Isolation,characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.     

Acute toxicity of emamectin benzoate (SLICE™) in fish feed to American lobster, Homarus americanus

SLICE? (active ingredient 0.2% emamectin benzoate (EMB)), a feed premix developed by Schering‐Plough Animal Health for the control of sea lice on cultured salmonids, is registered for use in several countries and is being prescribed on an emergency basis in Canada and the United States. The concentration of EMB in feed administered to farmed salmon ranges from 1 to 25 μg g^?1. To determine the acute toxicity of the compound to juvenile and adult American lobster (Homarus americanus), commercial salmon feed was coated with SLICE? at a range of concentrations and provided to the animals for 7 d in the laboratory. The LC₅₀ is estimated to be 644 μg g^?1 (95% CI=428, 1275) for adult lobsters and >589 μg g^?1 for stage V and VI juvenile lobsters. The consumption of medicated pellets by adult lobsters decreased significantly with increasing concentration of EMB. Adult lobsters that died during the study had a significantly greater concentration of emamectin B1a in their muscle tissue than those that survived. These results support the conclusion that salmon feed medicated with EMB at the concentrations used by the aquaculture industry is unlikely to pose an acute lethal threat to adult and small juvenile American lobsters.     

A comparative molecular study of the presence of “Candidatus arthromitus” in the digestive system of rainbow trout,Oncorhynchus mykiss (Walbaum), healthy and affected with rainbow trout gastroenteritis

Observations were made using histopathological techniques in conjunction with a nested polymerase chain reaction (PCR) protocol for the specific detection of “Candidatus arthromitus” on DNA extracted from wax‐embedded tissues and fresh digestive contents of rainbow trout. Samples positive for “Candidatus arthromitus” DNA included fish with rainbow trout gastroenteritis (RTGE), clinically normal cohabiting fish, and apparently healthy controls from RTGE positive and RTGE negative sites. The results obtained from the PCR were confirmed by nucleotide sequencing. “Candidatus arthromitus” DNA was found in distal intestine as well as in sections of pyloric caeca, suggesting that both these locations are appropriate for molecular detection of “Candidatus arthromitus” DNA in trout. Furthermore, rainbow trout fry distal intestinal samples from two different hatcheries where RTGE had not been reported were also positive. Differences in “Candidatus arthromitus” DNA detection between paraffin wax‐embedded and fresh digestive content samples from the same fish suggested that it may be predominantly epithelium‐associated in healthy trout. Parallel histopathological observations indicated that pyloric caeca are the preferred site for visualizing segmented filamentous bacteria (SFB) in trout with RTGE. The results of this study showed that the presence of SFB was not invariably associated with clinical disease and that more information is required to understand the role of these organisms.     

云龙石斑鱼胃排空特征和摄食消化特性研究

为阐明养殖新品种云龙石斑鱼(Epinephelus moara♀×E.lanceolatus♂)摄食胃排空特征和消化酶活性变化规律,本研究检测了体重为(680.35±39.84)g云龙石斑鱼摄食后胃内容物百分比,比较了线性模型、平方根模型和立方模型对胃排空曲线的拟合程度,分析了云龙石斑鱼摄食后血清中葡萄糖、皮质醇含量和肝脏消化酶活性变化,并对胃排空率与消化酶进行了相关性分析。结果发现,云龙石斑鱼摄食后,胃排空率呈典型的先慢后快再慢的消化类型。3种模型均能拟合云龙石斑鱼的胃排空数据,其中,立方模型拟合效果最佳。由立方模型可知,云龙石斑鱼胃80%排空(食欲基本恢复)时间为9.5 h,100%排空(食欲完全恢复)约为14.8 h。血清中葡萄糖、皮质醇含量及肝脏中消化酶(淀粉酶、糜蛋白酶和脂肪酶)活性在摄食胃排空过程中均呈先升高后降低的趋势。摄食后,血清中皮质醇、葡萄糖含量和肝脏内消化酶活性逐渐升高,0~3 h上升速率较慢,3~6 h内呈急剧上升的变化趋势,且在摄食后6 h,血清中皮质醇、葡萄糖含量和肝脏内消化酶活性达到最高(P<0.05),之后呈显著下降趋势,云龙石斑鱼瞬时排空速率与消化酶活性呈正相关(P<0.05)。综上,基于云龙石斑鱼胃排空特征和摄食消化特性,结合生产实践,在食欲基本恢复后开始投喂,投喂间隔为10 h左右,每日投喂2次,效果最佳。     

Digestive dynamics during chyme formation of Octopus maya (Mollusca,Cephalopoda)

In the present study, digestive dynamics, measured through the changes in the in situ pH along the digestive tract, was evaluated along with the glycogen concentration in the digestive gland (DG) and the proteolytic enzyme activity of the gastric juice (GJ) (both acid and alkaline proteases) to obtain useful information that will allow for the understanding of the DG's function and the role that it plays as an extracellular digestive enzyme source and for energy storage, providing new information on the digestive physiology of Octopus maya. The results showed that pH along the digestive tract changed according to the postprandial time following the food transit. At the beginning, the pH on crop (St) was 5, changing to 6 when the food arrived. Similar changes were observed on the caecum (Ce) and DG. Glycogen from DG is used as a source of energy during digestion and recovered 8 h after feeding. Maximum activities of digestive gland GJ enzymes were observed 6 h after feeding, indicating that chyme enzymes are still active when they arrive at the DG's lumen. The presence of food in the digestive tract modifies the pH, which, in combination with GJ, favours the activity of the released enzymes of O. maya.     

Experimental evaluation of the potential for crayfish plague transmission through the digestive system of warm-blooded predators

The crayfish plague pathogen (Aphanomyces astaci) can be transmitted through the digestive system of fish, but its dispersal through mammalian and bird digestive tracts has been considered unlikely, and direct experimental evidence remains scarce. We present a small-scale transmission experiment with European otter and American mink fed with infected crayfish, and experiments testing survival of cultures of five A. astaci strains at temperatures corresponding to those inside mammal and bird bodies. The pathogen was neither isolated from predator excrements nor transmitted to susceptible crayfish exposed to excrements. In agar-based artificial media, it occasionally survived for 15 min at 40.5°C and for 45 min at 37.5°C, but not so when incubated at those temperatures for 45 min and 75 min, respectively. The five tested strains differed in resistance to high temperatures, two (of genotype groups E and D) being more susceptible than other three (of groups A, B and D). Their survival to some extent varied when exposed to the same temperature after several weeks or months, suggesting that some yet-unknown factors may influence A. astaci resistance to temperature stress. Overall, we support the notion that passage through the digestive tract of warm-blooded predators makes A. astaci transmission unlikely.     

The Effect of Diet on Weight Gain,Shell Hardness,and Flavor of New-Shell American Lobster,Homarus americanus

Abstract

Harvested newly-shed American lobsters, Homants americanus, placed in pounds for long-term storage are prone to damage resulting from high-density confinement. It is beneficial for the new shell to harden as soon as possible after shedding. Artificial diets offer the potential for adding supplements to increase weight, muscle gain, and shell hardening. In this study, lobsters were fed 5 different diets: herring, fish racks, and prepared diets with three levels of vitamin D for a period of 38 days. After the feeding trial, lobsters were reweighed, and samples of the carapace and claw shell were removed and subjected to a compressive force/deformation test to measure shell strength. Results indicate a significant (P< 0.05) difference in weight gain, with the highest level of vitamin D actually inhibiting gain. Subsamples tested for compressive force yielded a positive correlation between the carapace measurement and feed type. Flavor tests on subsamples showed significant differences between all treatments; herring was the best, followed by pelleted feed and then fish racks. No off-flavors were detected in lobsters fed any of the three diet types.     

The Influence of Ovarian Fluid pH of Stripped Unfertilized Channel Catfish,Ictalurus punctatus,Eggs on the Hatching Success of Channel Catfish ♀ x Blue Catfish,Ictalurus furcatus♂, Hybrid Catfish Eggs

This study was designed to determine the effect of ovarian fluid pH of stripped unfertilized channel catfish, Ictalurus punctatus, eggs on fertilization and hatch rate of channel catfish ♀ x blue catfish, Ictalurus furcatus♂, hybrid catfish eggs. A significant correlation was established between ovarian fluid pH of stripped channel catfish eggs and hybrid embryo hatch rate (R² = 0.75, P = 0.01), suggesting ovarian fluid pH of stripped catfish eggs prior to fertilization can be predictive of the hatching success of hybrid catfish embryos. These data were used to categorize pH of stripped eggs: pH <7.0 as “low pH eggs,” pH 7.0–7.4 as “medium pH eggs,” or pH >7.4 as “high pH eggs” quality eggs. The range in percent hatch rate for these pH categories was <15%, 15–30%, and >30%, respectively. Higher calcium concentrations during incubation do not appear to improve hatching success of “low pH eggs.” The predictive model presented herein describes a quick method for evaluating stripped catfish eggs for hybrid fry production in catfish hatcheries.     

Gaffkemia in the California spiny lobster, Panulirus interruptus: Infection and immunization

Infection (gaffkemia) can be induced in the California spiny lobster (P. interruptus) with injections of Pediococcus homari (formerly Gaffkya homari). An LD_{5 0} of 10³ microbes per ml hemolymph is observed at 17°C. Prior immunization of lobsters (P. interruptus) with an avirulent strain of P. homari protects lobsters against 100 × LD_{5 0}. This is the first report of acquired immunity to P. homari in a susceptible species. The high incidence of gaffkemia in American lobsters (Homarus americanus) held in live holding facilities makes prophylactic measures essential in regions outside the enzootic area of the North Atlantic to avoid inadvertent transplantation of the disease to other susceptible populations of animals.     

Hatchery performance of early benthic juvenile American lobsters (Homarus americanus) fed enriched frozen adult Artemia diets

One of the main factors hindering aquaculture production of American lobsters (Homarus americanus) is the need for a cost‐effective and nutritionally sound diet. Live Artemia results in good growth, but is expensive and is a constant source of contamination. Frozen Artemia, although lower in cost, generally results in decreased survivorship and growth relative to live Artemia. The recent advent and mass production of enriched frozen Artemia products may provide for a cost‐effective and nutritionally complete food source for culturing American lobsters. Here, commercially available frozen adult Artemia enriched with either n‐3 fatty acids, or Spirulina was fed to juvenile American lobsters, and their growth and survivorship for three months was compared with that of animals fed unenriched frozen adult Artemia. Both enriched Artemia products produced survivorship superior to that for animals fed unenriched Artemia. Results for growth were equivocal although animals fed the Spirulina‐enriched Artemia had the greatest condition factor. Although more costly by the end of the experiment, enriched diets were more economically efficient than unenriched Artemia. This research demonstrates that enriched feedstuffs are cost‐effective over longer time intervals, and benefits may continue beyond the hatchery‐rearing phase. Enriched diets may also yield animals with a better condition factor, which may further influence their survivorship when released to the wild in enhancement programmes.     

Effects of lysine and methionine supplementation on growth,body composition and digestive function of grass carp (Ctenopharyngodon idella) fed plant protein diets using high‐level canola meal

A 76‐day feeding trial was carried out to evaluate the effects of Lysine and Methionine supplementation on growth and digestive capacity of grass carp (Ctenopharyngodon idella) fed plant protein diets using high‐level canola meal (CM). Fish with initial average weight 103.9 ± 0.6 g were fed three extruded diets. Fish meal (FM) diet was formulated as the normal control with 40 g kg^?1 FM and 300 g kg^?1 CM; CM diet was prepared by replacing all FM with CM (total 340 g kg^?1) without Lys or Met supplementation; CM supplement (CMS) diet was similar to CM diet but was supplemented with essential amino acids (EAA) to ensure the levels of Lys and Met similar to those in the FM diet. Feed intake, feed efficiency and specific growth rate of the grass carp fed CMS and FM diets were similar (P > 0.05), but higher than those of the grass carp fed CM diet (P < 0.05). The hepatosomatic index, relative gut length, intestosomatic index and intestinal folds height were significantly improved in fish fed FM and CMS diets as compared to CM diet (P < 0.05). Lower activities of trypsin, lipase and amylase in hepatopancreas were observed in fish fed CM diet (P < 0.05). Three hundred and forty gram per kilogram CM without Lys or Met supplementation significantly decreased trypsin, lipase and amylase mRNA levels in hepatopancreas (P < 0.05). These results indicated that the high supply of CM (340 g kg^?1) in plant protein (200 g kg^?1 soybean meal and 100 g kg^?1 cottonseed meal) diets decreased digestive ability through decreasing digestive enzyme activities and enzyme gene's expressions of grass carp, and these side effects can be reversed by supplementing Lys and Met. Therefore, CM could be high level used in a plant protein blend‐based extruded diet for grass carp as long as EAA were supplemented.     

The effects of seasonal,sex and size on the digestive enzyme activities in freshwater crayfish (Astacus leptodactylus Eschscholtz, 1823)

The effects of seasonal, sex and size on digestive enzyme activities of Astacus leptodactylus in natural habitat were investigated in the present study. The freshwater crayfish were sampled 584 individual as seasonally from Egirdir Lake. Ten male and 10 female individuals were sampled randomly from caught crayfish in each two different size ranges (4.5–7.0, 7.1–9.9 cm) and each season. The gastrointestinal tract, gastric, intestine and midgut gland were individually dissected on ice for enzyme analysis and stomach contents. At the end of the study, amylase activities were affected by interaction of season × sex × size in intestinal, season × sex in midgut gland. However, any interaction was not determined in amylase activities of the gastric. Lipase activities were affected by interaction of season × sex × size in intestinal and gastric, season × sex in midgut gland. The protease activities in each organ were affected by interaction of season, sex and size (p ? .05). In all organs, amylase and lipase activities were similar. However, the highest protease activity was in stomach while the lowest protease activity was the intestine. The protease activities were higher than amylase and lipase activities. There is a positive correlation between protease enzyme activities in gastric with gonad maturation and active feeding period. It can be said based on data of digestive system and high protease activity that A. leptodactylus is an omnivore species in need high protein. In addition, the reason for the low levels of lipase activity may be A. leptodactylus not prefer high fat foods or not fed high fat foods.     

健康和患病凡纳滨对虾幼虾消化道菌群结构的比较

凡纳滨对虾养殖过程中,早期阶段是病害易感阶段,而消化道菌群结构与对虾健康关系密切。因此,探讨幼虾的消化道菌群尤其是弧菌类细菌与对虾发病的关系对病害有效防治具有重要意义。本实验采用Illumina测序研究了凡纳滨对虾健康和患病幼虾的消化道细菌群落结构,并基于纯培养和16S r DNA,rec A和pyr H基因序列比对,分析了幼虾消化道中弧菌的主要种类组成。结果发现,健康幼虾消化道中α-变形菌纲和厚壁菌门丰度较高,而患病幼虾中γ-变形菌纲、拟杆菌门、放线菌门和β-变形菌纲较高,其中放线菌门丰度差异显著。基于科水平的响应比分析,发现患病幼虾消化道中动性球菌科和噬菌弧菌科的丰度显著降低,而弧菌科的丰度显著升高。相似度分析发现,驱动群落变异的OTU主要来源于弧菌属、海洋杆状菌属、冷杆菌属、假交替单胞菌属以及未分类至属的红杆菌科和微杆菌科。健康幼虾消化道弧菌组成以锡那罗亚州弧菌为主,而患病幼虾消化道弧菌组成以坎氏弧菌为主。尽管健康和患病幼虾消化道内细菌群落结构整体差异不显著,但一些重要类群丰度变化显著,其特征与已知的生态功能一致。     

Effect of Shrimp Stocking Density on Size-fractionated Phytoplankton and Ecological Groups of Ciliated Protozoa within Zero-water Exchange Shrimp Culture Systems

Zero‐water exchange shrimp culture systems are being investigated to reduce water and pathogens input and effluent output. The relative abundance and diversity of ciliates have been used as indicators of water quality and ecosystem dynamics in natural systems; however, their occurrence and role in shrimp production systems have not been investigated. The objectives of this research were to describe the succession of the ciliate community in a shrimp grow out system, to investigate the impact of increasing shrimp stocking density on the characteristics of the ciliated protozoa community, and to correlate this with shrimp performance. A growth trial with Litopenaeus vannamei was conducted outdoors with zero‐water exchange and three stocking densities (50, 75 and 100 shrimp/m²). In all treatments, free‐swimming ciliates were dominant in the early weeks, while substrate‐associated ciliates appeared later. The 100 shrimp/m² treatment exhibited a lower abundance of free‐swimming ciliates but higher abundance of hypotrichous ciliates in the second half of production and higher shrimp mortality and lower shrimp growth in the last 2 wk. These results provide a preliminary examination of the role of ciliates in shrimp culture systems and direction for future work in determining the role of protozoa as indicators of water quality.     

Potential virulence factors of bacteria associated with tail fan necrosis in the spiny lobster,Jasus edwardsii

Tail fan necrosis (TFN) is a common condition found in commercially exploited spiny lobsters that greatly diminishes their commercial value. Bacteria possessing proteolytic, chitinolytic and lipolytic capabilities were associated with TFN in spiny lobsters, Jasus edwardsii. In this study, 69 bacterial isolates exhibiting all the three enzymatic capabilities from the haemolymph and tail fans of J. edwardsii with and without TFN were further characterized and compared, including morphology, biofilm formation, antimicrobial activity, antimicrobial resistance, and production of siderophores, melanin and ammonia. The genomic patterns of the most common Vibrio crassostreae isolates were also compared between TFN‐affected and unaffected lobsters. Biofilm formation was stronger in bacterial isolates from both haemolymph and tail fans of TFN‐affected lobsters compared to those from the unaffected lobsters, while melanin production and siderophore production were stronger in the isolates from tail fans of lobsters with TFN. By contrast, the other characteristics of isolates were similar in lobsters with and without TFN. The Vib. crassostreae isolates from the affected lobsters had similar genomic patterns. Overall, the results indicate that in addition to proteolytic, chitinolytic and lipolytic activities, the bacteria associated with TFN commonly have enhanced activity of important virulence factors, including biofilm formation, melanin production and siderophore production.     

Food sources,digestive efficiency and resource allocation in the sea cucumber Holothuria forskali (Echinodermata: Holothuroidea): Insights from pigments and fatty acids

Various research projects in Europe and North Africa have recently intended to breed temperate holothurians to alleviate fishing pressure on natural populations. However, to date little is known about the nutritional requirements of East Atlantic and Mediterranean species. In this study, we propose a “natural population”‐oriented approach to characterize food sources, digestive efficiency and resources allocation based on the composition of pigments and fatty acids (FA) in gut contents and tissues (muscles, gonads and digestive tract walls) of wild individuals of the species Holothuria (Panningothuria) forskali (Delle Chiaje, 1823) sampled in Brittany (France). Our study reveals that neither green nor red algae enter the diet of H. forskali in spring and that the only fresh vegetal material found in gut contents is brown algae (very likely diatoms). The high nutritional quality of gut contents however contrasts with the detrital nature of the ingested food sources, suggesting that a trophic upgrading of organic matter occurs before digestion. In addition, unusual FA (i.e. only present in a few groups of living species) such as long‐chain monounsaturated FA (especially the FA 23:1ω9) were found in large proportions in muscles and gonad and their effect on sea cucumber fitness needs further investigation.     

Evaluation of the Ciliated Protozoa,Fabrea salina as a First Food for Larval Red Snapper,Lutjanus campechanus in a Large Scale Rearing Experiment

ABSTRACT

One of the major challenges of culturing of red snapper, Lutjanus campechanus, is providing an appropriate food source at onset of feeding. Ciliates are abundant in marine waters but their significance as a first food for fish larvae is poorly understood as many have no lorica to facilitate their identification in the gut of a larval fish. Fabrea salina is a naked heterotrich ciliate that can be mass cultured at densities up to 84 ± 10 ciliates/mL in 7 days. Its appropriateness as a first food for red snapper larvae was evaluated in a green-water setting using 1-m³ tanks. Larvae were stocked at 10/L, 36 h post-hatch, before first-feeding commenced. Larvae were fed either (1) copepod nauplii, 20–75 μm, only from days 1 to 10; (2) copepod nauplii from days 1 to 10 plus F. salina from days 1 to 5; or, (3) F. salina only from days 1 to 3 plus copepod nauplii from days 4 to 10. Copepod nauplii were added at 2/mL and ciliates were added at 5/mL. Survival after 28 days was 0.28 ± 0.15% for larvae given only copepod nauplii and 2.39 ± 2.75% for those given F. salina and copepod nauplii. Larvae given only F. salina did not survive past 4 days post-stocking. Larvae were more actively feeding in the tanks given F. salina and copepods as first foods with 34.6 ± 8.5% mean daily reduction in copepod nauplii compared to 15.8 ± 16.2% reduction when only nauplii were provided.     

Microfauna associated with amoebic gill disease in sea-farmed Atlantic salmon, Salmo salar L., smolts

A study of microfauna, associated with pathological changes in the gills of Atlantic salmon, Salmo salar L., was conducted over 2001-2002. Monthly samples of 1(+) salmon smolts were taken, protozoan populations were quantified and gill health was assessed histologically. Protozoan densities were correlated with pathological changes, in order to determine their possible role in lesions in the gills. The most severe gill tissue changes were observed in summer/autumn and the least in spring. A diverse polyphyletic protozoan community was observed colonizing the gills, including Neoparamoeba sp., other amoebae, scuticociliates, Ichthyobodo-like flagellates, trichodinid ciliates and prostomatean ciliates. The earlier gill tissue changes in the gill were not always associated with the presence of these microorganisms, whereas amoebae (other than Neoparamoeba sp.), Ichthyobodo-like flagellates and trichodinid ciliates correlated with augmenting gill lesions. Neoparamoeba sp. was present, but its abundance did not correlate with the disease. This study suggests that a diversity of protozoans including Ichthyobodo-like flagellates, trichodinid ciliates and amoebae other than Neoparamoeba sp. are involved in the aetiology of amoebic gill disease in the Irish situation.     

Complexity of river ciliate communities at a national park highlights the need for microbial conservation

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