue specific gene regulation. It is known to regulate ovarian aromatase, which plays a crucial role in ovarian development and mature. To understand the role of FoxL2/ovarian aromatase encoded gene Cyp19a1a during ovarian development and recrudescence, we identified cDNA characteristics of FoxL2 and Cyp19a1a, analyzed its temporal expression both at transcript and protein levels in the anadromous fish, Coilia nasus. Tissue distribution pattern revealed that FoxL2 mRNA expression level was highest in ovary, while Cyp19a1a mRNA was highest in brain. During the upstream migration cycle, in ovary, the FoxL2 mRNA temporal expression peaked at the multiplication stage (stage III in May), the Cyp19a1a mRNA expression peaked at the onset stage (stage I in March). It was found that their mRNA transcripts were maintained at high level during the migration stage (from stage I in March to stage VI in July). Additionally, the strongest immunolabeling positive signals of Cyp19a1a and FoxL2 proteins were mainly found in the cytoplasm of olfactory bulb cell, stratum granulare and neurogliocyte cells and development stage oocytes. Data indicated that FoxL2 and Cyp19a1a were inducible and functional in the C. nasus ovary development and migration process. Therefore, the present results can be regarded as evidence for indispensable roles of FoxL2 and Cyp19a1a in the ovary development and migratory behavior at gene expression patterns and encoded protein distribution level.

Molecular cloning and characteristics of DnaJa1and DnaJb1 in Coilia nasus: possible function involved in oogenesis during spawning migration.

BACKGROUND: Coilia nasus oogenesis/spawning migration is a well-defined synchronous arrangement process. DnaJs are indispensable molecular chaperones for oogenesis process. However, how DnaJs involved the anadromous spawning migration mechanism is outstanding and plausible. RESULTS: In this regard, two DnaJs (Cn-DnaJa1 and Cn-DnaJb1) are cloned from the Coilia nasus's ovary. Their structure both contains J domain, G/F domain and ZF domain. Their mRNA transcripts were found extensively expressed in all the sampled tissues and significantly highly in gonads, which probably mean that DnaJs involved in C. nasus's gonad development basal metabolic processes. In the process of spawning migration, Cn-DnaJa1 and Cn-DnaJb1 mRNA transcripts were also expressed with significant differences during oogenesis with highest levels in the development phase, and maintaining high levels during the multiplication, mature and spawning phase. Further study showed that the DnaJa1and DnaJb1protein have high distribution in the onset phase and mainly distributed in the oocyte cytoplasm especially during the migration development phase's. CONCLUSIONS: This experiment study demonstrated that DnaJs participate in reproductive regulation during the spawning migration process in C. nasus and possibly play a vital role in the ovary development process. These findings also provided a base knowledge for further molecular mechanism study of spawning migration.

Toxic function of CD28 involving in the TLR/MyD88 signal pathway in the river pufferfish (Takifugu obscurus) after exposed to tributyltin chloride (TBT-Cl).

Cluster of differentiation 28 (CD28) is a co-stimulatory receptor found on the surface of T cells. Takifugu obscurus is a kind of anadromous fish species. In this study, the full-length sequence of To-CD28 was obtained, including a 672-bp open reading frame that encodes a peptide chain of 223 amino acids. The phylogenetic analysis showed that To-CD28 is similar to the CD28 protein in Takifugu rubripes. The total hematocyte count distinctly decreased after TBT-Cl exposure, showing the adverse effects of TBT-Cl invasion and self-adjusting ability upon To-CD28 accumulation. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT-Cl. The tissue expression patterns indicated To-CD28 is a widely distributed receptor in T. obscurus. Its high expression in the liver and gill suggests that To-CD28 could be potentially functioned in TBT-Cl toxic process. The mRNA levels of To-CD28 and relative genes in the TLR-MyD88 signal pathway were significantly up-regulated under TBT-Cl exposure. The immunohistochemistry also showed that the To-CD28 protein signal was enhanced under TBT-Cl exposure, which proved that the positive protection of To-CD28 for maintaining homeostasis. Our study indicated that To-CD28 might participate in the toxicity mechanism upon TBT-Cl exposure and regulate homeostasis stability of T. obscurus.

Comparative transcriptomics analysis of the river pufferfish (Takifugu obscurus) by tributyltin exposure: Clues for revealing its toxic injury mechanism.

TBT residual in water had become a noticeable ecological problem for aquatic ecosystems. The river pufferfish (Takifugu obscurus) is a kind of an anadromous fish species and widely distributed in the East China Sea and the Yellow Sea. Because of the water contamination, the pufferfish wild resource had a sudden decline in recent years. Therefore, the study on the response of pufferfish to the TBT exposure may contribute to reveal toxic injury mechanism of T. obscurus under TBT exposure. In this study, the transcriptional library of T. obscurus liver and gill was constructed and sequenced by an improved Illumina HiseqX10 high-throughput sequencing platform under different concentrations of TBT acute stress. The blood cell numbers distinctly decreased after TBT exposure, showing the adverse effects of TBT invasion and self-adjusting ability of the pufferfish. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT. The obtained data were compared with the genome data of Takifugu rubripes and transcriptional resource database. On this basis, gene function annotation, analysis and classification were carried out by bioinformatics method, and differential genes related to toxic injury function were screened out. Meanwhile, new toxic related genes and related signal pathways were sought to provide new theoretical guidance for the pathogenesis of T. obscurus exposed to TBT. This study not only enriched the transcriptome data of T. obscurus under environmental stress, but also provided a new research method for the response mechanism of T. obscurus under the stimulation of environmental factors.

Developmental Expression of HSP60 and HSP10 in the Coilia nasus Testis during Upstream Spawning Migration.

Heat shock protein 60 (HSP60) and heat shock protein 10 (HSP10) are important chaperones, which have been proven to have essential roles in mediating the correct folding of nuclear encoded proteins imported to mitochondria. Mitochondria are known as the power house of the cell, with which it produces energy and respires aerobically. In this regard, the obtained HSP60 and HSP10 have typical characteristics of the HSP60/10 family signature. Their mRNA transcripts detected were highest during the developmental phase (in April), while the lowest levels were found in the resting phase (after spawning in late July). Additionally, the strongest immunolabeling positive signals were found in the primary spermatocyte, with lower positive staining in secondary sperm cells, and a weak or absent level in the mature sperm. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix. Data indicated that HSP10 and HSP60 were inducible and functional in the Coilia nasus testis development and migration process, suggesting their essential roles in this process. The results also indicated that HSP60 may be one indicator of properly working mitochondrial import and refolding in the fish testis. This study also provides an expanded perspective on the role of heat shock protein families in spawning migration biology.

Whole genome sequencing of Chinese clearhead icefish, Protosalanx hyalocranius.

Background: Chinese clearhead icefish, Protosalanx hyalocranius , is a representative icefish species with economic importance and special appearance. Due to its great economic value in China, the fish was introduced into Lake Dianchi and several other lakes from the Lake Taihu half a century ago. Similar to the Sinocyclocheilus cavefish, the clearhead icefish has certain cavefish-like traits, such as transparent body and nearly scaleless skin. Here, we provide the whole genome sequence of this surface-dwelling fish and generated a draft genome assembly, aiming at exploring molecular mechanisms for the biological interests. A total of 252.1 Gb of raw reads were sequenced. Subsequently, a novel draft genome assembly was generated, with the scaffold N50 reaching 1.163 Mb. The genome completeness was estimated to be 98.39 % by using the CEGMA evaluation. Finally, we annotated 19 884 protein-coding genes and observed that repeat sequences account for 24.43 % of the genome assembly. We report the first draft genome of the Chinese clearhead icefish. The genome assembly will provide a solid foundation for further molecular breeding and germplasm resource protection in Chinese clearhead icefish, as well as other icefishes. It is also a valuable genetic resource for revealing the molecular mechanisms for the cavefish-like characters.

Changes of gonadotropin-releasing hormone receptor 2 during the anadromous spawning migration in Coilia nasus.

BACKGROUND: An increase in the activity of the pituitary-gonad axis (PG-axis) and gonad development are essential for the onset of spawning migration in teleosts. In the fish Coilia nasus, gonad development and spawning migration up the Yangtze River occurs by the end of each summer. We hypothesized that gonadotropin releasing hormones receptor 2 (GnRH-R2), which together produce a signal that interacts with the PG-axis, may help to regulate spawning migration processes. RESULTS: In this regard, we (1) characterized the gonadosomatic index (GSI) in the anadromous fish C. nasus; (2) analyzed the GnRH-R2 mRNA expression levels in ovary and brain, and concentrations in the serum; and (3) identified the GnRH-R2 protein distribution in the brain and ovaries. We found strong relationships between all of these indices. CONCLUSIONS: The results indicate that GnRH-R2 could act together to promote spawning during the anadromous migration. There is some evidence that the GnRH-R2 gene expression levels and protein distributions change in association with the migratory behavior.

Molecular Characteristic, Protein Distribution and Potential Regulation of HSP90AA1 in the Anadromous Fish Coilia nasus.

Heat shock proteins play essential roles in basic cellular events. Spawning migration is a complex process, with significant structural and biochemical changes taking place in the adult gonad. To date, the molecular mechanisms underlying migration reproductive biology remain undetermined. In this regard, a full length HSP90AA1 comprising 2608 nucleotides from the anadromous fish Coilia nasus was characterized, encoding 742 amino acid (aa) residues with potential phosphorylation sites. HSP90AA1 mRNA transcripts were detected in all organs, especially in the gonad. Furthermore, the greatest transcript levels were found during the developmental phase, while the lowest levels were found during the resting phase. In addition, the strongest immunolabeling positive signal was found in the primary spermatocyte and oocyte, with lower positive staining in secondary germ cells, and a weak or absent level in the mature sperm and oocyte. Interestingly, HSP90AA1 was mainly located in the cytoplasm of germ cells. These results are important for understanding the molecular mechanism of anadromous migration reproductive biology. In combination with data from other fish species, the result of this present study may facilitate further investigations on the spawning migration mechanism.

Testes transcriptome profiles of the anadromous fish Coilia nasus during the onset of spermatogenesis.

RNA-Seq technology has been widely applied to transcriptomics, genomics and functional gene study. Here, we performed de novo transcriptome sequencing to produce 23,842,172 clean reads representing a total of 4,815,798,404 (4.8 Gb) nucleotides from comprehensive transcript dataset for testis of Coilia nasus. Over 20 million Illumina reads were assembled into 194,636 unigenes, and 42,642 annotated genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, SwissProt and KEGG) using the assembled gene models from catalogs of other species, we have sampled an extensive and diverse expressed gene catalog for C. nasus representing a large proportion of the genes involved in the onset of spermatogenesis. The results will provide a general clue to the potential spermatogenesis molecular mechanisms for this species.

Ovary transcriptome profiling of Coilia nasus during spawning migration stages by Illumina sequencing.

Coilia nasus is an anadromous kind of small to moderate size fish species, and limited transcriptomics research has been performed. In the present study, we performed Illumina sequencing to produce a 22,996,612 clean reads representing with a total of 4,599,079,601 (4.5Gb) nucleotides comprehensive transcript dataset for ovary of C. nasus. Over 20 million total reads were assembled into 63,141 unigenes, and 27,027 annotated genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, Swissprot and KEGG), we have sampled an extensive and diverse expressed gene catalog for C. nasus representing a large proportion of the genes expressed in the ovary development process. This approach will assist in the discovery and annotation of novel genes that play key roles in anadromous fish spawning migration process.

Characteristic of PGDS potential regulation role on spermatogenesis in the Chinese mitten crab Eriocheir sinensis.

Prostaglandin D synthase (PGDS) catalyzes the isomerization of PGH2 to produce PGD2 in the presence of sulfhydryl compounds. In this study, a full length PGDS gene comprising 1250 nucleotides from the Chinese mitten crab Eriocheir sinensis (Es-PGDS) was characterized, with a 615 bp open reading frame encoding 204 amino acid residues. Its deduced peptide has high homology with other species' PGDS protein. The Es-PGDS mRNA expression was tissue-related, with the highest expression observed in the hepatopancreas, accessory sex gland, testis and ovaries. We also detected the different stages of tissue expression and the enzyme activity for Es-PGDS in the testis and male crab hepatopancreas. The different expression patterns and its corresponding enzyme activity level indicated that PGDS is involving in the regulation of reproductive action during the period of rapid development in E. sinensis. Furthermore our research could arouse a heat debate on the PGDS reproductive function in invertebrate and further study will be needed to determine the molecular mechanism(s) linking PGDS functions to spermatogenesis and ontogenesis if this gene is to be exploited as a molecular biomarker in further studies of development.