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The wallago catfish (Wallago attu) is a new potential fish for aquaculture in Vietnam. Data related to the reproductive cycle of W. attu in captivity are, however, not available. To provide reliable indicators for oocyte maturation (OM) and the spawning season of the captive W. attu, this study investigated the temporal variation in hepatosomatic and gonadosomatic indices, oocyte diameter and color (greenish vs yellowish), germinal vesicle migration, and plasma concentrations of estradiol-17β (E2) and vitellogenin (Vtg) in female broodstock in association with changes in light density, temperature and amount of rainfall during the reproductive cycle. The results of this study displayed a clear seasonality in all the investigated parameters. The highest concentration of E2 (2.6 ± 3.5 ng/mL) was found in April, followed by a peak of Vtg (543 ± 43 ng/mL) in June. Meanwhile, the largest mean oocyte diameter (1.70 ± 0.02 mm) was observed in June. The shortest distance between the germinal vesicle and the edge of the oocyte (0.20 ± 0.01 mm) was recorded in July. Correspondingly, the amount of rainfall increased remarkably in July from 43.9 mm to over 200 mm in August. Taken together, we conclude that OM and the onset of the spawning season of captive W. attu occur in July and August, respectively. The percentage of greenish oocytes increased significantly over sampling time points. The changes in the color of oocytes combined with oocyte diameter could, therefore, be considered as promising indicators to predict the OM and spawning season of captive W. attu.

Background The genetic control of sex determination in teleost species is poorly understood. This is partly because of the diversity of mechanisms that determine sex in this large group of vertebrates, including constitutive genes linked to sex chromosomes, polygenic constitutive mechanisms, environmental factors, hermaphroditism, and unisexuality. Here we use a de novo genome assembly of New Zealand silver trevally (Pseudocaranx georgianus) together with sex-specific whole genome sequencing data to detect sexually divergent genomic regions, identify candidate genes and develop molecular makers. Results The de novo assembly of an unsexed trevally (Trevally_v1) resulted in a final assembly of 579.4 Mb in length, with a N50 of 25.2 Mb. Of the assembled scaffolds, 24 were of chromosome scale, ranging from 11 to 31 Mb in length. A total of 28,416 genes were annotated after 12.8 % of the assembly was masked with repetitive elements. Whole genome re-sequencing of 13 wild sexed trevally (seven males and six females) identified two sexually divergent regions located on two scaffolds, including a 6 kb region at the proximal end of chromosome 21. Blast analyses revealed similarity between one region and the aromatase genes cyp19 (a1a/b) (E-value < 1.00E-25, identity > 78.8 %). Males contained higher numbers of heterozygous variants in both regions, while females showed regions of very low read-depth, indicative of male-specificity of this genomic region. Molecular markers were developed and subsequently tested on 96 histologically-sexed fish (42 males and 54 females). Three markers amplified in absolute correspondence with sex (positive in males, negative in females). Conclusions The higher number of heterozygous variants in males combined with the absence of these regions in females support a XY sex-determination model, indicating that the trevally_v1 genome assembly was developed from a male specimen. This sex system contrasts with the ZW sex-determination model documented in closely related carangid species. Our results indicate a sex-determining function of a cyp19a1a-like gene, suggesting the molecular pathway of sex determination is somewhat conserved in this family. The genomic resources developed here will facilitate future comparative work, and enable improved insights into the varied sex determination pathways in teleosts. The sex marker developed in this study will be a valuable resource for aquaculture selective breeding programmes, and for determining sex ratios in wild populations.

Ectotherm species, such as marine fishes, depend on environmental temperature to regulate their vital functions. In finfish aquaculture production, being able to predict physiological responses in growth and other economic traits to temperature is crucial to address challenges inherent in the selection of grow-out locations. This will become an even more significant issue under the various predicted future climate change scenarios. In this study, we used the marine teleost silver trevally (

BackgroundThe genetic control of sex determinism in teleost species is poorly understood. This is partly because of the diversity of sex determining mechanisms in this large group, including constitutive genes linked to sex chromosomes, polygenic constitutive mechanisms, environmental factors, hermaphroditism, and unisexuality. Here we use a de novo genome assembly of New Zealand silver trevally (Pseudocaranx georgianus) together with whole genome sequencing to detect sexually divergent regions, identify candidate genes and develop molecular makers.ResultsThe de novo assembly of an unsexed trevally (Trevally_v1) resulted in an assembly of 579.4 Mb in length, with a N50 of 25.2 Mb. Of the assembled scaffolds, 24 were of chromosome scale, ranging from 11 to 31 Mb. A total of 28416 genes were annotated after 12.8% of the assembly was masked with repetitive elements. Whole genome re-sequencing of 13 sexed trevally (7 males, 6 females) identified sexually divergent regions located on two scaffolds, including a 6 kb region at the proximal end of chromosome 21. Blast analyses revealed similarity between one region and the aromatase genes cyp19 (a1a/b). Males contained higher numbers of heterozygous variants in both regions, while females showed regions of very low read-depth, indicative of deletions. Molecular markers tested on 96 histologically-sexed fish (42 males, 54 females). Three markers amplified in absolute correspondence with sex.ConclusionsThe higher number of heterozygous variants in males combined with deletions in females support a XY sex-determination model, indicating the trevally_v1 genome assembly was based on a male. This sex system contrasts with the ZW-type sex system documented in closely related species. Our results indicate a likely sex-determining function of the cyp19b-like gene, suggesting the molecular pathway of sex determination is somewhat conserved in this family. Our genomic resources will facilitate future comparative genomics works in teleost species, and enable improved insights into the varied sex determination pathways in this group of vertebrates. The sex marker will be a valuable resource for aquaculture breeding programmes, and for determining sex ratios and sex-specific impacts in wild fisheries stocks of this species.

Our previous work documented significant advancements in steroid-induced progression of oogenesis, demonstrating that co-treatment of female eels with 11-ketotestosterone (11KT) and estradiol-17β (E2) successfully induced uptake of vitellogenin by oocytes. Here we evaluate the effects of this steroid co-treatment on subsequent time to ovulation and egg quality in shortfinned eels artificially matured by hypophysation. Co-treatment with 11KT (1 mg) and E2 (0.2 or 2 mg) significantly reduced time to ovulation and therefore, the amount of pituitary homogenate required, without any detrimental effects on gonadosomatic index, oocyte diameter or the total weight of stripped eggs. E2 treatment resulted in promising increases in fertilization rates. These indicators suggest that co-treatment with 11KT and E2 holds promise for future artificial maturation practices in terms of minimising fish handling and stress, and of reducing the need for expensive pituitary preparations.

Silvering has been associated with advancing osmoregulatory ability. Given the demonstrated role of 11-ketotestosterone (11KT) in mediating many of the silvering-related changes, we investigated the role of 11KT in driving this advanced osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis). Yellow (non-migratory) eels with or without 11KT implants and blank-implanted silver (migratory) eels, either held in freshwater or subjected to seawater challenge, were sampled to determine serum [Na+] and [Cl−], pituitary prolactin mRNA levels, gill Na+/K+-ATPase activity and gill mRNA levels for Na+/K+-ATPase-α1 subunit and for Na+/K+/2Cl− co-transporter-1α-subunit. Developmental stage and 11KT treatment advanced the eels’ osmoregulatory ability. Thus, serum [Na+] and [Cl−] were affected by developmental stage and 11KT treatment upon seawater challenge. However, seawater challenge, not 11KT treatment or developmental stage, produced the strongest and the most consistent effects on A. australis osmoregulatory processes, inducing significant effects in all the relevant parameters we measured. In light of our results and in view of the eel’s marine ancestry, we contend that A. australis, or freshwater eels in general, are highly tolerant and able to adapt quickly to changing salinities even at the yellow stage, which may preclude a critical need for an advanced osmoregulatory ability at silvering.

Wild-caught hāpuku (Polyprion oxygeneios) spawn readily in captivity, but although first filial (F1) hāpuku complete vitellogenesis, females fail to undergo oocyte maturation and spawn or produce poor quality eggs. This study investigated whether administration of a synthetic agonist of gonadotropin-releasing hormone (GnRHa) could improve F1 hāpuku spawning and complete the life-cycle in captivity. Spawning trials were conducted over 2 years in 2013 and 2014, when F1 were aged five and six years. In 2013, females previously conditioned under a variable or constant temperature regime were implanted with GnRHa (100 &mu, g/kg&minus, 1) or blank implants constructed of powdered cellulose and cholesterol. Spawning was erratic and egg quality very poor in all tanks. No F2 offspring were produced by communal spawning. In contrast, viable F2 larvae were produced by strip-spawning and in vitro fertilization after a series of GnRHa injections. In 2014, two additional trials were conducted: females received ethylene-vinyl acetate copolymer (EVAc) matrix implants containing GnRHa (100 &mu, 1) or blank implants and in the second trial, two GnRHa doses (100 &mu, 1 and 50 &mu, 1) were tested. Eggs were first detected in all tanks 12&ndash, 17 days post-implantation when females received 100 &micro, 1 GnRHa implants, but not in the lower dose or control tanks. In summary, this study achieved induction of female spawning with GnRHa implants (target dose 100 &mu, 1) and the successful production of F2 hāpuku in captivity by strip-spawning.

&lsquo, Wreckfish&rsquo, a collective of species belonging to the family Polyprionidae, are an important commercial fishery and have significant aquaculture potential. Until now, genomic or transcriptomic information for any species within the genus Polyprion has either remained unpublished or is non-existent. Using Illumina HiSeq, we compared the transcriptomes of hāpuku (Polyprion oxygeneios) ovaries to explore developmental stage-specific variations underlying their reproductive physiology. We sought to identify differentially expressed genes and the associated shifts in biological pathways between previtellogenic and early vitellogenic ovaries. Ovarian tissue was repeatedly biopsied by gonopore cannulation from the same females (n = 3) throughout oogenesis. Reproductive status of initial biopsies was confirmed as being previtellogenic and that in biopsies collected eight weeks later as early vitellogenic. A de novo hāpuku transcriptome was assembled (146,189 transcripts) from RNA-Seq data without a reference genome. On average, each tissue sample contained 17.5 million trimmed reads. Gene annotation was 80% when using BLASTX against Genbank Non Redundant database. Fifty-three transcripts were differentially expressed within the FDR of 0.05 when previtellogenic and early vitellogenic ovaries were compared, this reduced to 35 differentially expressed genes when transcript duplications were pooled. Among these were genes tentatively associated with the electron transport chain, lipid metabolism, steroidogenesis and mineral/solute transportation. These data provide a snap-shot into stage-specific physiological events during oogenesis in the ovary of a teleost and an extensive molecular resource for research on species in the Genus Polyprion.

Molecular barcoding and next generation amplicon sequencing were used to investigate the diet of the larvae of two species: tiger (Epinephelus fuscoguttatus) and hybrid (E. lanceolatus × E. fuscoguttatus) grouper. Larvae were reared in open green-water ponds. These ponds had natural populations of wild zooplankton, and were also seeded with additional zooplankton. Zooplankton from water samples and larvae were collected daily for the first 12 days post hatch (dph) for hybrid grouper and the first 6 dph for tiger grouper. Next generation (NextGen) sequencing with degenerate cytochrome oxidase 1 oligonucleotide primers was performed on all samples. Resulting sequences suggest that both grouper species predated heavily on calanoid copepods – especially at first feeding and while still drawing energy from endogenous supplies. In addition, tiger grouper consumed mussel trochophores as a first feed. Grouper larvae diversified their diets at 6 dph and hybrid grouper were found to be consuming an unidentified insect as their primary food source from this point. Diets further diversified at 10 dph and the first substantial representation of ‘traditional’ aquaculture live feed was seen at 12 dph with the appearance of Artemia. Rotifers (Brachionus spp.) were consumed at a low level by tiger grouper larvae and at a negligible level by hybrid grouper despite daily addition to the pond systems. This study confirms that within the first 12 dph calanoid copepods are the key prey zooplankton for grouper larvae, and demonstrates that NextGen sequencing is a powerful tool for diet assessment in aquaculture.

Human chorionic gonadotropin (hCG) is widely used to induce spermatogenesis in male anguillids, but neither the efficacy of intramuscular (IM) versus intraperitoneal (IP) injection, nor the dose leading to a half-maximum response has been determined. We therefore conducted in vivo dose-response studies with single administrations of hCG in silver-staged male shortfinned eels and evaluated the gonadosomatic index (GSI), testicular histology and plasma 11-ketotestosterone (11KT) levels. Half-maximum effects of hCG on GSI and 11KT levels were deduced to require 0.47 and 0.85 IU hCG/g body weight, respectively. Gonadal responses were significantly stronger for IM than for IP injections. Subsequently, the effects of IP implants containing 11KT, a major mediator of gonadotropin action in the eel testis, were determined; our prediction that 11KT would mimic hCG effects was largely rejected - although testes responded dose-dependently within the 11-KT range tested, testicular growth was modest and germ cell development appeared to be associated primarily with limited spermatogonial proliferation. Expression of the luteinizing hormone receptor and both androgen receptors in the testes was significantly lower in 11KT-treated eels than in controls. We conclude that i ) IM hCG injections into male eels may be preferable over IP injections, ii ) that 11KT implants can only partly mimic the effects of hCG in male eels in vivo , and iii ) that hCG dose-response designs hold promise to evaluate treatment effects and compare between studies from different research groups. Statement of relevance/impact Induction of spermatogenesis in captive male eels using hCG.

The role of gonadotropins during early ovarian development in fish remains little understood. Concentrations of gonadotropins were therefore experimentally elevated in vivo by administration of recombinant follicle-stimulating hormone (rec-Fsh) or human chorionic gonadotropin (hCG) and the effects on ovarian morphology, sex steroid levels and mRNA levels of genes expressed in pituitary and ovary examined. Hormones were injected thrice at weekly intervals in different doses (20, 100 or 500 µg/kg BW for rec-Fsh and 20, 100 or 500 IU/kg BW for hCG). All treatments, especially at the highest doses of either rec-Fsh or hCG, induced ovarian development, reflected in increased oocyte size and lipid uptake. Both gonadotropins up-regulated follicle-stimulating hormone receptor (fshr) mRNA levels and plasma levels of estradiol-17β (E2). Exogenous gonadotropins largely decreased the expression of follicle-stimulating hormone β-subunit (fshb) and had little effect on those of luteinizing hormone β-subunit (lhb) in the pituitary. It is proposed that the effects of hCG on ovarian development in previtellogenic eels could be indirect as a significant increase in plasma levels of 11-ketotestosterone (11-KT) was found in eels treated with hCG. Using rec-Fsh and hCG has potential for inducing puberty in eels in captivity, and indeed, in teleost fish at large.

The ability to advance puberty in broodstock that have a long generation interval and mature at large size is a highly valuable tool in contemporary aquaculture enterprise. Juvenile male and female wreckfish 'hāpuku' (Polyprion oxygeneios), a candidate for commercialization in aquaculture, were subjected to treatment for 8weeks with two implants, one containing steroid (blank; estradiol-17β, E2; 11-ketotestosterone, KT; 17 α-methyltestosterone, MT), the other peptide (blank; gonadotropin-releasing hormone analog, GnRHa; kisspeptin, Kiss2-12). The expression of target genes (glycoprotein homone α-subunit, gpa; follicle stimulating-hormone β-subunit, fshb; luteinizing hormone β-subunit, lhb; GnRH receptor, gnrhr) in the pituitary was assayed by quantitative PCR. KT and MT decreased mRNA levels of all target genes in both male and female hāpuku, suggestive of a strong inhibitory tone by these steroid hormones. E2, GnRHa and Kiss2-12 were largely ineffective, regardless of whether they were administered alone or in combination with steroid implants. Clear differences in release and/or clearance rates between E2 and KT from implants were evident, in part explaining our observations. Advancement of puberty was not achieved, and we pose that different hormone doses and/or administration during more advanced stages of gonadogenesis need to be considered to move this field forward.

In order to better understand how photo-thermal conditions affect oogenesis in captive-bred F1 hāpuku, a wreckfish considered for aquaculture in New Zealand, juvenile (pre-pubertal) fish were assigned to one of two regimes: exposed to a constant temperature of 17°C (CT group) or to seasonally varying temperatures (VT group range: 10-17°C), both under simulated ambient photoperiod, for nearly 2years. Development in females was monitored through repeated gonadal biopsies (histology; target gene mRNA levels) and blood sampling (plasma levels of estradiol-17β; E2). Very little evidence of advancing oogenesis was found in the first year of study, when fish were in their 4th year. In the subsequent year, a proportion of fish reached the pre-spawning stage (fully-grown ovarian follicles); the proportion of females reaching this stage was notably higher in the VT (62%) than the CT (28%) group. Of the few females that did reach maturity in the CT group, significantly lower levels of plasma E2 were observed relative to those in fish from the VT group possibly indicating a temperature-induced endocrine impairment during oogenesis. Interestingly, females that did not reach the pre-spawning stage presented with a small transient, but significant increase in oocyte diameters and plasma E2, suggestive of a dummy run. Clear seasonality was observed for fish under both photo-thermal regimes, and this was reflected in plasma E2 levels and transcript abundances of aromatase, fshr and luteinizing hormone receptor in the ovary; these end points all peaked in maturing females during the late or post-vitellogenic stage. We conclude that captive female F1 hāpuku first mature as five-year-olds and that exposure to a decreased temperature is important for appropriate progression of oogenesis.

Attempts at artificial propagation and rearing of juvenile galaxiids (‘whitebait’) have been hindered by low fecundities and survival. To circumvent these issues, we subjected wild and captivity-acclimated giant kokopu (Galaxias argenteus), a hitherto unexplored galaxiid, to captive breeding protocols and evaluated the effects of salinity and temperature on egg fertilisation, incubation duration and hatching. Eggs sourced over several spawning seasons were subjected to different salinity (0, 9, 15, 20 and 30–35 PSU) and temperature (4, 10, 15 and 17°C) combinations. Average fertilisation rates were highest (95%) when gametes were activated in fresh water (0 PSU) and declined as salinity increased. Average hatch rates were highest (71–68%) when eggs were incubated in fresh water (0 PSU) at 10°C and decreased as salinity and temperature increased. Sequential stages of embryonic development were documented and several year classes of offspring were produced to form a captive breeding colony. We conclude that the giant kokopu has notable reproduction-related qualities that may be useful for future aquaculture exploitation of this species.

11-Ketotestosterone dramatically affects ovarian physiology in previtellogenic eels, notably increasing lipid accumulation and the expression of the follicle-stimulating hormone receptor. We therefore sought to combine androgen pre-treatment of female silver eels with “traditional” hypophysation in order to assess whether androgens constitute a valuable addition to induced spawning protocols. Eels were first implanted with different doses (0–30 mg) of 17-methyltestosterone to evaluate effects on oocyte size. Females (Trial I) were subsequently subjected to repeated injections with carp pituitary homogenate to artificially induce maturation. In a subsequent trial, we used 1 mg of androgen, implanted in either aromatizable (androstenedione) or non-aromatizable form (11-ketotestosterone) and again induced eels to mature artificially by repeated injections with carp pituitary homogenate. Androgen treatment significantly increased oocyte diameters prior to commencing carp pituitary homogenate treatment. Thereafter, less time, less money and less handling were required to reach the pre-ovulatory stage, while retaining comparable amounts of lipid in ovulated eggs. We contend that androgen pre-treatment has promise as a synchronizing and cost-effective measure for the artificial induction of maturation in anguillid eels.

To establish a method of gonad biopsy for ovarian tissue collection in the declining giant kokopu Galaxias argenteus (Gmelin 1789) as an alternative to lethal sampling in order to understand the species' reproductive biology.Six female giant kokopu weighing between 200 and 350 g were caught from the wild in early December of 2009 and transferred to a holding facility (Department of Zoology, University of Otago, Dunedin) where they were kept under a simulated natural photo-thermal regime for 10 months. Fish were repeatedly biopsied for ovarian tissue at near-monthly intervals (mean number of days between biopsies = 33) until ovulation.Ovarian samples were successfully collected from giant kokopu by biopsy for use in downstream analyses. Among a total of 23 biopsy events, a single death occurred when a two-layered suturing approach was used, highlighting the value of this method for study of the reproductive biology of valuable fish.This biopsy method may have implications for veterinary research on fish physiology, pathology, conservation and development, when repeated tissue samples need to be collected over a prolonged period of time or for general surgical manipulations on fish when accessing the coelom. Furthermore, this approach allows the implementation of a more powerful experimental design, as repeated measures reduces the variability of estimates due to the removal of inherent stage differences among individuals.

The androgen 11-ketotestosterone (11KT) is associated with the physiological and morphological changes that occur during the transformation of sedentary (‘yellow’) freshwater eels (Anguilla spp.) into their migratory form (‘silver’) prior to their spawning migration in the ocean. In this study, we investigate the possible role of 11KT in modulating behaviors consistent with downstream migration; i.e., downstream and salinity preference in the New Zealand shortfinned eel (A. australis). Unlike silvering, 11KT did not induce preference for downstream locations, scored as presence at the downstream ends of 35 m raceways. Likewise, there was no evidence for increased salinity preference in 11KT-treated yellow eels, scored as preference for sea water over fresh water in a choice experiment. However, the 11KT treatment induced higher frequency of movements between fresh water and sea water, which may indicate restlessness.