Acute toxicity of nitrite to mud crab Scylla serrata (Forsskål) larvae

Early larval stages of mud crab Scylla serrata were exposed to different concentrations of nitrite (40, 80 and 160 mg L⁻¹ and a control, without added nitrite) and three salinity levels (25, 30 and 35 g L⁻¹) using a static renewal method. No interactive effect of nitrite and salinity was detected. Estimated LT₅₀ in 96‐h toxicity tests decreased in all stages with increasing nitrite concentrations in all salinity levels. The 96‐h LC₅₀ values of nitrite‐N were 41.58, 63.04, 25.54, 29.98 and 69.93 mg L⁻¹ for zoea 1, 2, 3, 4 and 5 respectively. As the larvae grew, they showed a progressive increase in tolerance to nitrite. The toxicity of nitrite to larvae increased with exposure time. The median lethal concentration was not affected by salinity. The chloride component of salinity within 25–35 g L⁻¹ did not seem to be as effective in alleviating toxicity as has been reported in other crustacean species. Based on 96‐h LC₅₀ and an application factor of 0.1, the ‘safe level’ of rearing mud crab larvae was calculated to be 4.16, 6.30, 2.55, 2.99 and 6.99 mg L⁻¹ nitrite‐N for zoea 1, 2, 3, 4 and 5 respectively.     

Effects of tank colour on larval survival and development of mud crab Scylla serrata (Forskål)

Hatchery culture of mud crabs has not yet achieved commercial viability despite decades of research efforts. Further research is therefore needed to better understand larval culture requirements of the crab. Based on anecdotal observations, an experiment was carried out to test whether the background colour of the culture vessel affected larval culture success. Newly hatched larvae of Scylla serrata were reared in culture vessels of five colours, i.e., black, dark green, maroon, sky blue and white. Larval survival and development were monitored daily until all of them either moulted to the first crab stage or died. The results showed clear effects of background colour on larval survival. A general tendency of higher larval survival in darker‐coloured backgrounds was evident. In particular, overall zoeal survival for larvae reared in black vessels was significantly higher than those reared in white ones. Background colour also appeared to affect larval development. Larvae reared in darker backgrounds generally had shorter development times and more synchronized moulting. A significant delay in zoeal development was observed in larvae reared in white vessels. Dark backgrounds possibly facilitated more efficient feeding, reduced settlement of larvae at the bottom of the vessels as well as minimized stress. This result appears to be the first to demonstrate that background colour can significantly affect larval survival and development of a crustacean species.     

Influence of binder type on leaching rate and ingestion of microbound diets by mud crab, Scylla serrata (Forsskål), larvae

Microbound diets (MBD), composed of nutrients held within a matrix or binder, were shown to be readily ingested by larvae of the mud crab, Scylla serrata (Forsskål). In an effort to improve experimental MBD developed for S. serrata larvae, this study determined the effects of various binders on estimated larval ingestion of MBD as well as their leaching rates. Microbound diets with the same dietary compositions were bound with either agar, alginate, carrageenan, gelatin or zein. All diets contained ¹⁴C‐labelled rotifers and were fed to Zoea I, III, V larvae and megalopae. The ¹⁴C content of larvae fed ¹⁴C‐labelled MBD for 2 h was used to estimate the relative ingestion rates of MBD. For all larval stages tested, the estimated mean larval ingestion of MBD did not differ significantly between binder types (P>0.05). Determination of the amount of ¹⁴C‐labelled nutrient leaching from MBD bound with various binders after 30, 60 and 240 min of immersion showed that for all types of binders, ¹⁴C leaching occurred primarily within the first 30 min of immersion. Zein‐bound MBD consistently showed the lowest numerical leaching rate among the five binders tested for all immersion periods examined, and the differences compared with other binders were often significant, particularly after 60 and 240 min of immersion. Larval feeding experiments showed that MBD prepared with binders showing higher rates of leaching were not utilized to a greater degree by S. serrata larvae. Furthermore, unnecessary leaching from MBD particles wastes dietary nutrients and is likely to impact on water quality. On this basis, our results suggest that because of its consistently lower leaching rates, zein is probably a more suitable binder for MBD developed for S. serrata larvae.     

影响锯缘青蟹幼体成活的几个因素

本文对海捕和养殖的锯缘青蟹抱卵及胚胎发育情况进行了观察,并对幼体饵料营养强化、适口性、幼体的培育方式等进行了研究。结果表明：自然诲区捕获的亲蟹其抱卵率和幼体质量要好于人工养殖的亲蟹;胚胎发育的最适盐度为28～33,最适温度为26～30℃;幼体\期和大眼幼体时期摄食的饵料以边缘毛状的活体为佳,便于抱握;涵状幼体、期起应降低苗池中的直射光,投饵多餐少量．增加附着面积．可降低相互残杀率。     

Ontogeny of feeding apparatus and foregut of mud crab Scylla serrata Forsskål larvae

The development of the feeding apparatus of the mud crab Scylla serrata larvae was studied using electron microscopy for mandibles and light microscopy for other paired mouthparts and the foregut. The six paired mouthparts, which consisted of the mandibles, maxillules, maxillae, first maxillipeds, second maxillipeds and third maxillipeds, were dissected from specimens representing each larval stage. The first five paired appendages were already present in newly hatched larvae while third maxillipeds appeared only at the megalopa stage. Mandibles displayed complex incisor and molar processes at early zoeal stages, which became simple in morphology at megalopa. Mandibular palp buds were observed at the zoea 5 stage and these became fully developed as three‐segmented mandibular palps at the megalopa stage. Endopods of other paired mouthparts exhibited increased number of setae and size as the individual metamorphosed from zoeal stages to megalopa and crab instar. The foregut appeared as a continuous cavity at zoea 1 where the cardiopyloric valve was indistinct while the filter gland was clearly identifiable. Zoea 2 and succeeding zoeal stages exhibited a setose foregut; the gastric mill and its lateral and median teeth were prominent at zoea 3 stage. The significance of these morphological changes is discussed in terms of its implication in larval feeding management.     

Effects of acute and chronic toxicity of unionized ammonia on mud crab, Scylla serrata (Forsskål, 1755) larvae

While the effects of ammonia on fish and prawn larvae are well documented, little is known of its effect on mud crab (Scylla serrata) (Forsskål, 1755) larvae. Two experiments were conducted in 5 L hemispherical plastic bowls, containing 3 L of ultra‐filtered and settled seawater and various larval stages of mud crab to (1) determine the acute median lethal concentration (LC₅₀) of unionized ammonia and (2) to determine the chronic effects of unionized ammonia on survival and percentage moulting to zoea and megalop stages. The larval stages that exhibited the highest tolerance to ammonia over 24 h were zoea 1 (LC₅₀ of 4.05 mg L^?1 of unionized ammonia) and zoea 5 (LC₅₀ of 6.64 mg L^?1 of unionized ammonia). The megalop stage had the lowest total ammonia LC₅₀ at both 24 and 48 h, making it the larval stage most susceptible to total ammonia. Exposure to 6.54 mg L^?1 of unionized ammonia resulted in 100% death of all larvae within 24 h. The tolerance of S. serrata larvae to total ammonia did not appear to increase with ontogenetic development. The results indicate that the concentrations at which total ammonia produces an acute or chronic response in mud crab larvae are far higher than those experienced in current larval production systems (0–0.5 mg L^?1 of total ammonia) used as industry standards in Australia.     

Formalin as an alternative to trifluralin as prophylaxis against fungal infection in mud crab Scylla serrata (Forsskål) larvae

The toxicity of formalin and trifluralin to the larval stages of the mud crab Scylla serrata was compared in a static bioassay. Prophylactic doses of 5, 10, 15, 20 and 25 μg L⁻¹ formalin and 0.05, 0.1, 0.2, 0.4 and 0.8 μg L⁻¹ trifluralin were used. Toxicity was assessed on the basis of survival of larvae after 24, 48, 72 and 96 h exposure to the test chemicals and metamorphosis to the next larval stage. Result shows that larval survival in all stages was significantly reduced at concentrations of 20 and 25 μg L⁻¹ formalin whereas larvae were able to tolerate all trifluralin treatments. However, larvae became more tolerant to high formalin concentrations as the larval stage progressed. Survival was better at 5, 10 and 15 μg L⁻¹ formalin and in all trifluralin treatments than the control in almost all the larval stages. Faster metamorphosis was observed at 5 and 10 μg L⁻¹ formalin and 0.05, 0.1 and 0.2 μg L⁻¹ trifluralin concentrations. Doses of formalin and trifluralin obtained from the toxicity experiments were applied as prophylaxis to newly hatched larvae in white plastic basins. Prophylactic doses of 5 and 10 μg L⁻¹ formalin and 0.05 and 0.1 μg L⁻¹ trifluralin applied every other day were found to be effective in enhancing survival and larval development to megalopa compared with control. However, no megalopae survived to crab instar in all formalin treatments. Although the use of fungicides in rearing systems resulted in higher survival compared with controls, other strategies (i.e. maintenance of good water quality and hygienic practices in the hatchery) should be further investigated as an alternative to the use of chemicals in hatcheries.     

锯缘青蟹促雄腺发育的组织学研究

采用组织切片技术，以OlympusBH-2型显微镜对锯缘青蟹(Scylla serrata)促雄腺的发育进行观察。结果显示，促雄腺附着于射精管近末端，发育过程可分为4期：Ⅰ期促雄腺短小，腺细胞数量少；Ⅱ期促雄腺呈明显的索状；Ⅲ期促雄腺体积达到最大，一些部位膨大增生；Ⅳ期促雄腺不再生长，腺体急剧退化。腺细胞具有2种类型：A型细胞，核圆、异染色质少、胞质染色浅；B型细胞，核扁平、异染色质多，细胞染色深、界限不清晰，有时细胞质消失。促雄腺发育过程中，A、B型细胞数量、比例发生变化。B型细胞为A型细胞完成分泌后的存在形式，B型细胞比例反映了促雄腺分泌活动的状况。锯缘青蟹促雄腺发育和精巢成熟具有一定的相关性。     

Reproductive performance, lipids and fatty acids of mud crab Scylla serrata (Forsskål) fed dietary lipid levels

Natural food (NF, control), artificial diets (AD) containing total lipid levels of 10%, 12% and 14% (AD10, AD12 and AD14) and their combinations (AD10+NF, AD12+NF and AD14+NF) were fed for 112 days to pond‐sourced eyestalk‐ablated mud crab Scylla serrata (625±6.4 g) in tanks in order to determine their effects on reproduction and lipid profiles in broodstock tissues and zoeae. Crabs fed NF had the highest number of spawning followed by crabs fed AD10+NF and AD14+NF. Higher offspring production (number of zoeae) was obtained from crabs fed NF and AD+NF than from AD. As dietary total lipid levels increased, total lipid of broodstock ovaries, hepatopancreas, muscle and zoeae correspondingly increased in which AD+NF promoted higher levels than AD. Increased dietary total lipid levels enhanced lipid classes such as triacylglycerols and phosphatidyl choline levels in zoeae, all higher in crabs fed AD+NF than in AD. The major fatty acids in zoeae, particularly 16:0, 18:0, 18:1n‐9 and 20:4n‐6, 20:5n‐3 and 22:6n‐3, were higher in crabs fed AD+NF than in AD, the contents corresponding to broodstock dietary total lipid levels. A 10% total lipid in AD in combination with NF was sufficient to provide the essential lipids in crabs in the improvement of larval production and quality.     

Lipids and fatty acids in wild and pond-reared mud crab Scylla serrata (Forsskål) during ovarian maturation and spawning

Wild‐caught and pond‐reared female mud crab Scylla serrata at different stages of ovarian maturation were collected from Samar and Capiz, Philippines. Crabs were categorized into five stages according to the external morphological and microscopic appearance of the most advanced oocytes. The ovaries, hepatopancreas, muscle and newly spawned eggs (NSE) were analysed for lipid class components and fatty acids. Total lipid was higher in pond‐reared than in wild‐caught crabs but increased with ovarian maturation in both groups. Ovarian lipid peaked at the fully mature stage, coinciding with a decline in hepatopancreatic and muscle lipids. Lipid levels declined significantly in spent females. The tissues contained elevated highly unsaturated fatty acids such as arachidonic (20:4n‐6), eicosapentaenoic (20:5n‐3) and docosahexaenoic (22:6n‐3) acids, but at higher levels in late maturing and fully mature ovaries and in NSE. The type of lipid class and fatty acid components in mature ovaries as well as in NSE are generally considered to be indicative of their importance in reproductive physiology and embryonic and larval development.     

锯缘青蟹苗种培育的科技进展

锯缘青蟹是我国南方沿海各省传统养殖的海水蟹类,在当前农业产业结构调整中被列为主要发展品种之一。鉴于苗种是限制青蟹养殖业发展的主要因素,本文根据目前的研究现状对青蟹的种质、性成熟机制,产卵、孵化条件,幼体培育过程中营养条件、盐度、温度、光照、疾病等,以及从大眼幼体到仔蟹的中间培育技术等作了简要归纳,并对研究现状和进入产业化急需解决的问题进行了探讨。     

Ingestion of Brachionus plicatilis and Artemia salina nauplii by mud crab Scylla serrata larvae

Two feeding experiments were conducted to determine if Brachionus plicatilis and Artemia salina nauplii were ingested by mud crab Scylla serrata larvae. In the first experiment, larvae were fed with increasing densities of Artemia nauplii with or without Brachionus to determine consumption with increasing densities of Artemia and with increasing zoeal stage. This experiment also aimed to determine if the presence of Brachionus as an alternative prey influenced the intake of Artemia by the crab larvae. There was generally an increase in intake with increasing densities of Artemia and increased consumption of Artemia as the larvae grew. Consumption of Brachionus was consistently high in all zoeal stages. There was a significant reduction in the intake of Brachionus with increasing consumption of Artemia in the early zoeal stages (Z1, Z2, Z3), but at later stages (Z4, Z5) the intake of Artemia was no longer affected by the presence of Brachionus. In the second experiment, daily ingestion within instar of zoeal stages and megalopa were compared. There was an increased consumption of Artemia nauplii on the day before molting and increased ingestion of Brachionus on the day after larvae had molted, except at Z3. Megalopae exhibited a decline in Artemia nauplii intake on the days before metamorphosis to crablet.     

Ovarian maturation stages of the mud crab Scylla serrata

Ovarian maturation in adult wild‐sourced and pond‐grown Scylla serrata (Forsskål) was determined based on gross morphology and histological appearance. There were no significant differences noted in the histological features of both wild and pond‐reared S. serrata females. Ovarian maturation was classified into five stages: immature, early maturing, late maturing, fully mature and spent. The immature ovaries are thin and translucent to off white and contain oogonia, primary oocytes with large nuclei. The follicle cells were found around the periphery of the lobes and an area among groups of oogonia and oocytes. The follicle cells gradually enclosed the oocytes. The early‐maturing ovaries were yellow and small yolk globules started to appear in larger oocytes. In late‐maturing ovaries, the colour became light orange and lobules were apparent. Yolk globules occurred in the cytoplasm with larger globular inclusions towards the periphery, while follicle cells were hardly recognizable. Fully mature ovaries were orange to deep orange and had swollen lobules. Large yolk globules were apparent in the entire cytoplasm. Follicle cells were hardly seen. Spent ovaries were similar to the early‐maturing and late‐maturing stage in partially spawned females. The ovarian development was correlated closely to the gonadosomatic index, oocyte diameter, and ovarian histology. The classification of ovarian maturation provides baseline information for further studies on reproductive biology. Likewise, the information provides a guide for broodstock management in the hatchery.     

Quality assessment of newly hatched mud crab,Scylla serrata,larvae

Starvation and exposure to formalin were investigated as possible stress tests for evaluating the quality of mud crab, Scylla serrata, larvae. For the starvation stress test, newly hatched zoeae stocked in 150‐ml containers were either starved or fed rotifers. Similarly, newly hatched zoeae were stocked in containers with seawater of 0 (control), 20, 30 and 40 mg/L formalin for the formalin stress test. The zoeae from the same batches were used for seed production to monitor their performance and validate the results of stress tests. Starvation was found to be unsuitable for larval quality evaluation. However, the impact of initial food deprivation on the newly hatched larvae indicates that feeding immediately after hatching is necessary for mud crab larvae. Exposure of larvae to 40 mg/L formalin for 3 hr appeared to be a reliable and practical method for larval quality assessment as the survival of larvae in the mass production tanks validated the classification of good and poor quality batches in the stress tests. On this basis, a hatchery operator can decide which batch should be cultured further. Finally, there appears to be a link between the quality of larvae and the performance at the megalopa and early juvenile crabs.     

Stocking density and artificial habitat influence stock structure and yield from intensive nursery systems for mud crabs Scylla serrata (Forsskål 1775)

Intensive nursery systems are designed to culture mud crab postlarvae through a critical phase in preparation for stocking into growout systems. This study investigated the influence of stocking density and provision of artificial habitat on the yield of a cage culture system. For each of three batches of postlarvae, survival, growth and claw loss were assessed after each of three nursery phases ending at crab instars C1/C2, C4/C5 and C7/C8. Survival through the first phase was highly variable among batches with a maximum survival of 80% from megalops to a mean crab instar of 1.5. Stocking density between 625 and 2300 m⁻² did not influence survival or growth in this first phase. Stocking densities tested in phases 2 and 3 were 62.5, 125 and 250 m⁻². At the end of phases 2 and 3, there were five instar stages present, representing a more than 20‐fold size disparity within the populations. Survival became increasingly density‐sensitive following the first phase, with higher densities resulting in significantly lower survival (phase 2: 63% vs. 79%; phase 3: 57% vs. 64%). The addition of artificial habitat in the form of pleated netting significantly improved survival at all densities. The mean instar attained by the end of phase 2 was significantly larger at a lower stocking density and without artificial habitat. No significant effect of density or habitat on harvest size was detected in phase 3. The highest incidence of claw loss was 36% but was reduced by lowering stocking densities and addition of habitat. For intensive commercial production, yield can be significantly increased by addition of a simple net structure but rapidly decreases the longer crablets remain in the nursery.     

锯缘青蟹促雄腺进行全浆分泌的证据

采用透射电镜技术，对锯缘青蟹(Scylla serrata)促雄腺细胞的分泌活动进行了观察。结果表明，腺细胞具有2种类型：A型细胞核内常染色质丰富，具有大量的扁平嵴的线粒体、粗面内质网与核糖体，A型细胞进行分泌时，细胞质大量排出，属于全浆分泌方式；B型细胞异染色质绕核内膜分布，仅见极少量的细胞器或细胞质完全消失。B型细胞为A型细胞完成分泌后的存在形式。本研究为甲壳动物促雄腺的全浆分泌活动提供了直接的证据。     

Effects of Artemia enriched with eicosapentaenoic and docosahexaenoic acid on survival and occurrence of molting failure in megalop larvae of the mud crab Scylla serrata

ABSTRACT:   The present study was conducted to investigate the effect of eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) on the survival and the occurrence of molting failure to megalops of mud crab Scylla serrata larvae fed enriched Artemia . Survival rate, intermolt period, carapace width at the first crab stage, ratio of chela to carapace length at the fifth zoeal stage and the occurrence of molting failure to megalops were observed. Mud crab larvae were reared in 1-L plastic beakers and fed with Artemia enriched at five different levels of EPA (0.31% to 1.36% EPA; referred to as E25, E50, E75 and E100) or four different levels of DHA (0.75–0.95% EPA and 0.49–1.38% DHA; referred to as D25, D50, D75 and D100). As a negative control, larvae were fed Artemia enriched with oleic acid (OA). Mud crab larvae fed Artemia containing low (0.41–0.45% EPA and trace DHA) or high (1.36% and 0.95% EPA and 0.16% and 1.38% DHA) amounts of EPA and DHA showed a significantly lower survival rate and prolonged intermolt period ( P  < 0.05). Moreover, a high frequency of molting failure to the megalops stage (34 and 33%) occurs coincident with a high chela to carapace length ratio (43 and 44%) in mud crab larvae fed high amounts of EPA and DHA (E100 and D100), suggesting that both of these treatments contain EPA and DHA in excess. These results indicate that during Artemia feeding, EPA and DHA content should be adjusted to 0.71–0.87% and 0.49–0.72% for maintaining a high survival, accelerating the intermolt period, and producing larger carapace width in the first crab stage.     

低温驯化下锯缘青蟹肝胰腺的抗氧化效应及ATPase活性变化

采用生物化学方法对低温驯化的锯缘青蟹（Scylla serrata）肝胰腺中抗氧化酶（SOD、CAT和GPX）活性和脂类过氧化产物MDA含量以及Na^＋-K^＋ -ATPase、Mg^2＋ -ATPase、Ca^2＋ -ATPase和Ca^2＋ -Mg^2＋ -ATPase活性进行测定。结果显示，锯缘青蟹肝胰腺中抗氧化酶（SOD、CAT和GPX）活性随驯化温度的降低而降低；而MDA含量则是随驯化温度的降低而升高，其变化正好与抗氧化酶活性相反。Na^2＋ -K^＋ -ATPase、Mg^2＋ -ATPase和Ca^2＋ -Mg^2＋ -ATPase活性均是随驯化温度的降低而升高；而Ca^2＋ -ATPase活性没有表现出这一明显变化趋势，但低温驯化下仍然是酶活性升高。锯缘青蟹低温驯化下抗氧化酶活性降低表明其清除活性氧能力下降，从而导致MDA在肝胰腺中积累。ATPase活性低温驯化下升高，说明其表现出明显的温度补偿效应（低温使酶活性下降，同时又诱导酶合成增加，从而使酶浓度增加，其总的酶活性仍能维持机体正常的生理功能），但各种ATPase活性的温度补偿存在差异。     

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20–33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.