Does Ralstonia eutropha,rich in poly‐β hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly‐β‐hydroxybutyrate (PHB‐A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB‐A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin‐resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 10⁵ CFU/ml. Larvae pretreated 6 hr with PHB‐A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB‐A‐treated mussel larvae was 1.41‐ and 1.76‐fold higher than the non‐treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer β‐HB (1, 5, 25 and 125 mM) was tested in vitro in LB₃₅ medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of β‐HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.     

Effect of poly‐β‐hydroxybutyrate on growth,enzyme activity and intestinal microbial community of Chinese mitten crab,Eriocheir sinensis (Milne‐Edwards) juveniles

Poly‐β‐hydroxybutyrate (PHB) is microbial carbon and energy storage polymer, which can be degraded into water‐soluble β‐hydroxybutyric acid in the gastrointestinal tract of aquatic animals. A 60‐day culture experiment was performed using Chinese mitten crab, Eriocheir sinensis (Milne‐Edwards) juveniles with an average initial body weight of 0.74 ± 0.06 g which were fed a diet supplemented with 0%, 0.5%, 1%, 3% or 5% PHB. A PHB dietary supplementation of 1% and 3% significantly improved the body weight gain, moulting frequency and concomitantly reduced 2nd–3rd moulting intervals of the crabs (P < 0.05). The dietary PHB level positively related to hepatopancreatic pepsin, trypsin and lipase activity (P < 0.05). Increasing the dietary PHB also improved total superoxide dismutase activity, but reduced alkaline phosphatase and acid phosphatase activity in the serum of hemolymph (P < 0.05). A 16S rRNA gene analysis by denaturing gradient gel electrophoresis indicated that PHB supplementation led to a significantly higher range‐weighted richness, diversity and evenness of the gut bacterial community when dosed at 3% in the feed. The beneficial effects of PHB are discussed in terms of immune defense, metabolism and gut microbiota of the crabs.     

Immunostimulatory effects of dietary poly‐β‐hydroxybutyrate in European sea bass postlarvae

The stable production of high‐quality fry in marine aquaculture is still hampered by unpredictable mortality caused by infectious diseases during larval rearing. Consequently, the development of new biocontrol agents is crucial for a viable aquaculture industry. The bacterial energy storage compound poly‐β‐hydroxybutyrate (PHB) has been shown to exhibit beneficial properties on aquatic organisms such as enhanced survival, growth, disease resistance and a controlling effect on the gastrointestinal microbiota. However, the effect of PHB on the developing immune system of fish larvae has so far not been investigated. In this study, the effect of feeding PHB‐enriched Artemia nauplii on survival, growth and immune response in European sea bass (Dicentrarchus labrax) postlarvae was examined. Amorphous PHB was administered to 28‐day‐old sea bass postlarvae over a period of 10 days. The survival and growth performance were monitored, and the expression of 29 genes involved in immunity, growth, metabolism and stress‐response was measured. While the expression of the insulin‐like growth factor 1 (igf1), an indicator of relative growth, was upregulated in response to feeding PHB, the larval survival and growth performance remained unaffected. After 10 days of PHB treatment, the expression of the antimicrobial peptides dicentracin (dic) and hepcidin (hep) as well as mhc class IIa and mhc class IIb was elevated in the PHB fed postlarvae. This indicates that PHB is capable of stimulating the immune system of fish early life stages, which may be the cause of the increased resistance to diseases and robustness observed in previous studies.     

The role and mechanism of icmF in Aeromonas hydrophila survival in fish macrophages

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H₂O₂. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.     

clpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one‐sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV‐RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV‐RNAi strain failed to develop visible nodules at 5–8 dpi, with the swelling gradually disappearing. The burdens of clpV‐RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV‐RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.     

The effects of salinity on reproductive development and egg and larvae survival in the spotted scat Scatophagus argus under controlled conditions

In the present study, we report the first successful instance of controlled reproduction in Scatophagus argus, which has recently emerged as a new aquaculture resource. The controlled reproduction process for S. argus was optimized with regard to salinity acclimation. Gonadal maturation was affected by salinity in both sexes. Levels of plasma 17β‐estradiol (E₂) and 11‐ketotestosterone (11‐kT) were salinity dependent and increased significantly with the duration of acclimation. Plasma levels of gonadal steroids were higher in fish held at 25‰ salinity. The highest gonadosomatic indices (GSI), 15.1 ± 1.6 in the female and 6.4 ± 1.2 in the male, were also observed at 25‰ salinity. Nevertheless, the optimal salinity for S. argus embryonic development and larval culture was 15‰. Thus, the salinity requirement for gonadal maturation and early development are quite different. The use of advanced reproductive technologies combining salinity acclimation and stimulation of luteinizing hormone‐releasing hormone analog (LHRH‐A₂) resulted in a fertilization rate of 83.2%–91.3% and embryonic survival rates of over 90%. Embryos of S. argus at the 2‐cell, blastula, gastrula and pharyngula stages were observed. Most embryos hatched after 21.0 hr of incubation at 28.0 ± 1.0°C. The development of larvae into juveniles was completed at 40–45 days posthatch (dph). In this study, we provide information about the controlled reproduction of S. argus and identify the optimal environmental parameters for S. argus embryonic and larval culture, with the aim of developing reliable reproductive techniques for its mass production.     

Artificial substratum consisting of poly‐β‐hydroxybutyrate‐based biodegradable plastic improved the survival and overall performance of postlarval tiger shrimp Penaeus monodon

The use of artificial substratum consisting of poly‐β‐hydroxybutyrate (PHB)‐based biodegradable plastic for penaeid shrimp culture was investigated in the present study. The survival of postlarval tiger shrimp Penaeus monodon (30 ± 5 mg) provided with PHB substratum made out of PHB type DP9002 (Metabolix GmbH, Köln, Germany) was 88.7 ± 3.4% and this was significantly higher as compared to postlarvae provided conventional substratum consisting of polyvinylchloride (PVC) pipes (67.3 ± 6.5%). However, no significant weight improvement was observed for the postlarval tiger shrimp indicating that PHB could not be used as growth promoter. Nevertheless, a trend of improved robustness against adverse environmental conditions (lethal ammonium chloride concentration) and increased resistance to pathogenic Vibrio was observed in postlarval tiger shrimp provided with PHB substratum as compared to postlarvae provided with PVC substratum. Results indicate higher preference by postlarvae on PHB substratum over PVC substratum. Overall, this study indicates the potential of artificial substratum consisting of PHB‐based biodegradable plastic as replacement for conventional substratum consisting of PVC pipes in enhancing the survival of postlarval tiger shrimp and improving its performance against adverse environmental conditions and disease resistance.     

Poly‐beta‐hydroxybutyrate‐enriched Artemia sp. for giant tiger prawn Penaeus monodon larviculture

The beneficial effects of PHB as supplement for giant tiger prawn Penaeus monodon postlarvae using a short‐term enrichment strategy via Artemia were examined. The effects of co‐supplementing with a lipid emulsion were also evaluated to determine whether it yielded an additional benefit. Results on the average weight and larval development were not significantly different among postlarvae fed the different dietary treatments, indicating that PHB supplementation could not be used to stimulate growth in P. monodon postlarvae while such positive results have been reported in other aquaculture species. Nonetheless, significantly higher survival was obtained in postlarvae fed PHB‐enriched Artemia irrespective of lipid enrichment. In addition, PHB increased the survival of the postlarvae after exposure to a lethal dose of ammonia. Lipid supplementation nullified this effect. The cumulative mortality of postlarvae subjected to a sublethal concentration of ammonia for 24 h and subsequent exposure to pathogenic Vibrio campbelli showed that PHB but not lipids could effectively enhance the resistance of the postlarvae. Co‐supplementing lipids even significantly decreased this outcome. Our study indicates that PHB supplementation increases the quality of larval P. monodon and their chance of surviving under adverse environmental conditions. The short‐term co‐supplementation with lipid emulsion did not add to these effects.     

Comparative functional responses of native and high‐impacting invasive fishes: impact predictions for native prey populations

Comparative functional responses (FRs) can predict impacts of invasive species, including piscivorous fishes, via quantifying their depletion of native food resources as a function of prey density. The utility of FRs for predicting impacts on prey populations by invasive fishes of different trophic guilds was tested here by comparing the FRs of the invaders Cyprinus carpio and Carassius auratus, with three native, trophically analogous fishes, Barbus barbus, Squalius cephalus and Tinca tinca. Chironomid larvae and Gammarus pulex were used as prey items. Predictions, developed from studies on the foraging of C. carpio and C. auratus in the literature, were that the invaders would have significantly higher consumption rates for chironomids than the native fishes, but not for G. pulex. Mean consumption rates for chironomids were significantly lower for both invaders than B. barbus and S. cephalus, but were similar to T. tinca. Barbus barbus had a significantly lower consumption rate of G. pulex than both invaders, but there were no significant differences between S. cephalus, T. tinca and the invaders. All FRs were type II, with FR curves for the invaders preying upon chironomids never being significantly higher than the native fishes, contrary to predictions. For G. pulex, some significant differences were apparent between the invaders and native fishes, but again were contrary to predictions. These results indicated that when predation impacts of invasive fishes could also be a function of their population density and body sizes, these parameters should be incorporated into FR models to improve impact predictions.     

Effect of feed deprivation and refeeding on the MYP gene expression of the sea urchin (Strongylocentrotus intermedius)

To determine the main expression site of major yolk protein (MYP) gene and the mechanisms for adaptation to starvation and refeeding in Strongylocentrotus intermedius, MYP mRNA expression amounts were analysed using a real‐time RT‐PCR. The results showed that MYP could be transcribed in the intestine, stomach, gonad and coelomocytes, and that the intestine was the main expression site of MYP gene in non‐starved urchins. The MYP synthesis in the intestine decreased during 15 days of starvation (67.70%, 52.58% and 71.35% of the control at 5, 10 and 15 days of fasting respectively) and then increased dramatically by different amounts (the peaks were 2.71‐, 12.16‐ and 7.89‐fold that of the control respectively) during the refeeding stages. Nevertheless, the expression amounts in the gonads did not decline, but increased continuously during all periods of fasting (2.66‐, 3.72‐ and 13.19‐fold that of the control at 5, 10 and 15 days of starvation respectively) and during the refeeding stages. At the end of the recovery feeding experiment, the levels reached 9.58‐, 17.48‐ and 100.69‐fold that of the control. These data suggested that the ‘priority’ strategy for the sea urchin is to reduce MYP expression amounts in the intestine if food is limited and to increase MYP gene expression in the gonad to protect reproductive function.     

Energy allocation patterns in a multiple spawning sunfish: evidence for an income‐based reproductive strategy

Timing of reproduction is an important life‐history trait that varies among species as a function of energy allocation strategy, particularly as individuals are influenced by seasonal variations prior to and during the reproductive season. This study investigated size‐ and sex‐based seasonal patterns of energy allocation in bluegill, Lepomis macrochirus (Rafinesque), a species that spawns repeatedly throughout the summer, by quantifying gonad, liver and mesenteric fat masses. Results indicate that bluegill delay production of gonad tissue until the onset of the spawning season and use an ‘income spawning’ approach. Comparisons of energetic trends with crappies, Pomoxis annularis (Rafinesque) and P. nigromaculatus (Lesueur), a confamilial species of relatively similar size and morphology that spawns in the spring, highlight the flexibility of energy allocation patterns even among relatively similar fishes and shed light on bluegill life‐history strategies.     

Properties and activities of blood‐ or seawater‐contaminated wild‐caught Striped Jewfish (Stereolepis doederleini) sperm

Understanding the effects of environmental factors in sperm qualities will be helpful in the development of optimal artificial reproduction methods and contributes towards the knowledge base of better short‐ and long‐term fish semen preservation conditions The objectives of this study were to determine properties and activities of wild‐caught striped jewfish Stereolepis doederleini sperm contaminated with blood or seawater and compare them with data reported in the literature on other freshwater and marine fish species, for effective short‐ and long‐term storage of fish semen. Overall, we observed that the sodium, chloride, glucose, total protein concentrations of normal sperm were not significantly different from blood‐ or seawater‐contaminated sperm. The salinity and osmolality concentration of sperm contaminated with blood were lower than sperm contaminated with seawater and were not significantly different from normal sperm. In addition, the spermatozoa motility (SM) and duration of spermatozoa motility (DSM) in blood‐contaminated sperm were higher than seawater‐contaminated sperm and also not significantly different from normal sperm. The best condition for SM and DSM in normal sperm was dilution rate of 1:50. Sperm was immotile in distilled water, and cationic factors were shown to stimulate the initiation of spermatozoa activation. The maximum SM and DSM were observed in solution containing 0.4 M NaCl, 0.6 M KCl, 0.6 M CaCl₂ and 0.4 M MgCl₂. This study provides some basic and important knowledge about striped jewfish sperm sensitivity to a cationic condition. In this regard, Na⁺ is the major inhibitory factor of spermatozoa motility in this fish species.     

Dietary effects of poly‐β‐hydroxybutyrate on the growth performance,digestive enzyme activity,body composition,mineral uptake and bacterial challenge of rainbow trout fry (Oncorhynchus mykiss)

This study investigated the effects of diet supplementation with poly‐β‐hydroxybutyrate (PHB) on growth performance, digestive enzyme activity, lipid metabolism, mineral uptake and bacterial challenge of the rainbow trout fry (initial weight: 111.3 ± 13.7 mg) during a 6‐week experimental period. In the experimental set‐up, the commercial diet of rainbow trout fry was replaced with 0.5%, 1% and 2% PHB. The results of our work showed that the replacing of diets with PHB in experimental treatments decreases the weight of rainbow trout fry during the first 2 weeks before significantly increasing final weight at the end of the 6‐week period. PHB also improved digestive enzyme activity in experimental treatments. The highest total protease, pepsin activity and pancreatic enzyme secretion were observed with the 0.5% PHB treatment. A higher concentration of Na and K was observed in the whole body of the fry fed on 1% and 2% PHB‐supplemented diets. Enhanced survival rates occurred in all groups of fry after bath exposure to Yersinia ruckeri compared to rates in those fed the control diet. Our results suggest that the diet supplemented with PHB may improve growth performance, digestive enzyme activity and the functioning of the immune system. These positive effects could be considered for new applications in aquaculture.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

Differential leaf‐litter processing by native (Gammarus pulex) and invasive (Dikerogammarus villosus) freshwater crustaceans under environmental extremes

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Molecular and functional characterization of Raptor in mTOR pathway from Litopenaeus vannamei

Raptor, a member of the target of rapamycin complex 1 (TORC1), participates in the formation of complex proteins related to the mechanistic target of rapamycin (mTOR) signalling. In this study, a 5,020 bp cDNA of Raptor with an open reading frame (ORF) of 3,804 bp encoding for 1,267 amino acids was cloned from Litopenaeus vannamei. The protein contains three conserved domains: Raptor N, HEAT and WD40 domains. The expression of Raptor gene was detected by qRT‐PCR in different tissues of L. vannamei, including hepatopancreas, intestinal, stomach, eyestalk, gill and muscle. The mRNA expression profiles of Raptor in muscle were also analysed under suppression or stimulation of mTOR signalling pathway. The level of Raptor mRNA significantly increased either at 0.5–6 hr after an injection of rapamycin (RAPA) or after 3 days starvation. Leucine or arginine alleviated the up‐regulation of Raptor gene expression caused by RAPA or starvation. The Raptor gene was successfully suppressed using RNA interference (RNAi) technology, and the gene expression and the protein phosphorylation level of 4EBP1 and S6K were significantly decreased. The results of the study suggested that the expression of Raptor was sensitive to the immunology status of L. vannamei and participated in nutritional metabolism.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

The effects of the dietary biotin on zebrafish Danio rerio reproduction

An experiment was conducted to investigate the effects of dietary biotin on zebrafish Danio rerio reproduction. Biotin and avidin (biotin antagonist) were added to two isonitrogenous and isocaloric purified diets to provide molar avidin:biotin ratios of 0:1 (biotin‐sufficient diet) and 120:0 (biotin‐unsufficient diet). Each diet was fed to a group of males mean initial mass 0.262 g) and a group of females (mean initial mass 0.285 g) for 99 days. Following this feeding period, males fed biotin‐sufficient diet exhibited higher gonado‐somatic index, sperm density, sperm motility and sperm viability than those fed biotin‐unsufficient diet (P < 0.05). In the presence of biotin‐sufficient males, biotin‐sufficient females spawned more eggs (222.2 eggs) than biotin‐deficient females (18.8 eggs) (P < 0.05). The same pattern was observed with biotin‐deficient males (7.6 vs. 1.8 eggs) (P < 0.05). Biotin‐sufficient males generated a higher percentage of fertilized eggs (90% vs. 42%), hatching rate (62% vs. 27%), larvae survival (98% vs. 37%) and larvae length at 7 days post fertilization (4.4 mm vs. 4.2 mm) than biotin‐deficient males (P < 0.05) . Biotin status of the male is of high consideration for successful breeding in zebrafish, because it significantly impacts the reproductive performances of the female.     

Interactions between populations of the calanoid copepod Acartia tonsa Dana and the harpacticoid copepod Tisbe holothuriae Humes in mixed cultures of live feed for fish larvae

Copepods are considered high quality live feed for fish larvae due to their large within species size span, swimming pattern triggering hunting behaviour, and complete biochemical profile. In a continuous cultivation system aiming at the planktonic calanoid Acartia tonsa an intruding harpacticoid frequently appears. We experimentally evaluate how the interaction from the semi‐benthic Tisbe holothuriae is affecting our cultures and ask whether the two copepod‐species are expected to pose competitive exclusion or co‐exist long‐term. This is pursued by establishing ~40 days mono‐ and mixed copepod cultures reared in a stationary setup (S) theoretically advantageous for T. holothuriae and a rotating setup (R) with organisms kept in suspension by use of a plankton wheel, theoretically advantageous for A. tonsa. The carrying capacities of A. tonsa in either mono‐ or mixed cultures are not affected whether the copepods are subject to S or R treatment. Only exception is the S‐setup containing mixed culture were A. tonsa obtains a significantly lower carrying capacity compared with A. tonsa alone in R‐setup. The mono‐ and mixed cultures of T. holothuriae are, however, highly negatively affected by the R setup compared with S setup. A long‐term solution to limit the presence of T. holothuriae is to apply turbulence level in a classical stagnant tank setup to an extend that suppress T. holothuriae and simultaneously allow algae in suspension as food for A. tonsa, make their eggs sediment as well as minimize risk of benthic‐predation on eggs.