Toxicity of chlorothalonil to marine organisms

We evaluated the acute toxicity of the antifouling biocide 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile (chlorothalonil, TPN) to an alga (Skeletonema costatum), crustacea (Tigriopus japonicus, kuruma prawn Marsupenaeus japonicus), and a teleost fish (red sea bream Pagrus major) commonly found in the coastal waters of Japan. We also performed acute toxicity and early-life-stage tests for TPN using mummichog Fundulus heteroclitus and used the results to predict the chronic toxicity of TPN to red sea bream. The acute toxicity values of TPN (based on measured concentrations) for S.?costatum, T.?japonicus, M.?japonicus, red sea bream, and mummichog were 0.95, 16, 290, 35, and 61???g/l, respectively. The early-life-stage test with mummichog embryos revealed that hatchability and growth were the most sensitive indicators, and the lowest- and no-observed-effect concentrations, based on the measured concentrations, were 32 and 11???g/l, respectively. We estimated the chronic toxicity value for red sea bream to be 11???g/l based on these toxicity values. Considering the current measured concentrations of TPN in coastal waters of Japan, its environmental impact would appear to be limited.     

Bioprospection of Microalgae and Cyanobacteria as Biocontrol Agents Against Vibrio campbellii and Their Use in White Shrimp Litopenaeus vannamei Culture

Vibrio spp. are the most common and harmful shrimp pathogenic bacteria; however, microalgae and cyanobacteria have the ability to produce antimicrobial substances against these species. In this study, the organic and aqueous extracts of 28 species of marine microalgae and cyanobacteria were screened against Vibrio campbellii M1. Two of these phytoplankton species with antibacterial activity in aqueous extracts (Dunaliella tertiolecta and Skeletonema costatum) and nontoxic to brine shrimp Artemia franciscana nauplii were used to evaluate their anti‐Vibrio effect when used as green‐water cultures in Vibrio‐challenged white shrimp Litopenaeus vannamei cultures. No differences in mortality of juvenile L. vannamei were observed between treatments tested, suggesting that the pathogenicity of V. campbellii could be related to the growth stage of shrimp. The proximal composition of D. tertiolecta and S. costatum was in the recommended range for penaeid shrimp nutrition, allowing shrimp supplemented with these microalgae to have significantly greater total length and weight than control shrimp. Shrimp supplemented with S. costatum presented the highest values of organic mass (11.48 mg/organism) and growth rate (0.31 mg/d) in comparison to D. tertiolecta. These results indicate that microalgae are not only capable of producing antibacterial compounds against Vibrio but can also help shrimp nutrition.     

Use of microalgal‐enriched Diaphanosoma celebensis Stingelin, 1900 for rearing Litopenaeus vannamei (Boone, 1931) postlarvae

The present work investigates the effects of Chaetoceros calcitrans, Nannochloropsis oculata, Tetraselmis tetrahele and Isochrysis galbana diets on the lifespan, growth, neonate production and the nutritional profile of Diaphanosoma celebensis. In addition, the effects of enriched D. celebensis on the survival and growth of Litopenaeus vannamei postlarvae (PLs) was compared with Artemia. Results showed that significantly higher (P < 0.05) neonate production of D. celebensis was attained when fed with C. calcitrans compared to the other microalgae. In addition, D. celebensis fed on C. calcitrans had significantly higher levels (P < 0.05) of protein, lipid and carbohydrate compared to the other three microalgae. On the other hand, D. celebensis had a significantly (P < 0.05) longer lifespan when fed on N. oculata and T. tetrahele compared to those fed with C. calcitrans and I. galbana. Shrimp PLs fed D. celebensis enriched with C. calcitrans had higher survival and specific growth rate but it was not significantly different (P > 0.05) from PL fed only Artemia, indicating that D. celebensis has potential to be used as live feed for the hatchery rearing of L. vannamei PLs, in place of Artemia. This study illustrated that the quality of the D. celebensis production and proximate composition was highly correlated with the food type, and it can be used as a valuable live feed for shrimp larviculture.     

Ingestion,fecundity and population growth of Harpacticus sp. (Harpacticoida,copepod) fed on five species of algae

We investigated the ingestion, fecundity and population growth of Harpacticus sp. fed on monodiet or mixed diets to evaluate the effects of different algae on Harpacticus sp. Harpacticus sp. fed on diatoms (Skeletonema costatum and Chaetoceros curvisetus) had higher ingestion and pellet production. Time to attain 100% in proportions of gravid females differed, with quickest to slowest: S. costatum, C. curvisetus, Gymnodinium sp. and Heterosigma akashiwo, with the exception of Prymnesium parvum (≤16.67%). S. costatum or C. curvisetus produced higher populations than the other three diets, supported complete development to adulthood, and resulted in doubling copepod population within four days, while no population growth occurred for the other three diets. Mixed‐diet experiments showed that egg production and gross growth efficiencies reduced significantly when fed on H. akashiwo, Gymnodinium sp. or P. parvum mixed with Isochrysis galbana, in comparison with 100% I. galbana. Thus, S. costatum and C. curvisetus were beneficial foods while the other three diets were potentially toxic for Harpacticus sp. The data in this article provide further recognition of nutrient deficiency or toxicity of different algae on copepods.     

Species diversity of the marine diatom genus Skeletonema in Japanese brackish water areas

The genus Skeletonema includes phytoplankton species that are important primary producers in marine food chains. Brackish waters have been reported to be one of the important habitats of some species of Skeletonema. To elucidate the species diversity of Skeletonema in brackish waters, we investigated three Japanese brackish bodies of water: the coastal waters of Toyama Bay, a tidal area of the Chikugo River, and a constructed reservoir in Isahaya Bay. We used molecular analysis based on large subunit rDNA and fine morphological structure to identify species. Skeletonema costatum s.s. (sensu stricto) was isolated at salinities as low as 0.6, but Skeletonema dohrnii, Skeletonema subsalsum, and Skeletonema tropicum were not found at salinities below 11.0. S. costatum s.s. could survive transfer from a medium with a salinity of 15 to a salinity of 2, but S. dohrnii did not survive in the same experiment. Only S. costatum s.s. germinated from the sediment of a reservoir in which the salinity was 0.1–1.4; incubation conditions included temperatures of 10, 15, 20, 25, and 30 °C and salinities of 5 and 30. Skeletonema costatum s.s. was identified as the species most adaptable to low-level salinity variations throughout its lifecycle.     

Widespread distribution of cellulase and hemicellulase activities among aquatic invertebrates

Cellulase and hemicellulases (mannanase, xylanase, xyloglucanase, and licheninase) activities were measured in the extracts of 18 species of macrobenthos (an oligochaete, two polychaetes, two decapods, one amphipod, three bivalves, and nine gastropods) to check the relevance of macrobenthos to the breakdown process of hard-degradable carbohydrates derived from terrestrial plants. As a result, these enzyme activities were detected in all of the extracts of macrobenthos except for Tubificidae sp. High cellulase activities were detected in the extracts of the decapods (Neocaridina denticulate denticulate, Caridina multidentata) and the gastropods (Semisulcospira libertina, S. reiniana, Physa acuta, Terebralia palustris). However, only weak activities were detected in the polychaetes (Perinereis nuntia, P. aibuhitensis) and the bivalve (Ruditapes philippinarum). High mannanase and licheninase activities were detected in two decapod species (N. denticulate, C. multidentata), while high xylanase activities were seen in the gastropods (S. libertina, S. reiniana). High xyloglucanase activities were noted in the gastropod (T. palustris). These facts suggest that the activities of different types of hemicellulase were distributed rather unevenly among the macrobenthos. The present paper is the first to report the widespread distribution of cellulase and hemicellulase activities among ecologically important aquatic invertebrates, and the possible relevance of these species in the breakdown process of plant-derived hard-degradable carbohydrates.     

Broodstock conditioning of the Portuguese oyster (Crassostrea angulata,Lamarck, 1819): influence of different diets

The Portuguese oyster Crassostrea angulata shows great potential in oyster farming. The conservation of pure populations of this species is important for production diversification and biodiversity preservation. In this way, the zootechnological development for seed hatchery production is extremely important. Broodstock conditioning is a key step in the process of rearing bivalves in a hatchery. Many factors regulate the reproductive cycle, being food one of the most important ones. To evaluate the effect of different diets on C. angulata reproductive performance, broodstock were conditioned with different food regimes formulated fundamentally by flagellates (Diet 1 – Pavlova lutheri and Isochrysis galbana clone T‐ISO; Diet 2 – P. lutheri, T‐ISO and Skeletonema costatum) and constituted fundamentally by diatoms (Diet 3 – S. costatum and Chaetoceros calcitrans; Diet 4 – P. lutheri, S. costatum and C. calcitrans). During conditioning, samples of oysters were collected to evaluate condition index, gonadal development and biochemical composition. At the end of the conditioning period, oysters were induced to spawn to evaluate reproductive output (fecundity, fertilization rate and D‐larvae development). The diets had an impact on the gametogenesis process, energy storage and reproductive output performance, being the best results those obtained in broodstock fed with the diatoms‐predominant diets. However, those fed with diets majority flagellates had an unsuccessful performance. Holistic approaches incorporating all results in this study reveal and reinforce the idea that the diatom species used presented the nutritional requirements to C. angulata broodstock, being essential in the conditioning phase.     

Diet trials on tehuelche scallop Aequipecten tehuelchus (d'Orb) larvae

Laboratory spawned one week old veliger larvaeof the tehuelche scallop, Aequipectentehuelchus (D'Orb., 1846), were reared topedivelger stage under three different dietaryregimes in order to determine the best algalcombination under standard hatchery conditions.Veligers were cultured at mean densities of 2.7larvae/ml. Larval growth and survival wereassessed on larvae fed the following dietarytreatments: (1) Isochrysis galbana(T-Iso)+ Chaetoceros calcitrans, (2)Pavlova lutheri + C calcitrans, and (3)P. lutheri + I. galbana T-Iso +Ch. calcitrans. The highest growth rateswere detected on larvae fed diets includingP. lutheri.     

Influence of algal cell size on filtration and ingestion rates during different larval stages of the yellow neck clam,Paphia malabarica Chemnitz

Filtration rate and ingestion rate of different stages of Paphia malabarica larvae (D‐shape, (80 μm), Umbo (120 μm) and veliger (180 μm)) were determined in relation to feeding on various micro algae. The micro algae tested were Nannochloropsis salina, Isochrysis aff. galbana, Dicrateria inornata, Tetraselmis gracilis and Chaetoceros calcitrans at 5 or 10 × 10³ cells mL^?1. Both filtration and ingestion rate of micro algae tested were increased with increasing larval size; however, at all larval stages, C. calcitrans resulted in lower filtration and ingestion rate. Of the algal diets tested, P. malabarica larvae showed greatest filtration rate and ingestion rate with N. salina. Maximum filtration rate for N. salina was 15.7, 26.3 and 33.9 μL h^?1 and highest ingest rate was 15, 92 and 177 cells larva^?1 h^?1 in D shape larvae, umbo and pediveliger, respectively. Filtration rate and ingestion rate of N. salina were always higher than those other algal species tested because of its small cell size (2 μm).     

Suspended culture of the sea cucumber Apostichopus japonicus below a Pacific oyster raft – potential for integrated multi‐trophic aquaculture

To discuss the possibility of co‐culturing Pacific oyster with the sea cucumber Apostichopus japonicus, a field experiment was conducted in an oyster farm. Apostichopus japonicus juveniles (mean wet weight, 0.08 g) were cultured below an oyster raft and at a control station for 216 days, and the wet weight and stable isotope ratios (δ¹³C, δ¹⁵N) were analysed together with settling organic matter (OM) collected using sediment traps. All sea cucumbers cultured below the raft survived (survival rate, 100%), while at the control station one individual disappeared (96%). During 216 days, the juveniles at the oyster and control stations grew to a mean weight of 5.5 and 2.6 g, attaining respective specific growth rates of 2.0% and 1.6% (paired t‐test, P < 0.001). Settlement rates of carbon and nitrogen at the oyster station were ~5 times larger than those at the control station. The stable isotope analysis showed that settling OM at both stations originated from coastal phytoplankton and that phytoplankton represented the primary food source for A. japonicus. The rapid growth of A. japonicus at the oyster station was concluded to be due to the abundant supply of oyster biodeposits, which could be ingested by this species.     

A preliminary study on the potential value of a novel integrated aquaculture system on water purification

Intensive and semi-intensive sea cucumber aquaculture produces effluents that impact the quality of the local aquatic environment and sea cucumber growth; such effluents are an important cause of coastal water eutrophication. Integrated aquaculture has been shown to efficiently reduce the release of nutrients during aquaculture and to provide economic benefits. In this study, we report our investigation of a polyculture system utilizing Styela clava (stalked sea squirt) and Stichopus japonicus (Japanese sea cucumber) under two feeding modes. To examine purification of the water in the system, nutrients in the water and the sediment, attached heterotrophic bacteria, and the survival rates and growth rates of S. clava and S. japonicus were determined. Tank cultivation trials showed that, under the same feeding modes, the concentration of nutrients in the polyculture system was approximately 50–60 % of the sum of the nutrients in the S. japonicus mixture and S. clava monoculture systems. The abundance of attached heterotrophic bacteria in the polyculture system was remarkably lower than that of the mixed culture system under the same feeding mode. S. japonicus in the polyculture system grew better than those in the mixed culture system under the same feeding mode. These results demonstrate that the S. clava–S. japonicus integrated culture system offers a means of constraining or reversing the pollutive impacts of coastal sea cucumber aquaculture.     

Optimisation of larval culture of the mussel <Emphasis Type="Italic">Mytilus</Emphasis><Emphasis Type="Italic">edulis</Emphasis> (L.)

The blue mussel Mytilus edulis is a commercially important species whose fishery and culture generally relies on natural spat collection. Hatchery-production could provide an alternative source of seed, enabling reliable expansion of the industry. Mussel spawning and larval rearing trials were carried out to optimise elements of hatchery production. Culturing fertilised eggs at low density (20–200 eggs cm⁻²) rather than high density (400–720 eggs cm⁻²) significantly improved the quality of first veliger larvae and differences in this improvement were evident between the eggs from different females (maternal effects). Veliger larval growth at 17 or 21°C was significantly faster than growth at 14°C. Feeding veliger larvae an identical total ration either daily or at 2–3 day intervals did not significantly affect their growth. Different microalgal diets (1: Isochrysis sp. (clone T-ISO), 2: Chaetoceros calcitrans forma pumilus, 3: C. muelleri, 4: mixed Isochrysis sp. (clone T-ISO) and C. calcitrans f. pumilus, and 5: mixed Isochrysis sp. (clone T-ISO) and C. muelleri) were tested on veliger larval growth and mixed diets outperformed single-species diets.     

中华绒螯蟹蜕皮激素受体基因（Ers-EcR）的克隆和组织表达分析

蜕皮激素受体(ecdysteroid receptor,EcR)介导调控甲壳动物蜕皮生长、附肢再生等重要生命活动。为了解EcR在人工控制甲壳动物的繁殖和生长中的作用,采用RACE方法结合同源克隆技术,首次从中华绒螯蟹Y-器官中克隆得到蜕皮激素受体基因全长cDNA序列(Ers-EcR,登录号:KF736985),并进行了结构解析和组织表达分析。结果发现,Ers-EcR编码基因全长2 176 bp,开放阅读框为1 638 bp,编码545个氨基酸,具有DNA结合域(DBD)和配体结合域(LBD)等典型的核受体超家族结构域,但不具有信号肽结构。其中,DBD含有8个保守的Cys残基,可以形成2个锌指结构(C156-C159-C173-C176、C192-C198-C208-C211),是典型的DBD特征。多重序列比对分析表明,Ers-EcR氨基酸序列与拳手招潮蟹同源性最高,达到91%。荧光定量PCR结果显示成体中华绒螯蟹ErsEcR基因在Y-器官和肌肉组织中表达量最高,在血液、肠道、卵巢、眼柄、心脏和肝胰腺中有一定表达,在鳃、胸神经节和精巢表达量较低。这表明Ers-EcR基因在中华绒螯蟹各组织器官中的表达不具有典型的特异性,提示Ers-EcR基因可能参与体内多种生命活动的调控。     

Testing the potential for improving quality of sediments impacted by mussel farms using bioturbating polychaete worms

Biodeposits from farmed mussels severely influence the biogeochemistry of sediments by increasing the levels of organic matter (OM). Mitigation of such negative impacts is important for the development of sustainable aquaculture operations. As a step towards developing methods for remediation of coastal sediments affected by mussel farming, the effects of the polychaete, Hediste diversicolor was evaluated experimentally. In a series of field‐ and laboratory experiments we tested hypotheses about the effects of polychaetes on sediment oxygen consumption, nutrient fluxes and sulphide pools under different polychaete densities and sedimentation regimes. The experimental results support the idea that polychaetes can mitigate negative effects on the benthic environment beneath mussel farms. H. diversicolor oxidized the sediment and generally enhanced the oxygen consumption, and thus the decomposition of OM. The accumulation of pore water sulphides were reduced and fluxes of nutrients across the sediment‐water interface increased. Additional calculations suggest that the effects of polychaetes were mainly indirect and driven by increased microbial activity due to the borrowing activity of the polychaetes. Trends of increasing decomposition with increasing polychaete density suggest that the decomposition could be further enhanced by higher densities. Overall, we concluded that H. diversicolor is a potentially strong candidate for remediation of mussel farm sediments. The results show that sediments inhabited by H. diversicolor have high assimilative capacity of OM and oxygen conditions are significantly improved following the addition of polychaetes at naturally occurring densities. However, technological developments are needed in order to allow the approach to be used in practice.     

Use of Nannochloropsis sp. isolated from the East China Sea in larval rearing of sea cucumber (Apostichopus japonicus)

The supply of microalgae to hatcheries is a limiting factor for the mass larval production of sea cucumber (Apostichopus japonicus) in Fujian Province, China. In this study, Nannochloropsis sp. isolated from the East China Sea was tested as food for A. japonicus larvae. The first trial compared the effect of mono‐, bi‐ and trialgal diets comprising three microalgae (Chaetoceros muelleri, Dunaliella tertiolecta and Nannochloropsis sp.) on A. japonicus larval growth, survival, settlement and juvenile growth. The results showed that there were no significant differences in survival and settlement between larvae fed with Nannochloropsis sp. and other diets. All diet treatments yielded similar juvenile sea cucumber output. In the second trial, A. japonicus larvae were fed equally four times daily at three different rations (5000, 20 000 and 40 000 cells mL^?1 day^?1). Larvae fed 20 000 cells mL^?1 day^?1 were significantly larger than larvae in other groups and experienced the highest survival rate. In the third trial, A. japonicus larvae were fed 20 000 cells mL^?1 day^?1 in three different frequency (2, 3 and 4 meals day^?1). The greatest body length was observed in larvae that received 3 meals day^?1. Survival and settlement of larvae fed 2 meals day^?1 were significantly lower than other two groups. These results suggest that Nannochloropsis sp. can be used as a diet for the large‐scale production of A. japonicus seed, and larvae fed three times daily at a ration of 20 000 cell mL^?1 day^?1 are recommended for hatchery production of A. japonicus.     

Cell growth, effect of filtrate and nutritive value of the tropical Prasinophyte Tetraselmis tetrathele (Butcher) at different phases of culture

A local algal isolate Tetraselmis tetrathele was studied to determine the suitable levels of three growth factors (nutrient medium, light intensity and photoperiod) for mass culture. Significantly higher population cell count (141.92 × 10⁴) were found in 4‐day‐old cultures (exponential phase) enriched with Conwy medium and exposed continuously to 93.31–103.5 μmole photon m^?2 s^?1 light intensity compared with two other media (F, SEAFDEC/AQD) under the same light conditions. Cultures exposed to higher levels of photosynthetic photon flux density and continuous photoperiod had a significantly higher cell count regardless of nutrient media used. The chemical composition of T. tetrathele varied as the culture aged. Significantly higher crude protein (49.6%) and crude ash (15.9%) were found in cultures harvested at exponential than at stationary phase of growth. In contrast, significantly higher carbohydrate (55.2%) was noted as the culture aged. There was an increase in the percentage of saturated, monoenoic and n‐3 highly unsaturated fatty acids at the stationary phase of growth. Addition of filtrate from various phases of growth and from 10‐day‐old cultures at various concentrations stimulated cell growth of T. tetrathele. This study identified the optimum growth conditions for mass culture of T. tetrathele and demonstrated its change in the chemical composition as the culture aged.     

Diet trials on tehuelche scallop Aequipecten tehuelchus (d''Orb) larvae

Laboratory spawned one week old veliger larvaeof the tehuelche scallop, Aequipectentehuelchus (D'Orb., 1846), were reared topedivelger stage under three different dietaryregimes in order to determine the best algalcombination under standard hatchery conditions.Veligers were cultured at mean densities of 2.7larvae/ml. Larval growth and survival wereassessed on larvae fed the following dietarytreatments: (1) Isochrysis galbana(T-Iso)+ Chaetoceros calcitrans, (2)Pavlova lutheri + C calcitrans, and (3)P. lutheri + I. galbana T-Iso +Ch. calcitrans. The highest growth rateswere detected on larvae fed diets includingP. lutheri.     

仿刺参体腔液的抗菌特性

为了解仿刺参体腔液的抗菌谱以及不同二价金属离子对仿刺参体腔液抗菌活性的影响,实验采用生长曲线测定法分别测定了仿刺参体腔细胞破碎液上清和体腔液上清对哈维氏弧菌、灿烂弧菌、希瓦氏菌、假交替单胞菌、金黄色葡萄球菌、溶壁微球菌、停乳链球菌、拟诺卡式菌生长的影响,并通过该法以溶壁微球菌为受试菌测定了海洋环境中常见的Fe2+、Ca2+、Cd2+、Mg2+、Mn2+、Zn2+对仿刺参体腔液上清抗菌活性的影响。结果显示,仿刺参体腔细胞破碎液上清对受试的8株细菌的生长均无抑制作用,体腔液上清对溶壁微球菌的生长有明显的抑制作用,而对其他7株受试菌的生长无明显影响;Mn2+和Zn2+可明显增强体腔液上清对溶壁微球菌的生长抑制作用,而Fe2+、Ca2+、Cd2+、Mg2+对体腔液上清的抗菌活性无明显影响。研究表明,仿刺参体腔液在体外状态下只具有窄谱抗菌活性,与抗菌相关的免疫因子主要存在于体腔液上清中;体腔液中的抗菌免疫因子对海洋环境中的常见二价金属离子有一定的适应性,而且适当浓度的Mn2+和Zn2+可能具有促进仿刺参抗菌应答能力的作用。     

三疣梭子蟹心激肽基因克隆及在低盐适应中的功能验证

采用RACE技术克隆获得三疣梭子蟹(Portunus trituberculatus)心激肽(CCAP)基因。该基因全长606 bp, 5￠端非编码区72bp,3￠端非编码区108bp,开放阅读框426bp,编码141个氨基酸,预测分子量15.6kD,理论等电点9.55。同源性分析表明,三疣梭子蟹CCAP与拟穴青蟹(Scylla paramamosain)和蓝蟹(Callinectes sapidus)的CCAP同源性较高,分别为85%和82%。系统进化树分析显示,三疣梭子蟹与蓝蟹首先聚为一支,之后再与拟穴青蟹相聚。组织表达分析发现, CCAP基因在胸神经节中的相对表达量最高,其次是脑和眼柄组织。通过分析CCAP基因在低盐胁迫过程中的表达规律发现,低盐可显著改变CCAP在胸神经节中的表达模式,在24 h, 48 h和72 h实验组的表达量显著高于对照组(P0.05),分别为对照组的1.73, 2.16和2.19倍。体外注射CCAP多肽可降低三疣梭子蟹在低盐条件下的死亡率,并诱发钠钾ATP酶(Na~+/K~+-ATPase)和V型ATP酶(V-ATPase)酶活力显著提高。本实验结果暗示CCAP通过调控Na~+/K~+-ATPase和V-ATPase活力以达到盐度适应的作用。