Organ health and development in larval kingfish are unaffected by ocean acidification and warming.

Anthropogenic CO2 emissions are causing global ocean warming and ocean acidification. The early life stages of some marine fish are vulnerable to elevated ocean temperatures and CO2 concentrations, with lowered survival and growth rates most frequently documented. Underlying these effects, damage to different organs has been found as a response to elevated CO2 in larvae of several species of marine fish, yet the combined effects of acidification and warming on organ health are unknown. Yellowtail kingfish, Seriola lalandi, a circumglobal subtropical pelagic fish of high commercial and recreational value, were reared from fertilization under control (21 °C) and elevated (25 °C) temperature conditions fully crossed with control (500 µatm) and elevated (1,000 µatm) pCO2 conditions. Larvae were sampled at 11 days and 21 days post hatch for histological analysis of the eye, gills, gut, liver, pancreas, kidney and liver. Previous work found elevated temperature, but not elevated CO2, significantly reduced larval kingfish survival while increasing growth and developmental rate. The current histological analysis aimed to determine whether there were additional sublethal effects on organ condition and development and whether underlying organ damage could be responsible for the documented effects of temperature on survivorship. While damage to different organs was found in a number of larvae, these effects were not related to temperature and/or CO2 treatment. We conclude that kingfish larvae are generally vulnerable during organogenesis of the digestive system in their early development, but that this will not be exacerbated by near-future ocean warming and acidification.

Expressional regulation of gonadotropin receptor genes and androgen receptor genes in the eel testis.

Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11ß-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro, and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish.

Does silvering or 11-ketotestosterone affect osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis)?

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.

Ocean warming has a greater effect than acidification on the early life history development and swimming performance of a large circumglobal pelagic fish.

Ocean warming and acidification are serious threats to marine life; however, their individual and combined effects on large pelagic and predatory fishes are poorly understood. We determined the effects of projected future temperature and carbon dioxide (CO2 ) levels on survival, growth, morphological development and swimming performance on the early life stages of a large circumglobal pelagic fish, the yellowtail kingfish Seriola lalandi. Eggs, larvae and juveniles were reared in cross-factored treatments of temperature (21 and 25°C) and pCO2 (500 and 985 µatm) from fertilisation to 25 days post hatching (dph). Temperature had the greatest effect on survival, growth and development. Survivorship was lower, but growth and morphological development were faster at 25°C, with surviving fish larger and more developed at 1, 11 and 21 dph. Elevated pCO2 affected size at 1 dph, but not at 11 or 21 dph, and did not affect survival or morphological development. Elevated temperature and pCO2 had opposing effects on swimming performance at 21 dph. Critical swimming speed (Ucrit ) was increased by elevated temperature but reduced by elevated pCO2 . Additionally, elevated temperature increased the proportion of individuals that responded to a startle stimulus, reduced latency to respond and increased maximum escape speed, potentially due to the more advanced developmental stage of juveniles at 25°C. By contrast, elevated pCO2 reduced the distance moved and average speed in response to a startle stimulus. Our results show that higher temperature is likely to be the primary driver of global change impacts on kingfish early life history; however, elevated pCO2 could affect critical aspects of swimming performance in this pelagic species. Our findings will help parameterise and structure fisheries population dynamics models and improve projections of impacts to large pelagic fishes under climate change scenarios to better inform adaptation and mitigation responses.

Ovarian development of captive F1 wreckfish (hapuku) Polyprion oxygeneios under constant and varying temperature regimes - Implications for broodstock management.

In order to better understand how photo-thermal conditions affect oogenesis in captive-bred F1 hapuku, 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-17ß; 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 hapuku first mature as five-year-olds and that exposure to a decreased temperature is important for appropriate progression of oogenesis.

Effects of neuropeptides and sex steroids on the pituitary-gonadal axis of pre-pubertal F1 wreckfish (hapuku) Polyprion oxygeneios in vivo: Evidence of inhibitory effects of androgens.

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 'hapuku' (Polyprion oxygeneios), a candidate for commercialization in aquaculture, were subjected to treatment for 8weeks with two implants, one containing steroid (blank; estradiol-17ß, 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 ß-subunit, fshb; luteinizing hormone ß-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 hapuku, 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.

Mapping the sex determination locus in the hapuku (Polyprion oxygeneios) using ddRAD sequencing.

BACKGROUND: Hapuku (Polyprion oxygeneios) is a member of the wreckfish family (Polyprionidae) and is highly regarded as a food fish. Although adults grow relatively slowly, juveniles exhibit low feed conversion ratios and can reach market size in 1-2 years, making P. oxygeneios a strong candidate for aquaculture. However, they can take over 5 years to reach sexual maturity in captivity and are not externally sexually dimorphic, complicating many aspects of broodstock management. Understanding the sex determination system of P. oxygeneios and developing accurate assays to assign genetic sex will contribute significantly towards its full-scale commercialisation. RESULTS: DNA from parents and sexed offspring (n = 57) from a single family of captive bred P. oxygeneios was used as a template for double digestion Restriction-site Associated DNA (ddRAD) sequencing. Two libraries were constructed using SbfI - SphI and SbfI - NcoI restriction enzyme combinations, respectively. Two runs on an Illumina MiSeq platform generated 70,266,464 raw reads, identifying 19,669 RAD loci. A combined sex linkage map (1367 cM) was constructed based on 1575 Single Nucleotide Polymorphism (SNP) markers that resolved into 35 linkage groups. Sex-specific linkage maps were of similar size (1132 and 1168 cM for male and female maps respectively). A single major sex-determining locus, found to be heterogametic in males, was mapped to linkage group 14. Several markers were found to be in strong linkage disequilibrium with the sex-determining locus. Allele-specific PCR assays were developed for two of these markers, SphI6331 and SphI8298, and demonstrated to accurately differentiate sex in progeny within the same pedigree. Comparative genomic analyses indicated that many of the linkage groups within the P. oxygeneios map share a relatively high degree of homology with those published for the European seabass (Dicentrarchus labrax). CONCLUSION: P. oxygeneios has an XX/XY sex determination system. Evaluation of allele-specific PCR assays, based on the two SNP markers most closely associated with phenotypic sex, indicates that a simple molecular assay for sexing P. oxygeneios should be readily attainable. The high degree of synteny observed with D. labrax should aid further molecular genetic study and exploitation of hapuku as a food fish.

Effects of 11-ketotestosterone and temperature on inhibin subunit mRNA levels in the ovary of the shortfinned eel, Anguilla australis.

Members of the transforming growth factor-b (TGFb) superfamily are important during early oogenesis in mammals. In this study, we tested whether documented effects of 11-ketotestosterone (11KT) on previtellogenic eel ovaries are mediated through affecting the expression of key ovarian TGFb genes. Furthermore, we investigated whether 11KT effects interacted with temperature. Accordingly, three thermal regimes were compared and their interaction with 11KT-mediated actions on expression of TGFb superfamily genes (chiefly inhibin subunits) evaluated in the eel (Anguilla australis). Inhibin subunit mRNA levels were also measured in ovarian explants cultured in vitro with 11KT and in ovaries from eels collected from the wild. In wild eels, inhibin-bA mRNA levels were higher in early than in previtellogenic eels; inhibin-a expression did not differ between stages, whereas that of inhibin-bB first decreased, then recovered with advanced developmental stage. Temperature was ineffective in modulating any of the end points, at least as long as a Q10 adjustment was made to correct for 'metabolic dose'. However, 11KT affected the expression of inhibin-a compared to control fish, while those of inhibin-b subunit genes remained unaffected. In contrast, 11KT dramatically reduced mRNA levels of inhibin-b subunits in vitro, but had inconsistent effects on inhibin-a transcript abundance. We conclude that 11KT affects ovarian inhibin subunit gene expression, but effects are not in keeping with the changes seen during early oogenesis in eels from the wild. We further contend that in vivo temperature experiments are easily biased and that Q10 corrections may be required to identify 'true' temperature effects.

Androgen-specific regulation of FSH signalling in the previtellogenic ovary and pituitary of the New Zealand shortfinned eel, Anguilla australis.

The evidence for androgens having a pivotal role in the functioning of the female reproductive axis--such as initiating puberty or vitellogenesis--is mounting. However, the use of aromatizable androgens and the tissue-specific focus of most studies often make it unclear if androgenic effects throughout the axis proceed via androgen or estrogen signalling mechanisms. In this study, we assessed the effects of 11-ketotestosterone (11KT, a non-aromatizable androgen) on the pituitary and ovary of previtellogenic (PV) freshwater eels Anguilla australis, comparing them with eels naturally undergoing early vitellogenesis (EV). We found that 11KT treatment produces molecular and morpho-physiological phenotypes that were generally intermediate between PV and EV. Most notably, we demonstrated that 11KT induces effects on follicle-stimulating hormone (FSH) signalling in the pituitary and ovaries that are in opposition to each other. Thus, 11KT significantly reduced fshß subunit expression in the pituitary. At the same time, 11KT dramatically increased mRNA levels of ovarian FSH receptor and plasma levels of estradiol-17ß, very likely sensitizing the previtellogenic follicle to the FSH signal. Androgens therefore may be important in facilitating puberty in the eel.

The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale.

BACKGROUND: Quantitative real-time PCR (qPCR) has been the method of choice for the quantification of mRNA. Due to the various artifactual factors that may affect the accuracy of qPCR, internal reference genes are most often used to normalize qPCR data. Recently, many studies have employed computer programs such as GeNorm, BestKeeper and NormFinder in selecting reference genes, but very few statistically validate the outcomes of these programs. Thus, in this study, we selected reference genes for qPCR of liver and ovary samples of yellow (juvenile), migratory (silver) and 11-KT treated juveniles of New Zealand shortfinned eels (Anguilla australis) using the three computer programs and validate the selected genes statistically using REST 2009 software and the Mann-Whitney test. We also tested for the repeatability of use for the best reference genes by applying them to a data set obtained in a similar experiment conducted the previous year. RESULTS: Out of six candidate genes, the combination of 18 s and eef1 was found to be the best statistically validated reference for liver, while in ovary it was l36. However, discrepancies in gene rankings were found between the different programs. Also, statistical validation procedures showed that several genes put forward as being the best by the programs were in fact, regulated, making them unsuitable as reference genes. Additionally, eef1 which was found to be a suitable--though not the top ranked--reference gene for liver tissues in one year, was regulated in another. CONCLUSIONS: Our study highlights the need for external validations of reference gene selections made by computer programs. Researchers need to be vigilant in validating and reporting the rationale for the use of reference gene in published studies.

Elevated hormonal stress response and reduced reproductive output in Yellow-eyed penguins exposed to unregulated tourism.

The endangered, endemic Yellow-eyed penguin (Megadyptes antipodes) is one of the flagship species for New Zealand's wildlife tourism, and recently concern has been raised that tourism-related pressures may be becoming too great. We compared two neighbouring breeding areas exposed to different levels of human disturbance. Penguins at the site exposed to unregulated tourism showed significantly lower breeding success and fledging weights than those in an area visited infrequently for monitoring purposes only. High parental baseline corticosterone concentrations correlated with lower fledgling weights at both sites. Stress-induced corticosterone concentrations were significantly higher at the tourist-exposed site, suggesting birds have been sensitized by frequent disturbance. Consequences are likely to include reduced juvenile survival and recruitment to the tourist site, while the changed hormonal stress responses may ultimately have an effect on adult fitness and survival. For maintenance of attractive Yellow-eyed penguin-viewing destinations we recommend that tourists stay out of breeding areas and disturbance at penguin landing beaches is reduced.

Effects of artificial eggs on prolactin secretion, steroid levels, brood patch development, incubation onset and clutch size in the yellow-eyed penguin (Megadyptes antipodes).

Several studies have shown that the transition from egg laying to incubation behavior in birds is associated with changes in plasma levels of prolactin and steroid hormones. However, any effect of the tactile and visual input provided by eggs at initiating these hormonal changes has not been fully investigated in wild birds. A few days before yellow-eyed penguins, Megadyptes antipodes, started egg laying, we placed an artificial egg into their nests or under cages next to their nest. We then investigated the effect of the tactile and/or visual stimulus of such an artificial egg on prolactin secretion, steroid hormone levels (total androgen, estradiol and progesterone), brood patch development, incubation onset and clutch size in these penguins. Prolactin levels rose in females in response to having an artificial egg in the nest, while they declined considerably in males. Total androgen concentrations in males were less than 7% of those of control males and the levels prior to egg placement. Brood patch width increased in both males and females. Additionally, an egg in the nest caused yellow-eyed penguin pairs to attend and sit prone on their nest more frequently. Females that initiated egg laying 1 or 2 days after placement of the artificial egg in the nest, laid a full clutch of two eggs, while most other females that were exposed to an artificial egg in their nest, laid only a single egg. In contrast, the visual stimulus of an artificial egg alone (that was placed under a cage) did not influence hormone levels, brood patch development, incubation behavior or clutch size. The stimulation of an egg in the nest influences prolactin and total androgen levels in yellow-eyed penguins, particularly in males. While brood patch development and incubation behavior were initiated and egg laying was terminated in response to an artificial egg in the nest, the exact endocrine mechanisms underlying these physiological and behavioral changes remain poorly understood. We encourage further studies on other bird species taking an experimental approach to investigate the direct influence of hormones in initiating brood patch development and incubation behavior. Moreover, such experimental studies will widen our understanding of the endocrine mechanisms that regulate clutch size.

Effects of artificial social stimuli on the reproductive schedule and hormone levels of yellow-eyed penguins (Megadyptes antipodes).

The effects of social stimuli on avian reproductive behaviors such as breeding schedules and courtship behaviors are well known due to numerous field studies. However, studies that have simultaneously examined the effects of social stimuli on reproductive behavior and the mediating endocrine mechanisms have been largely restricted to captive populations, which may not be representative of free-living populations. This study, conducted over two breeding seasons, aimed to simultaneously measure the effects of experimentally increasing auditory stimuli on the breeding schedule and endocrinology (levels of total androgen, estradiol, progesterone and prolactin) on free-living yellow-eyed penguins (Megadyptes antipodes). The yellow-eyed penguin is the least colonial of all penguins, nesting far apart from each other under dense vegetation, and, therefore, is presumed to experience much lower levels of social stimuli than other penguins. Egg laying was significantly more synchronous and tended to be earlier when birds were exposed to playbacks of the calls of conspecifics in 1 year of the study. We also found that levels of total androgen and estradiol of males in 1 year, and prolactin in another year, were proportionally higher among treated birds compared control birds that received no artificial auditory stimuli. These results show that even among supposedly solitary nesters, social stimuli could still play a role in influencing reproductive behavior and physiology. For the first time in free-living seabirds, we have demonstrated that behavioral responses to increased social stimuli are associated with hormonal changes.

Hormonal correlates of parental behavior in yellow-eyed penguins (Megadyptes antipodes).

Penguins show varying degrees of brood reduction behavior, from obligate brood reducers to brood maximizers, and we hypothesize that this is associated with differences in prolactin secretion. To address this hypothesis, we determined the breeding season prolactin profile of the yellow-eyed penguin (Megadyptes antipodes) for comparison with those of other penguin species found in the literature. We also measured sex steroid plasma concentrations to better characterize the reproductive cycle of the species. Plasma concentrations of prolactin increased from early in the season, reaching a peak during late incubation, and remained elevated up to the guard period. This general pattern was similar to that of other penguins for which we have corresponding data. However, we found that throughout the laying period, prolactin titers in yellow-eyed penguins remained elevated while they fell to basal levels after the laying of the first egg in macaroni penguins, which corresponds to differences in incubation behavior during this time. We conclude, therefore, that differences in the brood reduction behavior in penguins, may be reflected in the pattern of PRL concentrations around the time of egg laying.