Influence of photoperiod and temperature manipulation on gonadal development and spawning in Caspian roach (Rutilus rutilus caspicus): Implications for artificial propagation

Caspian roach (Rutilus rutilus caspicus), a spring spawning teleost, were subjected to various photoperiod and temperature regimes to study the feasibility of shifting the timing of spawning for artificial propagation purposes. A total of 650 female reproductively mature R. rutilus caspicus were subjected to different photoperiod and temperature regimes including four light regimes (natural light (NL), 16 hr of light (L):8 hr of darkness (D), 9L:15D, 11L:13D), each affected by three temperature regimes (14, 20 and 24°C) for 70 days. Five fish per tank were randomly sampled on Feb. 10, Feb. 20, March 28, April 15 and April 30 (natural spawning time). Ovarian tissue sections were studied using light microscope and transmission electron microscope (TEM). The levels of 17‐β estradiol (E2) and 17α‐hydroxyprogesterone (OHP) were also measured in the serum samples. In late winter (March 28th), the gonadal maturation and spawning were accelerated in fish treated with the long day length (16L/8D) and warm temperature (20°C). While, the maturation of oocytes and spawning delayed in fish exposed to low temperature (14°C) and short day length (9L/15D and 11L/13D). Photoperiod seems to play a more important role in the ovarian development of the R. rutilus caspicus compared to temperature; since even among the fish treated with the lowest temperature (14°C), those exposed to a longer day length (16L/8D), matured and spawned earlier than the others. Considering that the earliest spawning occurred in R. rutilus caspicus treated with 16L/8D at 20°C and the latest spawning occurred in fish exposed to low temperature and short photoperiod, it can be concluded that temperature and photoperiod play an important role in accelerating oocyte maturation and spawning.     

Maltoporin (LamB protein) contributes to the virulence and adhesion of Aeromonas veronii TH0426

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A. veronii TH0426 mutant strain (ΔlamB) with an in‐frame deletion removed nucleotides 10–1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD₅₀ value of the mutant ΔlamB to zebrafish and mice was 13.7‐fold and 5.6‐fold higher than those of the wild‐type strain, respectively. The toxicity of wild‐type strain to EPC cells was 2.1‐fold and threefold higher than those of ?lamB when infected for 1 and 2 hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ?lamB significantly decreased for 5.6‐fold and 1.8‐fold separately. In addition, motility detection result indicated that ?lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ?lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A. veronii TH0426, and it can be known that the lamB gene of A. veronii plays a crucial role in the pathogenesis.     

Screening of a high bioflocculant‐producing bacterial strain from an intensive fish pond and comparison of the bioflocculation effects with Rhodococcus erythropolis

A high bioflocculant‐producing bacterial strain BX‐13 isolated from an intensive fish pond by multiple subcultures and measured through primary screening and rescreening using kaolin. The strain BX‐13 was identified to belong to the same branch as Bacillus subtilis by morphological, physiological and biochemical, 16S rDNA gene sequencing, Blast homology search and multiple sequence alignments. The bioflocculation potential of BX‐13 and Rhodococcus erythropolis was compared, using water from the intensive pond where BX‐13 was isolated. The results showed the number of colonies in the BX‐13 treatment was higher than that of the R. erythropolis treatment from day 10 to the end of the experiment (p < 0.05). The numbers of both BX‐13 and R. erythropolis in the water body peaked on day 15, with 2.23 × 10⁷ cfu/L and 9.90 × 10⁶ cfu/L respectively. The bioflocculation volume (BFV) in water increased and then stabilized in both BX‐13 and R. erythropolis treatments. The BFV in BX‐13 peaked on day 25 (52.13 ml/L) and R. erythropolis peaked on day 30 (22.63 ml/L). At the end of the experiment, the BFV of the BX‐13 treatment was higher than the R. erythropolis treatment (p < 0.05). In conclusion, B. subtilis BX‐13 has a very strong flocculation effect, and merits further research into its application for water quality regulation in aquaculture systems.     

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability,isolated from freshwater aquaculture pond

Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca²⁺ 0.8, Mg²⁺ 0.2, Mn²⁺ 0.4, Fe²⁺ 0.6, Al³⁺ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics.     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

Genomic comparison of virulent and non‐virulent Streptococcus agalactiae in fish

Streptococcus agalactiae infections in fish are predominantly caused by beta‐haemolytic strains of clonal complex (CC) 7, notably its namesake sequence type (ST) 7, or by non‐haemolytic strains of CC552, including the globally distributed ST260. In contrast, CC23, including its namesake ST23, has been associated with a wide homeothermic and poikilothermic host range, but never with fish. The aim of this study was to determine whether ST23 is virulent in fish and to identify genomic markers of fish adaptation of S. agalactiae. Intraperitoneal challenge of Nile tilapia, Oreochromis niloticus (Linnaeus), showed that ST260 is lethal at doses down to 10² cfu per fish, whereas ST23 does not cause disease at 10⁷ cfu per fish. Comparison of the genome sequence of ST260 and ST23 with those of strains derived from fish, cattle and humans revealed the presence of genomic elements that are unique to subpopulations of S. agalactiae that have the ability to infect fish (CC7 and CC552). These loci occurred in clusters exhibiting typical signatures of mobile genetic elements. PCR‐based screening of a collection of isolates from multiple host species confirmed the association of selected genes with fish‐derived strains. Several fish‐associated genes encode proteins that potentially provide fitness in the aquatic environment.     

Virulence regulation of cel‐EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel‐EIIB protein‐mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel‐EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel‐EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel‐EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel‐EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel‐EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel‐EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel‐EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel‐EIIB strain (p < 0.05). In conclusion, cel‐EIIB protein‐mediated cel‐PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Molecular characterization of transformer‐2 gene in the mud crab Scylla paramamosain and its putative role in sexual development

Transformer‐2 (Tra‐2) is an mRNA splicing factor that acts in concert with Tra to regulate the female sexual development in Drosophila melanogaster. In decapods, as no Tra homologues have been identified, the role of Tra‐2 in sexual development is more often discussed accordingly. In the present study, the full‐length cDNA of Tra‐2 of the mud crab Scylla paramamosain (SpTra‐2) was cloned and characterized. The deduced amino acid sequence of SpTra‐2 contained an RNA‐recognition motif (RRM) flanking by two RS‐rich regions, which are the hallmarks of the Tra‐2 proteins. The SpTra‐2 mRNA was highly abundant in the hepatopancreas, muscles and androgenic glands of the male crabs, implying a potential role in male sexual development. The hypothesis was supported by the RNAi experiments, finding that the injection of SpTra‐2 dsRNA in vivo led to a decreased expression of SpIAG, a key gene in crustacean male sexual differentiation. The relationship between SpTra‐2 and SpIAG was further affirmed by eyestalk ablation (ESA) experiments, which caused SpTra‐2 expression earlier than SpIAG expression in both androgenic glands and hepatopancreas. In addition, treatment with SpTra‐2 dsRNA also reduced the mRNA level of SpSxl, but had no effects on SpDmrt expression, suggesting the sex determination system in S. paramamosain is different from that in D. melanogaster.     

Aeromonas shuberti as a cause of multi‐organ necrosis in internal organs of Nile tilapia,Oreochromis niloticus

A disease with white spots in internal organs of Nile tilapia occurred in Zhanjiang, southern China. Multiple, white nodules, 0.8–2.2 mm in diameter, were scattered throughout the liver, spleen and kidney of diseased fish. Signs of nodules reproduced after artificial infection with the isolated strain. Isolated bacteria were Gram‐negative, facultative anaerobic, motile, short rod‐shaped, with a length of 1.2–2.2 μm. Morphological and biochemical tests, as well as phylogenetic analysis, all strongly indicated that the isolate from tilapia is identical to Aeromonas schubertii (A. schubertii) which temporary named LF1708 strain. Antibiotic sensitivity assays showed the LF1708 is sensitive to 24 of 27 tested antibiotics. Pathogenicity test revealed that the isolate at the dose of 3.75 × 10⁶ CFU/g killed 100% of experimental tilapia within 2 days and the dose of 1 × 10⁷ CFU/g killed 100% of experimental zebrafish within 1 day. Histopathology of diseased tilapia infected with A. schubertii showed numerous necrotic lesions widely distributed in spleen, liver and kidney, and infiltration with a large number of bacteria. To our knowledge, this was the first report that associated A. schubertii with mortality in tilapia.     

Effects of γ‐aminobutyric acid supplementation on the growth performance,serum biochemical indices and antioxidant status of pharaoh cuttlefish,Sepia pharaonis

This study evaluated the effects of dietary γ‐aminobutyric acid (GABA) on the growth performance, serum biochemical indices and antioxidant status of pharaoh cuttlefish, Sepia pharaonis. Cuttlefish were cultured in open‐culturing cement pool systems for 8 weeks. Six practical diets supplemented with graded levels of GABA (0, 20, 40, 60, 80 and 100 mg/kg) were formulated. Each diet was randomly assigned to triplicate groups of 60 cuttlefish (mean weight: 10.33 g), the cuttlefish were fed two times per day to apparent satiation. The results showed that the specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) significantly increased with dietary GABA supplementation (p < .05). The survival rate (SR) and protein content in muscle significantly increased when 58.9 mg/kg GABA supplied. Moreover, the nitric oxide (NO) content and acid phosphatase (ACP) activity in serum were significantly increased with dietary GABA supplementation (p < .05), while the activity of aspartate aminotransferase (AST) in serum decreased significantly when supplied with GABA at 58.9 mg/kg (p < .05). In addition, dietary GABA improved antioxidation activity by significantly increasing the activities of superoxide dismutase (SOD) and catalase (CAT) but decreasing malondialdehyde (MDA) levels in the liver and gill (p < .05). On the basis of the quadratic regression analysis of FE, the optimum content of dietary GABA in S. pharaonis was estimated to be 55.3 mg/kg. The findings of this study demonstrated that dietary GABA had a positive effect on the growth performance, serum biochemical indices and antioxidant status of S. pharaonis.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

The effects of elevated pCO2 on growth,shell production and metabolism of cultured juvenile abalone,Haliotis iris

Reduced seawater pH and elevated pCO₂ are important considerations in tank‐based abalone aquaculture, while sea‐based farms may be at risk to ocean acidification reductions in pH. Juvenile Haliotis iris (5–13 and 30–40 mm shell length) were reared in two, 100‐day experiments at ambient pH_nbs (~ 8.1, 450 μatm CO₂), pH 7.8 (~1000 μatm CO₂) and pH 7.6 (~1600 μatm CO₂). Seawater pH was measured and adjusted automatically by bubbling CO₂ into water in replicated flow through tanks. Two separate trials were run, in winter (8.8°C) and summer (16.5°C). Survival and growth were monitored every 30 days, and post experiment measurements of morphometrics and respiration rate undertaken. Growth of shell length and wet weight were negatively affected by reduced pH, with a 2 to 3‐fold reduction in growth of both size classes between ambient and pH 7.6 treatments in the summer experiment. For small juveniles, growth reductions were in conjunction with decreases to shell weight, while large juveniles showed greater resilience in shell production. No changes to respiration rate occurred, suggesting that juveniles may maintain physiological functioning while tolerating dissolution pressure or that they are unable to upregulate metabolism to compensate for pH effects. These data show that CO₂ driven reductions in pH can impact growth, metabolism and biomineralization of abalone, and indicate that water quality and ocean acidification are of importance in aquaculture of the species.     

Siltation increases the susceptibility of surface‐cultured eastern oysters (Crassostrea virginica) to parasitism by the mudworm Polydora websteri

Mudworm (Polydora websteri) parasitism is known to result in unsightly mud blisters in eastern oysters (Crassostrea virginica), resulting in reduced product quality and increased vulnerability to illness and environmental stress. While typically a concern only for bottom‐grown oysters, an abnormal severe outbreak of P. websteri in surface‐cultured oysters in New Brunswick, Canada, was reported in 2013, along with an anecdotally reported concurrent increase in siltation. Although heavier loads of silt are reported to increase P. websteri infestations in oysters, studies report mixed effects of siltation to this regard. Here, we report the results of a field experiment testing the effect of siltation on P. websteri recruitment in surface‐grown oysters at an aquaculture site in Richibucto, New Brunswick. Live oysters of similar size were deployed at the aquaculture site and were left to collect P. websteri recruits under one of two siltation treatments (high vs. low) for 50 days. Results suggested that P. websteri recruitment correlated with metrics of oyster size (shell weight, length, width, depth and surface area). When P. websteri counts were standardized for oyster shell morphometry, P. websteri recruitment was significantly higher in oysters exposed to the high siltation treatment, accumulating approximately 1.5× as many P. websteri as oysters exposed to the low siltation treatment. Our results suggest that higher amounts of siltation on surface‐cultured oysters can result in increased rates of P. websteri parasitism. Enhanced cleaning regimes may help to alleviate the impacts of P. websteri in surface‐grown oysters, although other mitigation strategies exist.     

Dietary β‐glucan modulate haematological parameters,cytokines and gene expression in TLR and ERK pathways of rainbow trout (Oncorhynchus mykiss) during infection by Aeromonas salmonicida

To explore the role of β‐glucan (0, 0.05%, 0.1% and 0.2%) in resisting bacteria, rainbow trout (Oncorhynchus mykiss) was challenged with Aeromonas salmonicida after 42‐day β‐glucan administration, and then sampled at 0, 2, 4 and 6 days post infection (dpi). The data showed that 0.2% β‐glucan reduced the accumulated mortality rates compared with the ICG (infected control group) (p < .05). The white blood cells, red blood cells and haemoglobin were higher in the 0.2% β‐glucan group (BG) than the ICG (p < .05). 0.1% and 0.2% β‐glucan elevated serum total antioxidative capability and glutathione activity but alleviated the increase of serum alkaline phosphatase activity and glucose concentration (p < .05) during infection. Serum TNF‐α, IL‐1β, IL‐8 and IgM in three BGs elevated remarkably on 6 dpi compared with the ICG (p < .05). Expression of tnf, il1b and cxcl8 of the head kidney in the 0.1% and 0.2% BGs was higher than the ICG on 4 dpi while ighm expression in the 0.2% BG was higher than in the ICG on 2 and 6 dpi (p < .05). 0.1% and 0.2% β‐glucan increased the expression of tlr5m, tlr5s, tmek and myd88 in the spleen after infection (p < .05). Similarly, 0.2% β‐glucan up‐regulated the expression of tmek, myd88, oncmyk‐dab and c3 in head kidney (p < .05). Overall, 0.2% dietary β‐glucan effectively decreased accumulated mortality rate by modulating the biochemistry process, cytokines, and activating TLR and ERK signalling pathways during A. salmonicida infection.