Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft‐shell farming system

Soft‐shell crab, a newly moulted crab having non‐calcified and hydrated soft exoskeleton, farming has been increasing tremendously in recent years throughout the world due to its high market price and consumer preferences. However, high aggressiveness and cannibalistic behaviour of mud crab limit its culture to an individual chamber system which ultimately makes the soft‐shell mud crab farming space and labour‐intensive. Therefore, the aim of this study was to improve the efficiency of soft‐shell crab production by autotomy of the claw and/or walking legs to reduce the moulting duration and intensify the culture system through double stocking in the existing individual chamber culture system. To achieve our objectives, a series of experiment were conducted at both indoor and on‐farm levels by removing the claw and/or walking legs of Scylla olivacea for two consecutive moulting cycles. Results showed that both claw ablation significantly (p < .01) shortened the moulting duration and increased the carapace width, weight gain, specific growth rate, feeding efficiency and yield parameter compared to the non‐ablated crabs more prominently at 1st moulting than the 2nd moulting in both indoor and on‐farm trials. Ablation of all walking legs while keeping the claw intact did not significantly influence (p > .05) the moulting duration and carapace width, but significantly (p < .01) improved the specific growth rate, weight gain, feeding efficiency and yield parameters at 1st moulting compared to the non‐ablation and unilateral three walking leg ablation groups. However, ablation of all appendages (both claw and walking legs) showed significant (p < .01) improvements of carapace width, weight gain, specific growth rate, feeding efficiency and yield parameters, and shortened the moulting duration up to 23 days from 34 days during 1st moulting and up to 27 days from 40 days at 2nd moulting. The survivals of different autotomized mud crabs were statistically non‐significant (p > .05) to the non‐autotomized crabs in various indoor and on‐farm trials. Besides improving the production performances, ablation of bilateral claw and all appendages allowed to double the stocking density in the existing individual chamber‐based soft‐shell crab farming that enhanced the production about threefold higher than the existing individual chamber‐based culture system. Finally, our results suggest that autotomy could be a promising technology for the commercial farming of soft‐shell mud crab in near future.     

Morphological deformities in mud crab Scylla serrata juveniles exposed to antibiotics during the larval stage

The effects of antibiotics on the external deformities, growth and survival of mud crab Scylla serrata larvae and juveniles were determined. Zoeae were exposed to oxytetracycline (OTC) (0, 3.0, 6.0, 9.0, 12 mg L^?1) and furazolidone (FZD) (0, 0.5, 1.0, 1.5, 2.0 mg L^?1) in the first and second experiments, respectively, until the late megalopa. The crab instars were grown in nursery tanks for 1 month. Larvae survived until megalopa only at 3.0 and 6.0 mg L^?1 OTC or 0.5 and 1.0 mg L^?1 FZD. These four concentrations were run simultaneously in another experiment. Morphological deformities in zoea 5 were bent dorsal, rostral and furcal spines. There was no significant difference (P > 0.05) on the deformities of zoea 5 in 3.0 and 6.0 mg L^?1 OTC and 0.5 and 1.0 mg L^?1 FZD. Significantly (P < 0.05) higher survival and faster growth were attained in 3.0 mg L^?1 OTC and 0.5 mg L^?1 FZD. Deformities observed in juveniles were fused frontal and lateral spines, asymmetrical and depressed tip of abdominal flap and gap between sternites. High percentage occurrence of deformities was observed in the 6.0 mg L^?1 OTC and 1.0 mg L^?1 FZD in the first and third experiments, respectively. There was no significant difference (P > 0.05) observed in the survival of juveniles in OTC and FZD treatments. However, growth was significantly (P < 0.05) faster in lower concentrations of the two antibiotics. The study shows the effects of OTC and FZD in the morphology of mud crab. Therefore, there is a need to eliminate the use of antibiotics and find alternatives.     

Dietary protein requirement of giant mud crab Scylla serrata juveniles fed iso-energetic formulated diets having graded protein levels

The protein requirement of juvenile mud crab Scylla serrata (body weight=0.25±0.051 g, carapace width=9.3±0.04 mm) fed with different iso-energetic, iso-lipidic diets with graded protein levels (15–55% crude protein at 5% intervals) was determined. The feeding trial was conducted for a period of 63 days to determine the minimum and optimum protein requirement of juvenile S. serrata . The crabs fed with 15% and 20% dietary protein levels showed 100% and 12.5% of mortalities respectively. The mortalities observed in the above treatments were associated with the prolonged intermoult duration (46 and 32 days respectively). All other treatments recorded 100% survival. The best growth performance as well as the nutrient turn-over was recorded in crabs fed with 45% crude protein in the diet. Second-order polynomial regression of specific growth rate (SGR) as well as body protein gain vs. dietary protein levels suggested that 46.9–47.03% dietary protein is required for the best growth response and protein deposition in juvenile S. serrata . An extrapolation of 'SGR' and 'daily protein gain' upon the 'dietary protein level' axis ( Y =0) showed that 14.7–16.2% dietary protein is necessary for the minimum maintenance metabolism.     

Dietary astaxanthin supplementation for hatchery‐cultured red king crab,Paralithodes camtschaticus,juveniles

We conducted large‐scale production trials in Seward, Alaska, USA to investigate effects of dietary astaxanthin supplementation on survival, growth and shell colouration of recently settled juvenile (C1–C4) red king crabs (Paralithodes camtschaticus). We supplemented a control diet of commercial crustacean feeds with astaxanthin, and fed these diets to juvenile king crabs at densities of 2000 and 4000 crabs m^?2 for 56 days. We assessed survival and growth by counting crabs and individually measuring carapace width and weighing crabs at the start and end of the experiment, and quantified crab colour (hue, saturation, brightness) in digital photographs. Diets containing astaxanthin had higher survival, suggesting that astaxanthin may provide nutritional or immune system benefits. Crabs had lower hue, higher saturation and lower brightness values when fed diets containing astaxanthin, suggesting that red king crab colouration is plastic and responds to diet. Astaxanthin is likely an important dietary component for hatchery or laboratory reared red king crab juveniles, and should be considered for aquaculture and other rearing of this and possibly other crustacean species.     

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.     

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.     

Extension of nursery culture of Scylla serrata (Forsskål) juveniles in net cages and ponds

To address the preference of mud crab farmers for larger size Scylla serrata juveniles (5.0–10 g body weight or BW; 3.0–5.0 cm internal carapace width or ICW), a study was conducted to compare the growth and survival of crab juveniles (2.0–5.0 g BW; 1.0–3.0 cm ICW) produced a month after stocking of megalopae in net cages when reared further in net cages installed in earthen ponds or when stocked directly in earthen ponds. In a 3 × 2 factorial experiment, three stocking densities (1, 3 and 5 ind m⁻²), two types of rearing units (net cages or earthen pond) were used. Megalopae were grown to juvenile stage for 30 days in net cages set inside a 4000 m² brackishwater pond and fed brown mussel (Modiolus metcalfei). Crab juveniles were then transferred to either net cages (mesh size of 1.0 mm) or earthen ponds at three stocking densities. After 1 month, no interaction between stocking density and rearing unit was detected so data were pooled for each stocking density and rearing unit. There were no significant differences in the growth or survival rate of crab juveniles across stocking density treatments. Regardless of stocking density, survival in net cages was higher (77.11±6.62%) than in ponds (40.41±3.59%). Growth, however, was significantly higher for crab juveniles reared in earthen ponds. The range of mean BW of 10.5–16.0 g and an ICW of 3.78–4.33 cm obtained are within the size range preferred by mud crab operators for stocking grow‐out ponds.     

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.     

Effect of selected feed meals and starches on diet digestibility in the mud crab,Scylla serrata

The present study examined the capacity of the mud crab, Scylla serrata to digest experimental diets that contained different animal and plant‐based feed meals or different levels or types of starch. The apparent dry matter digestibility (ADMD) coefficients for all feed meals tested in the first part of this study, except meat meal, were similar (78–88%). Crude protein digestibility (ACPD) coefficients for all feed meals were relatively high, with values ranging from 86% to 96%. Cotton seed meal, poultry meal, canola meal, fishmeal, soybean meal and lupin meal had similar gross energy digestibility (AGED) values (P>0.05) ranging from 84% to 89%. In the second part of this study, the impact of selected starches on the digestibility of fishmeal‐based formulated diets was assessed. The apparent starch digestibility (ASD) of wheat starch decreased significantly as the inclusion level was increased from 15% to 60%, however, there was no significant effect on ACPD values. At a 30% inclusion level, the ASD of diets containing different starches decreased in the order corn>wheat>potato=rice. Moreover, ACPD values were significantly higher (P<0.05) in the diets containing corn or rice starch than in those containing wheat or potato starches.     

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。     

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.     

环境因子变化对锯缘青蟹后期幼体及仔蟹变态存活率的影响

通过实验对锯缘青蟹Z、M、C1～C4仔蟹在不同温度、盐度及干露时间的条件下，观察变态及存活率的差异。结果表明：1．温度改变对Z5变态率有影响，30—26℃温度突变其变态率为53％，30-28～25℃温度渐变变态率为60％，对照组(30℃)为57％；温度改变对M的变态存活率和变态时间间隔无影响．但C1变态为C2的时间间隔比对照组延长24～48h。2．盐度改变对青蟹Z5及C1-C4。仔蟹存活和变态的影响不同,在直接由盐度33移至28、21、15、8的不同条件下，Z5和M的盐度忍耐下限分别为21和15；C1-C4仔蟹在以上的盐度变化条件下均能成活、变态，但C4仔蟹在28、33高盐度下部分个体蜕壳不遂或蜕壳后死亡。3．蟹苗及仔蟹耐干露能力随着个体长大而增强；同规格蟹苗在干露6～18h条件下，变态时间间隔随着干露时间的增加延长24．48h；M在潮湿海绵和不透风的条件下放置12h，可正常变态，离开海绵则在6h内全部死亡；但C1-C4仔蟹即便离开海绵18h，也能正常存活变态。     

土霉素在锯缘青蟹体内的药物代谢和消除规律

采用高效液相色谱法检测土霉素,研究土霉素口灌给药途径下在锯缘青蟹体内的药代动力学。锯缘青蟹口灌给药土霉素50 mg/kg后,其血浆、肌肉和肝胰脏中的药峰浓度分别为16.78±1.98 mg/L、9.39±2.12μg/g和32.12±6.12μg/g,达峰时间分别为4 h、8 h和4 h。血浆中土霉素浓度-时间关系曲线符合一级吸收的二室开放动力学模型。土霉素在锯缘青蟹体内分布广泛,其表观分布容积(Vd)为2.129 L/kg;分布半衰期(t1/2α)和消除半衰期(t1/2β)分别为3.200 h和47.856 h,总体清除率(CLs)为0.063 mL/(kg.h)。肌肉和肝胰脏中土霉素浓度与时间关系的药动学参数采用统计矩原理分析,其消除半衰期(t1/2 z)分别为60.145 h和71.009 h,总体清除率(CLz)分别为0.054 g/(kg.h)和0.037 g/(kg.h)。土霉素在精巢和卵巢中达峰时间分别为8 h和12 h,峰浓度分别为9.83μg/g和10.26μg/g。给药后24 d时,血浆、肌肉、肝胰脏、精巢和卵巢中土霉素含量都已低于0.10μg/g。土霉素在锯缘青蟹体内消除比较缓慢。     

Improved hatchery‐rearing techniques for juvenile production of blue swimming crab,Portunus pelagicus (Linnaeus, 1758)

The effects of tank colour, larval stocking density, antibiotic administration and water exchange on survival and moulting of blue swimming crab, Portunus pelagicus, were determined. Circular 4‐m³ experimental larval‐rearing tanks were used in triplicate for all treatments. White, dark grey, blue and brown were tested as tank background colours. The stocking densities tested were 10, 20, 40, 60, 80 and 100 larvae L^?1. The effect of oxytetracycline was estimated by comparing a treatment with oxytetracycline to a treatment without oxytetracycline administration. The daily water exchange rates tested were 0%, 25%, 50% and 100% of the tank volume. In all treatments, the larvae were fed with Artemia nauplii, rotifers and encapsulated Spirulina. The highest percentage survival was observed in the dark‐grey tanks when the stocking density of larvae was 20 larvae L^?1. No larva reached the juvenile crab size in white tanks. No significant difference in survival was found between treated and non‐treated larvae with oxytetracycline when the daily water exchange rate was more than 50%.     

Chemical constituents in the abdominal muscle of cultured mud crab Scylla serrata in relation to seasonal variation and maturation

ABSTRACT:     Seasonal changes in levels of chemical constituents in the abdominal muscle of male and female mud crab Scylla serrata were investigated. The gonadosomatic index in the female crab was higher in October and August, of which the muscle yield, glycogen, and total amount of adenosine 5'-triphosphate (ATP)-related compounds (ARC) in the October sample were the highest throughout the year, but the total amount of free amino acids (FAA) was the lowest. The total FAA and individual FAA such as glycine, alanine, and arginine increased in the August and November female samples, while that in the male specimens was higher in January, March, and August. The total ARC in both crabs was lower in the samples collected from winter than in other seasons whereas glycinebetaine was higher in winter and early spring. Glutamic acid, glycine, alanine, arginine, glycinebetaine, and adenosine monophosphate might be responsible for the taste of mud crab. Their level together was relatively higher in the August and January male samples, and in the August and November female specimens. However, the correlation between the taste component content and degree of gonad maturation was low.     

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.     

Effects of temperature and salinity on the survival and development of mud crab, Scylla serrata (Forsskål), larvae

The combined effects of temperature and salinity on larval survival and development of the mud crab, Scylla serrata, were investigated in the laboratory. Newly hatched larvae were reared under 20 °C temperature and salinity combinations (i.e. combinations of four temperatures 25, 28, 31, 34 °C with five salinities 15, 20, 25, 30, 35 g L⁻¹). The results showed that temperature and salinity as well as the interaction of the two parameters significantly affected the survival of zoeal larvae. Salinity at 15 g L⁻¹ resulted in no larval survival to the first crab stage, suggesting that the lower salinity tolerance limit for mud crab larvae lies somewhere between salinity 15 and 20 g L⁻¹. However, within the salinity range of 20–35 g L⁻¹, no significant effects on survival of zoeal larvae were detected (P>0.05). The combined effects of temperature and salinity on larval survival were also evident as at low salinities, both high and low temperature led to mass mortality of newly hatched larvae (e.g. 34 °C/15 g L⁻¹, 34 °C/20 g L⁻¹ and 25 °C/15 g L⁻¹ combinations). In contrast, the low temperature and high salinity combination of 25 °C/35 g L⁻¹ resulted in one of the highest survival to the megalopal stage. It was also shown that at optimal 28 °C, larvae could withstand broader salinity conditions. Temperature, salinity and their interaction also significantly affected larval development. At 34 °C, the mean larval development time to megalopa under different salinity conditions ranged from 13.5 to 18.5 days. It increased to between 20.6 and 22.6 days at 25 °C. The effects of salinity on larval development were demonstrated by the fact that for all the temperatures tested, the fastest mean development to megalopa was always recorded at the salinity of 25 g L⁻¹. However, a different trend of salinity effects was shown for megalopae as their duration consistently increased with an increase in salinity from 20 to 35 g L⁻¹. In summary, S. serrata larvae tolerate a broad range of salinity and temperature conditions. Rearing temperature 25–30 °C and salinity 20–35 g L⁻¹ generally result in reasonable survival. However, from an aquaculture point of view, a higher temperature range of 28–30 °C and a salinity range of 20–30 g L⁻¹ are recommended as it shortens the culture cycle.