Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> from tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis> sp.) transmitted to a new host,bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>), in China

Several outbreaks of Streptococcus agalactiae infection of bighead carp (Aristichthys nobilis) were observed in China. The molecular epidemiology and pathogenicity of S. agalactiae in bighead carp and tilapia (Oreochromis sp.) is poorly understood. In the present study, we identified S. agalactiae strains isolated from diseased bighead carp using the API 20 Strep kit and 16S rDNA sequencing and determined whether these strains came from tilapia. Of the 46 identified S. agalactiae strains, 24 strains were successfully isolated from diseased bighead carps, 20 S. agalactiae strains were isolated from tilapia, and two S. agalactiae strains were isolated from tiger frog (Hoplobatrachus chinensis). The results of molecular typing, including multilocus sequence typing, molecular serotyping, surface protein gene detection, and virulence-related gene detection showed that the 44 strains from bighead carp and tilapia were highly similar, whereas different from tiger frog GBS strains. Remarkably, the bighead carp strain Hn1404 showed high virulence in bighead carp and zebrafish. Moreover, this strain was pathogenic to Nile tilapia (Oreochromis niloticus). In addition, comparative genomic analysis showed that isolate Hn1404 had a close relationship with the bighead carp and tilapia S. agalactiae strains. All the analyses of the genetic characteristics of bighead carp and tilapia strains showed that tilapia S. agalactiae strains could be transmitted to other fish species such as bighead carp.     

Environmental factors on virulence of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Aeromonas hydrophila are known for being opportunistic pathogens, harboring various virulence factors and triggering lesions and death in fish. The disease caused by bacteria can make fish inappropriate for human consumption, besides representing a risk to public health. The pathogenesis can be influenced by environmental variables, affecting fish productivity and mortality. The present study aimed to determine whether A. hydrophila harbor the virulence genes aerolysin, hydrolipase, elastase, lipase, cytotonic enterotoxin (ast), lateral flagellum (laf), and polar flagellum (fla) and to evaluate the influence of environmental variables on in vitro growth, in vivo virulence and expression of some of these genes. Polymerase chain reaction (PCR)-based screening for the presence of these virulence genes was performed on 35 isolates. Six isolates containing different profiles of virulence genes were tested for in vitro growth under different conditions of pH, temperature, and ammonia and for in vivo virulence under these same environmental conditions. RT-qPCR was used to quantify the expression of aerolysin, lipase, and fla genes. All the tested environmental factors influenced the growth of A. hydrophila, while pH and ammonia concentrations influenced the bacterial virulence. The expression of the fla gene increased when bacteria were grown in higher ammonia concentration. The mortality established by Aeromonas is influenced by several environmental factors pinpointing the importance of its control in fish farming to avoid higher economic loses associated to bacterial disease outbreaks.     

Immunomodulatory effects of <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> leaf extract and its derivative compound,rhodomyrtone, on head kidney macrophages of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 μg mL^?1 of R. tomentosa and 1 μg mL^?1 of rhodomyrtone for 4 and 24 h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.     

Dietary supplementation of yeast (<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>) improves growth,stress tolerance,and disease resistance in juvenile Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

The yeast Saccharomyces cerevisiae is one of the commonest probiotics incorporated in aquafeeds. An 84-day feeding trial was conducted to evaluate the effects of varying dietary inclusions of S. cerevisiae, 0% (control), 3% (YF3), 5% (YF5), and 7% (YF7), on growth, stress tolerance, and disease resistance in juvenile (body mass ~?21 g) Nile tilapia (Oreochromis niloticus). Fish were randomly distributed in groups of 20 into 12 1-m³ hapas and fed isoenergetic (~?17 kJ g^?1 gross energy) and isonitrogenous (~?300 g kg^?1 crude protein) diets at 3% of their bulk weight daily. Specific growth rates were significantly higher for the yeast-fed fish (0.77–0.78% day^?1) than for the control fish (0.60% day^?1) and resulted in significantly higher mean final weights for the yeast-treated groups. Protein and lipid retention efficiencies were also significantly higher in the yeast-fed fish than in the control group. In subsequent stress challenge trials, the yeast-fed fish had greater tolerance to acute heat as well as hypoxia exposure than the control fish. Survival rates of the yeast-treated groups following sudden exposure to elevated water temperature (40 °C) ranged from 82.5 to 100% compared to 15% for the control. Mean survival rates following a 24-h hypoxia exposure were also generally significantly higher for the yeast-fed fish. The probiotic groups recorded a relative percent survival (RPS) of 75% after a 14-day Aeromonas hydrophila infection challenge. The results of this study indicate that S. cerevisiae as an additive in Nile tilapia diets has beneficial impacts on growth, stress tolerance, and disease resistance.     

Morphology,sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154–334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574–964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal.     

Diethylstilbestrol arrested spermatogenesis and somatic growth in the juveniles of yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>), a fish with sexual dimorphic growth

In fish, spermatogenesis and somatic growth are mainly regulated by hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-somatic (HPS) axes, respectively. Xenoestrogens have been reported to impair spermatogenesis in some fishes, and arrest somatic growth in some others, whereas, whether xenoestrogens are capable of disrupting spermatogenesis and somatic growth simultaneously in fish that exhibits sexual dimorphic growth is little known, and the underlying mechanisms remain poorly understood. In this study, male juveniles of yellow catfish (Pelteobagrus fulvidraco), which exhibits a sexual dimorphic growth that favors males, were exposed to diethylstilbestrol (DES) for 28 days. After exposure, DES significantly disrupted the spermatogenesis (decreased gonadal-somatic index (GSI) and germ cell number) and arrested the somatic growth (declined body weight) of the catfish juveniles. Gene expression and plasma steroid analyses demonstrated the suppressed mRNA levels of genes in HPG axis (gnrh-II, fshβ, and lhβ in the brain and dmrt1, sf1, fshr, cyp17a1, cyp19a1a, and cyp11b2 in the testis) and decreased 17β-estrodial (E2) and 11-ketotestosterone (11-KT) levels in plasma. Further analysis revealed the arrested germ cell proliferation (cyclin d1), meiosis (dmc1, sycp3), and enhanced apoptosis (decreased bcl-2 and elevated bax/bcl-2 ratio) in the testis. Besides, DES also suppressed the mRNA levels of genes in HPS axis (ghrh, gh, and prl in the brain and ghr, igf1, igf2a, and igf2b in the liver). The suppressed HPG and HPS axes were thus supposed to disturb spermatogenesis and arrest somatic growth in yellow catfish. The present study greatly extended our understanding on the mechanisms underlying the toxicity of DES on spermatogenesis and somatic growth of fish.     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.     

New insights into the evolution,hormonal regulation,and spatiotemporal expression profiles of genes involved in the Gfra1/Gdnf and Kit/Kitlg regulatory pathways in rainbow trout testis

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20β progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.     

Effect of growth hormone overexpression on gastric evacuation rate in coho salmon

Growth hormone (GH) transgenic (T) coho salmon consistently show remarkably enhanced growth associated with increased appetite and food consumption compared to non-transgenic wild-type (NT) coho salmon. To improve understanding of the mechanism by which GH overexpression mediates food intake and digestion in T fish, feed intake and gastric evacuation rate (over 7 days) were measured in size-matched T and NT coho salmon. T fish displayed greatly enhanced feed intake levels (~ 2.5-fold), and more than 3-fold increase in gastric evacuation rates relative to NT coho salmon. Despite the differences in feed intake, no differences were noted in the time taken from first ingestion of food to stomach evacuation between genotypes. These results indicate that enhanced feed intake is coupled with an overall increased processing rate to enhance energy intake by T fish. To further investigate the molecular basis of these responses, we examined the messenger RNA (mRNA) levels of several genes in appetite- and gastric-regulation pathways (Agrp1, Bbs, Cart, Cck, Glp, Ghrelin, Grp, Leptin, Mc4r, Npy, and Pomc) by qPCR analyses in the brain (hypothalamus, preoptic area) and pituitary, and in peripheral tissues associated with digestion (liver, stomach, intestine, and adipose tissue). Significant increases in mRNA levels were found for Agrp1 in the preoptic area (POA) of the brain, and Grp and Pomc in pituitary for T coho salmon relative to NT. Mch and Npy showed significantly lower mRNA levels than NT fish in all brain tissues examined across all time-points after feeding. Mc4r and Cart for T showed significantly lower mRNA levels than NT in the POA and hypothalamus, respectively. In the case of peripheral tissues, T fish had lower mRNA levels of Glp and Leptin than NT fish in the intestine and adipose tissue, respectively. Grp, Cck, Bbs, Glp, and Leptin in stomach, adipose tissue, and/or intestine showed significant differences across the time-points after feeding, but Ghrelin showed no significant difference between T and NT fish in all tested tissues.     

A growth comparison among three commercial tilapia species in a biofloc system

Despite a worldwide boom in tilapia aquaculture, South Africa has failed to follow suit, resulting in a small, very much undeveloped local industry. Much of the justification for this reality lies on the inadequacy of the species used and the stringent legislation preventing the use of the Nile tilapia, Oreochromis niloticus. With momentum gained in recent years, the local industry lobbying for the legalization of the O. niloticus has made great progress and its use in certain systems has recently been facilitated. This study was conducted to compare the growth performance between the indigenous Mozambique tilapia, Oreochromis mossambicus, another African alternative gaining momentum in Zambia, the Three Spot tilapia, Oreochromis andersonii, and the globally reputable O. niloticus. The study was conducted in a biofloc technology (BFT) system, showing great potential as an environmentally friendly technology. The trial was run for 10 weeks with 60 fish/tank stocked into four repeats tanks per species. Fish were sampled on six occasions with a regression fitted to the mass data. Oreochromis niloticus showed a significantly higher growth rate with an average daily gain of 0.693 ± 0.018 g/day and the lowest feed conversion ratio (FCR) of 1.01 ± 0.05, followed by O. mossambicus with average daily gain of 0.405 ± 0.025 g/day and FCR of 2.24 ± 0.16, and then O. andersonii with an average daily gain of 0.185 ± 0.025 g/day and an FCR of 2.53 ± 0.28, respectively. From this study, O. niloticus therefore appears to be the most adequate species for use in BFT systems in South Africa.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Importance of <Emphasis Type="Italic">Gammarus</Emphasis> in aquaculture

In recent years, there have been significant increases in the production and breeding of aquaculture species; for example, the gilt-head (sea) bream and seabass in salt water and trout, carp, and tilapia in fresh water., Amphipods in the genus Gammarus are used as a rich source of protein to feed various pet animals such as aquarium fish, lizards, and turtles, as well as in aquatic ecotoxicology and water quality assessment. Gammarus meal is used as a partial replacement (10–20%) of fish meal. However, the reproduction and rearing of Gammarus in intensive or semi-intensive conditions has not yet been carried out. Gammarus products (live, dried flakes, or powdered) transported to the markets are constituted from wild caught Gammarus. Due to the high price of these products, Gammarus aquaculture potentially has great economic benefits. Therefore, there is a need to focus on the importance of Gammarus in aquaculture. In the present review, the ecology and distribution of Gammarus, its importance in aquatic ecotoxicology and water quality assessment, the chemical analysis of Gammarus and factors affecting chemical composition of it, and the importance of Gammarus in fish nutrition are discussed. In addition, the economic importance and culture methods of Gammarus are also reviewed. This review will be beneficial for scientific investigators and fish-crustacean farmers.     

Evaluation of potential probiotics isolated from saline tilapia in shrimp aquaculture

Integration of tilapia to shrimp culture is currently being practiced to minimize the growth of pathogenic luminous bacteria. The microorganisms that are associated in tilapia may contribute to the inhibition of the growth of Vibrio harveyi through the production of secondary metabolites. In this study, two Bacillus strains (MJA1.1, MJA2.1) isolated from mucus of tilapia were evaluated for their possible application in shrimp culture. The inhibitory property of these isolates against V. harveyi was determined in vitro using co-culture assay in a liquid medium. Also qualitative extracellular enzyme assay was conducted to assess whether the bacterial isolates produce extracellular enzymes. Furthermore, the potential use of these isolates as shrimp feed additive was tested. Thereafter, shrimps were exposed to lethal dose of ammonia (140 mg l^?1) to test the effects of the isolates in vivo. The results showed that in vitro co-culture assay after 72 h caused a significant decline in the population of V. harveyi in treatments with potential probiotic isolates. Both isolates showed protease, amylase, and cellulase activities. Although no significant difference was observed in growth, survival was significantly higher in shrimp fed with diets added with either of the isolates. The shrimp exposed to lethal dose of ammonia demonstrated better survival when supplemented with the probionts compared to the control group. Thus, the efficiency of the isolates in inhibiting V. harveyi population and the improvement of survival and resistance of cultured shrimp to ammonia stress indicate their potential as probionts for shrimp culture.