Photoreception and transcriptomic response to light during early development of a teleost with a life cycle tightly controlled by seasonal changes in photoperiod.

Light cues vary along the axis of periodicity, intensity and spectrum and perception of light is dependent on the photoreceptive capacity encoded within the genome and the opsins expressed. A global approach was taken to analyze the photoreceptive capacity and the effect of differing light conditions on a developing teleost prior to first feeding. The transcriptomes of embryos and alevins of Atlantic salmon (Salmo salar) exposed to different light conditions were analyzed, including a developmental series and a circadian profile. The results showed that genes mediating nonvisual photoreception are present prior to hatching when the retina is poorly differentiated. The clock genes were expressed early, but the circadian profile showed that only two clock genes were significantly cycling before first feeding. Few genes were differentially expressed between day and night within a light condition; however, many genes were significantly different between light conditions, indicating that light environment has an impact on the transcriptome during early development. Comparing the transcriptome data from constant conditions to periodicity of white light or different colors revealed overrepresentation of genes related to photoreception, eye development, muscle contraction, degradation of metabolites and cell cycle among others, and in constant light, several clock genes were upregulated. In constant white light and periodicity of green light, genes associated with DNA replication, chromatin remodeling, cell division and DNA repair were downregulated. The study implies a direct influence of light conditions on the transcriptome profile at early developmental stages, by a complex photoreceptive system where few clock genes are cycling.

Impact of freshwater rearing history on Atlantic salmon gill response to viral stimulation post seawater transfer.

Land-based recirculating aquaculture systems (RAS) have risen in prevalence in recent years for Atlantic salmon production, enabling intensive production which allows increased growth and environmental control, but also having the potential for reducing water use and eutrophication. The Atlantic salmon has an anadromous life history with juvenile stages in freshwater (FW) and on-growing in seawater (SW), enabled by a transformational process known as smoltification. The timing of smoltification and transfer of smolts from FW to SW is critical under commercial production with high mortalities during this period. The impact of FW rearing system on immune function following seawater transfer (SWT) is not well understood. In this study parr were raised in either RAS or a traditional open-LOCH system until smolting and then transferred to a common marine environment. Two-weeks post-SWT fish were immune stimulated with a viral mimic (poly I:C) for 24 h to assess the ability to mount an antiviral immune response, assessed by whole transcriptome analysis of gill tissue, an important immune organ in fish. We show that unstimulated smolts reared in the LOCH had higher immune gene expression than those reared in RAS as determined by functional analysis. However, following stimulation, smolts reared in the RAS mounted a greater magnitude of response with a suite of immune genes displaying higher fold induction of transcription compared to LOCH reared smolts. We suggest RAS smolts have a lower steady state immune-associated transcriptome likely due to an unvarying environment, in terms of environmental factors and lack of exposure to pathogens, which shows a compensatory mechanism following stimulation allowing immune 'catch-up' with those reared in the LOCH. Alternatively, the RAS fish are experiencing an excessive response to the immune stimulation.

Development of genomic markers associated to growth-related traits and sex determination in lumpfish (Cyclopterus lumpus).

Cleaner fish species have gained great importance in the control of sea lice, among them, lumpfish (Cyclopterus lumpus) has become one of the most popular. Lumpfish life cycle has been closed, and hatchery reproduction is now possible, however, current production is reliant on wild caught broodstock to meet the increasing demand. Selective breeding practices are called to play an important role in the successful breeding of most aquaculture species, including lumpfish. In this study we analysed a lumpfish population for the identification of genomic markers linked to production traits. Sequencing of RAD libraries allowed us to identify, 7193 informative markers within the sampled individuals. Genome wide association analysis for sex, weight, condition factor and standard length was performed. One single major QTL region was identified for sex, while nine QTL regions were detected for weight, and three QTL regions for standard length. A total of 177 SNP markers of interest (from QTL regions) and 399 high Fst SNP markers were combined in a low-density panel, useful to obtain relevant genetic information from lumpfish populations. Moreover, a robust combined subset of 29 SNP markers (10 associated to sex, 14 to weight and 18 to standard length) provided over 90% accuracy in predicting the animal's phenotype by machine learning. Overall, our findings provide significant insights into the genetic control of important traits in lumpfish and deliver important genomic resources that will facilitate the establishment of selective breeding programmes in lumpfish.

Efficacy testing of an immersion vaccine against Aeromonas salmonicida and immunocompetence in ballan wrasse (Labrus bergylta, Ascanius).

The development of effective vaccines is a critical step towards the domestication of emerging fish species for aquaculture. However, traditional vaccine delivery through intraperitoneal (i.p.) injection requires fish to reach a minimum size and age and therefore cannot provide protection at early developmental stages when infection may occur. This study investigated the effectiveness of immersion vaccination with respect to immunocompetence in a cleaner fish species (ballan wrasse, Labrus bergylta, Ascanius) used in Atlantic salmon farming as an alternative means to control sea lice. The species is susceptible to atypical strains of Aeromonas salmonicida (aAs) at early life stages (<15 g), when i.p. vaccination is not applicable. While immersion vaccination is currently used in commercial hatcheries, the optimal fish size for vaccination, and efficacy of the vaccine delivered by this route has not yet been established. Importantly, efficacy depends on the capability of the species immune system to recognise antigens and process antigens to trigger and produce an adaptive immune response, (process known as immunocompetence). In this study, the efficacy of a polyvalent autogenous vaccine administered by immersion in juvenile ballan wrasse and the subsequent immune response induced was investigated after prime and booster vaccination regimes. In addition, temporal expression (0-150 days post hatch) of adaptive immune genes including major histocompatibility complex (MHC II CD74 molecule) and immunoglobulin M (IgM) was assessed using quantitative PCR (qPCR). Prime and/or boost vaccination by immersion of juvenile ballan wrasse (0.5 g and 1.5 g corresponding to 80 and 170 days post hatch (dph), respectively) did not provide significant protection against aAs vapA V after bath challenge under experimental conditions. Despite no evident protection >80 dph, MHC II and IgM transcripts were first reported at 35 and 75 dph, respectively, suggesting a window of immunocompetence. The results provide important new information on the onset of adaptive immunity in ballan wrasse and highlight that immersion vaccination in the species for protection against aAs should be performed at later developmental stages (>1.5 g) in the hatchery.

A high-density linkage map and sex-linked markers for the Amazon Tambaqui Colossoma macropomum.

BACKGROUND: Tambaqui (Colossoma macropomum, Cuvier, 1818) is the most economically important native freshwater fish species in Brazil. It can reach a total length of over 1 m and a weight of over 40 kg. The species displays a clear sex dimorphism in growth performance, with females reaching larger sizes at harvest. In aquaculture, the production of monosex populations in selective breeding programmes has been therefore identified as a key priority. RESULTS: In the present study, a genetic linkage map was generated by double digest restriction-site associated DNA (ddRAD) sequencing from 248 individuals sampled from two F1 families. The map was constructed using 14,805 informative SNPs and spanned 27 linkage groups. From this, the tambaqui draft genome was improved, by ordering the scaffolds into chromosomes, and sex-linked markers were identified. A total of 235 markers on linkage group 26 showed a significant association with the phenotypic sex, supporting an XX/XY sex determination system in the species. The four most informative sex-linked markers were validated on another 206 sexed individuals, demonstrating an accuracy in predicting sex ranging from 90.0 to 96.7%. CONCLUSIONS: The genetic mapping and novel sex-linked DNA markers identified and validated offer new tools for rapid progeny sexing, thus supporting the development of monosex female production in the industry while also supporting breeding programmes of the species.

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius).

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time-consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V-specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost-effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

Novel atypical Aeromonas salmonicida bath challenge model for juvenile ballan wrasse (Labrus bergylta, Ascanius).

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n = 50; approx. 2 g) using a bath challenge, and fish were monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish, histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence.

Response of triploid Atlantic salmon (Salmo salar) to commercial vaccines.

While triploid Atlantic salmon represent a practical and affordable solution to the issues associated with sexual maturation in the salmonid aquaculture industry, empirical evidence suggests triploids are more susceptible to disease and vaccine side-effects than diploids. With vaccination now part of routine husbandry, it is essential their response be studied to confirm their suitability for commercial production. This study tested the response of triploid and diploid Atlantic salmon to vaccination with commercially available vaccines. Triploid and diploid Atlantic salmon siblings were injected with one of three commercial vaccines (or sham-vaccinated) and monitored for performance throughout a commercial production cycle. Sampling at smolt and harvest was undertaken along with individual weight and length assessments through the cycle. Antibody response to Aeromonas salmonicida vaccination was similar in both ploidy, with a positive response in vaccine-injected fish. For both adhesions and melanin, analysis found that higher scores were more likely to occur as the anticipated severity of the vaccine increased. In addition, for adhesion scores at smolt and melanin scores at smolt and harvest, triploids were statistically more likely to exhibit high scores than diploids. Triploids maintained a significantly higher body weight during freshwater and until 11 months post-seawater transfer, with diploids weighing significantly more at harvest. Growth, represented by thermal growth coefficient (TGC), decreased in both ploidy as the severity of adhesions increased, and regression patterns did not differ significantly between ploidy. Vertebral deformity prevalence was consistently higher in triploids (smolt 12.3 ± 4.5%; harvest 34.9 ± 5.9%) than diploids (smolt 0.8 ± 0.5%; harvest 15.9 ± 1.9%), with no significant difference between vaccine groups in each ploidy. This study demonstrates that triploids respond as well to vaccination as diploids and provides further supporting evidence of triploid robustness for commercial aquaculture.

GnRHa implants and size pairing effects on plasma and cephalic secretion sex steroids in Arapaima gigas.

Arapaima gigas, one of the world's largest freshwater fish, is considered an emerging species for aquaculture development in Brazil given its high growth rate and meat quality. However, the lack of reproductive control in captivity has limited the expansion of Arapaima farming. This study aimed to test the effects of hormonal induction using mGnRHa implants and size pairing on broodstock reproduction through the analyses of sex steroids. To do so, broodstock of different sizes (large, small or mixed) were paired and implanted. Plasma and cephalic secretion profiles of testosterone (T), 11-ketotestosterone (11-KT) and 17ß-oestradiol (E2) were analysed. Compared to control (non-implanted), implanted broodstock showed a significant increase in plasma 11-KT (large and small males) and T (large and mixed females) post GnRHa implantation. In females, a significant increase in plasma T levels was shown, however, E2 remained unchanged after implantation. Despite the lack of clear spawning induction, this study showed the potency of GnRHa on sex steroid production regardless of pairing groups. Interestingly, significant correlations between blood plasma and cephalic secretion levels of 11-KT in males and T in females were observed, indicating the possible release of pheromones through the cephalic canals of A. gigas.

Investigating the kisspeptin system in the hermaphrodite teleost gilthead seabream (Sparus aurata).

The kisspeptin system, a known regulator of reproduction in fish, was investigated during two key phases within the gilthead seabream (Sparus aurata) life cycle: protandrous sex change and larval ontogeny. Seabream specific partial cDNA sequences were identified for two key targets, kissr4 and kiss2, which were subsequently cloned and qPCR assays developed. Thereafter, to examine association in expression with sex change, a group of adult seabream (2+ years old) undergoing sex change were sampled for gene expression at two different periods of the annual cycle. To study the kisspeptin system ontogeny during early life stages, transcript levels were monitored in larvae (till 30 days-post-hatch, DPH) and post-larvae (from 30 till 140 DPH). During sex change, higher expression of kissr4 and kiss2 was observed in males when compared to females or individual undergoing sex change, this is suggestive of differential actions of the kisspeptin system during protandrous sex change. Equally, variable expression of the kisspeptin system during early ontogenic development was observed. The higher expression of kissr4 and kiss2 observed from 5 DPH, with elevations at 5-20 and 90 DPH for kissr4 and at 5, 10, 20, and 60 DPH for kiss2, is coincident with the early ontogeny of gnrh genes previously reported for seabream, and possibly related with early development of the reproductive axis in this species.

Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins.

BACKGROUND: Arapaima gigas (Schinz, 1822) is the largest freshwater scaled fish in the world, and an emerging species for tropical aquaculture development. Conservation of the species, and the expansion of aquaculture requires the development of genetic tools to study polymorphism, differentiation, and stock structure. This study aimed to investigate genomic polymorphism through ddRAD sequencing, in order to identify a panel of single nucleotide polymorphisms (SNPs) and to simultaneously assess genetic diversity and structure in wild (from rivers Amazon, Solimões, Tocantins and Araguaia) and captive populations. RESULTS: Compared to many other teleosts, the degree of polymorphism in A. gigas was low with only 2.3% of identified RAD-tags (135 bases long) containing SNPs. A panel of 393 informative SNPs was identified and screened across the five populations. Higher genetic diversity indices (number of polymorphic loci and private alleles, Shannon's Index and HO) were found in populations from the Amazon and Solimões, intermediate levels in Tocantins and Captive, and very low levels in the Araguaia population. These results likely reflect larger population sizes from less urbanized environments in the Amazon basin compared to Araguaia. Populations were significantly differentiated with pairwise FST values ranging from 0.086 (Amazon × Solimões) to 0.556 (Amazon × Araguaia). Mean pairwise relatedness among individuals was significant in all populations (P < 0.01), reflecting a degree of inbreeding possibly due to severe depletion of natural stocks, the species sedentary behaviour and possible sampling biases. Although Mantel test was not significant (P = 0.104; R2 = 0.65), Bayesian analysis in STRUCTURE and discriminant analysis of principal components (DAPC) showed populations of Amazon and Solimões to be genetically differentiated from Araguaia, with Tocantins comprising individuals from both identified stocks. CONCLUSIONS: This relatively rapid genotyping by sequencing approach proved to be successful in delineating arapaima stocks. The approach and / or SNP panels identified should prove valuable for more detailed genetic studies of arapaima populations, including the elucidation of the genetic status of described discrete morphotypes and aid in delivery of conservation programs to maintain genetic diversity in reservoirs across the Amazon region.

Temperature-induced testicular germ cell loss and recovery in Nile tilapia Oreochromis niloticus.

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37 °C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27 °C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing.

Transcriptomic Analysis of Marine Gastropod Hemifusus tuba Provides Novel Insights into Conotoxin Genes.

The marine gastropod Hemifusus tuba is served as a luxury food in Asian countries and used in traditional Chinese medicine to treat lumbago and deafness. The lack of genomic data on H. tuba is a barrier to aquaculture development and functional characteristics of potential bioactive molecules are poorly understood. In the present study, we used high-throughput sequencing technologies to generate the first transcriptomic database of H. tuba. A total of 41 unique conopeptides were retrieved from 44 unigenes, containing 6-cysteine frameworks belonging to four superfamilies. Duplication of mature regions and alternative splicing were also found in some of the conopeptides, and the de novo assembly identified a total of 76,306 transcripts with an average length of 824.6 nt, of which including 75,620 (99.1%) were annotated. In addition, simple sequence repeats (SSRs) detection identified 14,000 unigenes containing 20,735 SSRs, among which, 23 polymorphic SSRs were screened. Thirteen of these markers could be amplified in Hemifusus ternatanus and seven in Rapana venosa. This study provides reports of conopeptide genes in Buccinidae for the first time as well as genomic resources for further drug development, gene discovery and population resource studies of this species.

Comparative ploidy response to experimental hydrogen peroxide exposure in Atlantic salmon (Salmo salar).

While research into the growth, survival, nutrition and, more recently, disease susceptibility of triploid Atlantic salmon has expanded, there remains an overall lack of studies assessing the response of triploids to chemical treatments. It is essential that the response of triploids to disease treatments be characterised to validate their suitability for commercial production. This study aimed to investigate and compare the stress and immune responses of triploid and diploid Atlantic salmon following an experimental treatment with hydrogen peroxide (H2O2). A dose response test was first undertaken to determine a suitable test dose for both diploid and triploid Atlantic salmon. Following this, diploids and triploids were exposed to H2O2 (1800 ppm) for 20 min, as per commercial practices, after which blood glucose and lactate, and plasma cortisol and lysozyme were measured, along with the expression of oxidative stress and immune-related genes. In the first 6 h post-exposure to H2O2, comparable mortalities occurred in both diploid and triploid Atlantic salmon. Cortisol, glucose and lactate were not significantly influenced by ploidy suggesting that, physiologically, triploid Atlantic salmon are able to cope with the stress associated with H2O2 exposure as well as their diploid counterparts. Exposure to H2O2 significantly elevated the expression of cat and sod2 in diploid livers and gr, il1ß and crp/sap1b in diploid gills, while it significantly decreased the expression of saa5 and crp/sap1a in diploid gills. In triploids, the expression levels of cat, hsp70, sod1, saa5, crp/sap1a and crp/sap1b in liver was significantly higher in fish exposed to H2O2 compared to control fish. The expression of gr, sod1 and il1ß in triploid gills was also elevated in response to H2O2 exposure. This study represents the first experimental evidence of the effects of H2O2 exposure on triploid Atlantic salmon and continues to support their application into commercial production.

Early nutritional intervention can improve utilisation of vegetable-based diets in diploid and triploid Atlantic salmon (Salmo salar L.).

The present study investigated nutritional programming in Atlantic salmon to improve utilisation of a vegetable-based diet. At first exogenous feeding, fry were fed either a marine-based diet (Diet Mstimulus, 80% fishmeal (FM)/4% fish oil (FO)) or a vegetable-based diet (Diet Vstimulus, 10% FM/0% FO) for 3 weeks. Subsequently, all fish were then fed under the same conditions with a commercial, marine-based, diet for 15 weeks and thereafter challenged with a second V diet (Diet Vchallenge, 10% FM/0% FO) for 6 weeks. Diploid and triploid siblings were run in parallel to examine ploidy effects. Growth performance, feed intake, nutrient utilisation and intestinal morphology were monitored. Fish initially given Diet Vstimulus (V-fish) showed 24 % higher growth rate and 23 % better feed efficiency compared with M-fish when later challenged with Diet Vchallenge. There was no difference in feed intake between nutritional histories, but increased nutrient retentions highlighted the improved utilisation of a V diet in V-fish. There were generally few significant effects of nutritional history or ploidy on enteritis scores in the distal intestine after the challenge phase as only V-triploids showed a significant increase (P<0·05) in total score. The data highlighted that the positive effects were most likely a result of nutritional programming and the ability to respond better when challenged later in life may be attributed to physiological and/or metabolic changes induced by the stimulus. This novel study showed the potential of nutritional programming to improve the use of plant raw material ingredients in feeds for Atlantic salmon.

A comparison of disease susceptibility and innate immune response between diploid and triploid Atlantic salmon (Salmo salar) siblings following experimental infection with Neoparamoeba perurans, causative agent of amoebic gill disease.

Few studies have focussed on the health and immunity of triploid Atlantic salmon and therefore much is still unknown about their response to commercially significant pathogens. This is important if triploid stocks are to be considered for full-scale commercial production. This study aimed to investigate and compare the response of triploid and diploid Atlantic salmon to an experimental challenge with Neoparamoeba perurans, causative agent of amoebic gill disease (AGD). This disease is economically significant for the aquaculture industry. The results indicated that ploidy had no significant effect on gross gill score or gill filaments affected, while infection and time had significant effects. Ploidy, infection and time did not affect complement or anti-protease activities. Ploidy had a significant effect on lysozyme activity at 21 days post-infection (while infection and time did not), although activity was within the ranges previously recorded for salmonids. Stock did not significantly affect any of the parameters measured. Based on the study results, it can be suggested that ploidy does not affect the manifestation or severity of AGD pathology or the serum innate immune response. Additionally, the serum immune response of diploid and triploid Atlantic salmon may not be significantly affected by amoebic gill disease.

Effects of temperature on feed intake and plasma chemistry after exhaustive exercise in triploid brown trout (Salmo trutta L).

The physiological effect of temperature on feed intake and haematological parameters after exhaustive swimming in diploid and triploid brown trout (Salmo trutta) was investigated. Trout were exposed to an incremental temperature challenge (2 °C/day) from ambient (6 °C) to either 10 or 19 °C. Feed intake profiles did not differ between ploidy at 10 °C; however, triploids had a significantly higher total feed intake at 19 °C. After 24 days, each temperature-ploidy group was exposed to exhaustive swimming for 10 min. The haematological response differed between ploidy, with the magnitude of the response affected by temperature and ploidy. Post-exercise, acid-base and ionic differences were observed. Plasma lactate increased significantly from rest for both temperature and ploidy groups, but glucose increased significantly at higher temperature. Post-exercise, triploids at 19 °C had significantly higher osmolality and cholesterol than diploids, but differences were resumed within 4 h. Elevated alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in fish at higher temperature suggested greater tissue damage; however, both ploidy responded similarly. Despite no significant differences in deformity prevalence, the type and location of deformities observed differed between ploidy (decreased intervertebral space with higher prevalence in tail area and fin regions for diploids, while vertebral compression, fusion in cranial and caudal trunks for triploids). These results suggest triploids have greater appetite than diploids at elevated temperature and that triploids suffer similar blood disturbances after exercise as diploids. These findings have implications for the management of freshwater ecosystems and suggest that stocking triploid brown trout may offer an alternative to diploid brown trout.

A comparison of the response of diploid and triploid Atlantic salmon (Salmo salar) siblings to a commercial furunculosis vaccine and subsequent experimental infection with Aeromonas salmonicida.

Sterile triploid fish represent a solution to the problems associated with sexual maturation and escapees in aquaculture. However, as disease outbreaks continue to cause significant economic losses to the industry, it is essential that the response of triploids to disease and disease treatments be characterised. The aim of this study was to compare the response of triploid Atlantic salmon to a commercial furunculosis vaccine with that of diploid fish, and to assess the vaccine efficacy in the two ploidies through an experimental infection with Aeromonas salmonicida. Diploid and triploid Atlantic salmon were injected intraperitoneally with either phosphate buffered saline, liquid paraffin adjuvant or a commercial furunculosis vaccine. Following vaccination, growth, adhesion scores and a variety of assays to assess immune function, such as respiratory burst and antibody response, were measured. Vaccination did not have a significant effect on the weight of either ploidy prior to challenge at 750° days. Adhesion scores were significantly higher in vaccinated fish compared to unvaccinated fish, although no effect of ploidy was observed. Ploidy significantly affected respiratory burst activity following vaccination, however, with triploids exhibiting higher activity than diploids. Combined with lower white blood cell numbers observed in the triploids, it may be that this low cell number is compensated for by increased cellular activity. Ploidy however, did not have a significant effect on complement activity or antibody response, with significantly higher antibody levels detected in all vaccinated fish compared to unvaccinated controls. In addition, both ploidy groups were well protected following challenge with no difference in the relative percentage survival. Based on these results, it appears that ploidy does not affect the severity of adhesions that result post-vaccinate or in the fish's immune response following vaccination, and the furunculosis vaccine performs equally well in both diploid and triploid Atlantic salmon.

Influence of tidal cycles on the endocrine control of reproductive activity in common snook (Centropomus undecimalis).

The aim of our study was to confirm the role of tidal pattern on the coordination of oocyte maturation and spawning in common snook Centropomus undecimalis. To do so, we studied oocyte maturation during the spawning season in relation to the tidal pattern in both males and females by means of histology and hormonal profiling along the pituitary-gonadal axis. Plasma LH levels, as well as transcript levels of gonadotropin genes (fshß and lhß) from the pituitaries of sexually mature male and female common snook were analyzed using a heterologous ELISA and quantitative RT-PCR, respectively. The fshß and lhß cDNAs were isolated and phylogenetic analysis of the deduced amino acid sequences revealed strong identity with other teleosts (75-90%). A strong link was found between tide and follicular development irrespective of the time of the day: female snook sampled on the rising tide were all found to have oocytes in the Secondary Growth Stage whereas females sampled at high tide or on the falling tide had oocytes in the later stages of maturation and ovulation. In addition, LH plasma and mRNA levels of fshß and lhß increased during the later stages of vitellogenesis peaking at ovulation in females. Plasma estradiol and testosterone significantly increased in late vitellogenesis (Secondary Growth Stage) and oocyte maturation (Eccentric Germinal Vesicle Step) respectively. Among male common snook sampled, no correlation was identified between tide and gonadal development. In addition, lhß mRNA expression in males peaked at the mid germinal epithelium stage as for testosterone and 11-KT in the blood while fshß expression and plasma LH levels peaked at late germinal epithelium stage. This study confirms the role played by tidal cycle on the entrainment of the later stages of oogenesis of common snook and provides a better understanding of the link between environmental and endocrine control of reproduction in this species.

Comparative study of pineal clock gene and AANAT2 expression in relation to melatonin synthesis in Atlantic salmon (Salmo salar) and European seabass (Dicentrarchus labrax).

The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 &2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway.