Mhd Ikhwanuddin

Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass, Lates calcarifer challenged with Vibrio harveyi.

The present study was aimed to address the possible evaluation of Azadirachta indica (neem) leaf-supplemented diets on innate immune response in Asian seabass, Lates calcarifer fingerlings against Vibrio harveyi infection. Fish were fed for two weeks diets containing six graded levels of neem leaf at 0 g, 1 g, 2 g, 3 g, 4 g and 5 g per kg feed. Fish fed neem leaf-supplemented diet displayed significant differences (p < 0.05) in weight gain, specific growth rate (SGR) and feed conversion ratio (FCR) compared to the control group fed without neem leaf-supplemented diet. Various innate immune parameters were examined pre-challenge and post-challenge. Fish was injected intraperitoneally with a lethal dose of V. harveyi containing 10(8) cells mL(-1). Supplementation of neem leaf diet significantly increased phagocytic activity, superoxide anion production, serum lysozyme, serum bactericidal activity, serum anti-protease activity throughout the experimental period when compared with the control group. Dietary doses of neem leaf diet significantly influenced the immune parameters, haematological parameters and blood biochemical indices of treated fish. The results suggested that fish fed neem leaf-supplemented diet improved the immune system and increased survival rate in L. calcarifer fingerlings against V. harveyi infection.

A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective

Study of broodstock maturation diets is important in order to increase the quality of berried females, which indirectly improve the larval quantity in the hatchery production of cultured species. This paper reviewed the studies on the maturation diets for mud crab broodstock, genus Scylla and compared independently to identify their effect on reproductive performance and larval quality. The broodstock is usually caught from the wild and held in the spawning or maturation tank for further use of hatchery seed production. Mud crab broodstock was fed either natural diet, artificial diet or mixed diet. Trash fishes were commonly used as a natural feed for mud crab broodstock; meanwhile artificial diets are from formulated fish meal and various kinds of feed. The results indicated that mud crab broodstock has a high dietary requirement for lipids, fatty acids and protein which are to be used during the maturation and breeding processes. However, the natural diet produce better larval quality compared to the artificial diet. The mixed diet is the better diet which resulted in better reproductive performances such as growth, survival, fecundity and maturation processes. This review also discusses the problems in the previous studies for the potential future research to develop very high quality and cost-effective formulated diet for the enhancement of broodstock and seed production technology. Information from this review can be useful in developing a better quality of crustacean broodstock’s diet for commercial hatchery production.

Size distribution, length–weight relationship and size at the onset of sexual maturity of the orange mud crab, Scylla olivacea, in Malaysian waters

ABSTRACT The size distribution, length–weight relationship and size at the onset of sexual maturity of the orange mud crab (Scylla olivacea) from four geographically distinct locations (Taiping, Setiu, Kota Marudu and Lundu) representing Malaysian waters were analysed and estimated. Scylla olivacea was found in the size range of 47–134 mm carapace width. Males were significantly smaller in size but heavier than females. Geographical variation in carapace width and body weight were significant, but no interaction was found between sexes and locations. As shown by the length–weight relationships of S. olivacea, the males exhibited positive growth allometry whereas the females exhibited negative growth allometry. Males mature physiologically prior to attaining morphometric sexual maturity. Females, however, achieve physiological and morphometric sexual maturity in synchrony. No significant variation was found in the estimates of size at the onset of sexual maturity of males and females among different locations. We recommend the use of the third right walking leg merus length and carapace width to estimate the size at the onset of sexual maturity (morphometric maturity) for S. olivacea. Data obtained in this study serve as important baseline data for future mud crab resource management in Malaysia and were used to recommend minimum landing sizes for S. olivacea in each respective location based on the largest size at the onset of sexual maturity estimates were suggested.

Transcriptome Analysis and Differential Gene Expression on the Testis of Orange Mud Crab, Scylla olivacea, during Sexual Maturation

Adequate genetic information is essential for sustainable crustacean fisheries and aquaculture management. The commercially important orange mud crab, Scylla olivacea, is prevalent in Southeast Asia region and is highly sought after. Although it is a suitable aquaculture candidate, full domestication of this species is hampered by the lack of knowledge about the sexual maturation process and the molecular mechanisms behind it, especially in males. To date, data on its whole genome is yet to be reported for S. olivacea. The available transcriptome data published previously on this species focus primarily on females and the role of central nervous system in reproductive development. De novo transcriptome sequencing for the testes of S. olivacea from immature, maturing and mature stages were performed. A total of approximately 144 million high-quality reads were generated and de novo assembled into 160,569 transcripts with a total length of 142.2 Mb. Approximately 15–23% of the total assembled transcripts were annotated when compared to public protein sequence databases (i.e. UniProt database, Interpro database, Pfam database and Drosophila melanogaster protein database), and GO-categorised with GO Ontology terms. A total of 156,181 high-quality Single-Nucleotide Polymorphisms (SNPs) were mined from the transcriptome data of present study. Transcriptome comparison among the testes of different maturation stages revealed one gene (beta crystallin like gene) with the most significant differential expression—up-regulated in immature stage and down-regulated in maturing and mature stages. This was further validated by qRT-PCR. In conclusion, a comprehensive transcriptome of the testis of orange mud crabs from different maturation stages were obtained. This report provides an invaluable resource for enhancing our understanding of this species’ genome structure and biology, as expressed and controlled by their gonads.

Gonadal development in males of the orange mud crab, Scylla olivacea (Herbst, 1796) (Decapoda, Brachyura, Portunidae)

This study describes the morphological and histological characteristics of the male reproductive system of the orange mud crab, Scylla olivacea (Herbst, 1796). Three maturation stages were determined on the basis of the vas deferens diameter, the gonad colour and the gonadosomatic index (GSI). Significant differences were observed in the GSI value as the crabs matured. All germ cells were present in the histological sections of the testes, and these decrease significantly in size as they progress from spermatogonia, spermatocytes, and spermatids to spermatozoa. Spermatophores are found in the anterior vas deferens (AVD) and median vas deferens (MVD) of all maturation stages but not in the posterior vas deferens (PVD), with a significantly smaller size in immature and maturing specimens. Thus, the classification of S. olivacea gonads into various maturation stages based solely on histological characteristics (i.e., the presence of spermatophores) is impossible. Therefore, the combination of both microscopic and macroscopic features is essential for determining the gonad maturation stages of male S. olivacea .

Use of Abdomen Looseness as an Indicator of Sexual Maturity in Male Mud Crab Scylla spp.

ABSTRACT Stock assessment in wild populations and research involving reproductive biology of crabs rely greatly on the ability to accurately determine sexual maturity status. Although determination of sexual maturity in male mud crabs was commonly based on the presence of spermatophores in the vas deferens, preliminary studies on the mud crab Scylla spp. showed that this method is inconclusive and was reevaluated in this study. In addition, the feasibility of using male abdomen looseness as maturity indicator was explored. Spermatophores were present in the vas deferens of all Scylla spp. regardless of maturity status and body size. Spermatophores also significantly increased in size as the crabs reached sexual maturity. Examination of the vas deferens external morphology (e.g., color and size) consistently differentiated immature from mature males. The size at maturity (CW50) was successfully estimated using three maturity indicators, that is, the looseness of the abdomen, the observation of mating scars, and the vas deferens development. The estimated sizes obtained using all three indicators showed no significant differences, but varied significantly among species. A positive relationship was observed between body size and sexual maturity in all three Scylla species. Determination of sexual maturity using the presence of mating scars as indicator was inconsistent as some crabs that were mature in terms of looseness of abdomen and vas deferens development showed no presence of mating scars. On the other hand, male crabs with loose abdomens were found to be sexually matured with enlarged, milky white vas deferens. This finding suggests that abdomen looseness is an accurate sexual maturity indicator in males. Using looseness of abdomen as sexual maturity indicator in the male Scylla spp., therefore, is feasible and superior to conventional methods, with the advantages of being practical, easy to carry out, and does not require sacrificing of the crabs.

Comparative Transcriptome Analysis Provides Insights into Differentially Expressed Genes and Long Non-Coding RNAs between Ovary and Testis of the Mud Crab (Scylla paramamosain)

The molecular mechanism underlying sex determination and gonadal differentiation of the mud crab (Scylla paramamosain) has received considerable attention, due to the remarkably biological and economic differences between sexes. However, sex-biased genes, especially non-coding genes, which account for these differences, remain elusive in this crustacean species. In this study, the first de novo gonad transcriptome sequencing was performed to identify both differentially expressed genes and long non-coding RNAs (lncRNAs) between male and female S. paramamosain by using Illumina Hiseq2500. A total of 79,282,758 and 79,854,234 reads were generated from ovarian and testicular cDNA libraries, respectively. After filtrating and de novo assembly, 262,688 unigenes were produced from both libraries. Of these unigenes, 41,125 were annotated with known protein sequences in public databases. Homologous genes involved in sex determination and gonadal development pathways (Sxl-Tra/Tra-2-Dsx/Fru, Wnt4, thyroid hormone synthesis pathway, etc.) were identified. Three hundred and sixteen differentially expressed unigenes were further identified between both transcriptomes. Meanwhile, a total of 233,078 putative lncRNAs were predicted. Of these lncRNAs, 147 were differentially expressed between sexes. qRT-PCR results showed that nine lncRNAs negatively regulated the expression of eight genes, suggesting a potential role in sex differentiation. These findings will provide fundamental resources for further investigation on sex differentiation and regulatory mechanism in crustaceans.

The final spawning ground of Tachypleus gigas (Müller, 1785) on the east Peninsular Malaysia is at risk: a call for action.

Tanjung Selongor and Pantai Balok (State Pahang) are the only two places known for spawning activity of the Malaysian horseshoe crab - Tachypleus gigas (Müller, 1785) on the east coast of Peninsular Malaysia. While the former beach has been disturbed by several anthropogenic activities that ultimately brought an end to the spawning activity of T. gigas, the status of the latter remains uncertain. In the present study, the spawning behavior of T. gigas at Pantai Balok (Sites I-III) was observed over a period of thirty six months, in three phases, between 2009 and 2013. Every year, the crab’s nesting activity was found to be high during Southwest monsoon (May–September) followed by Northeast (November–March) and Inter monsoon (April and October) periods. In the meantime, the number of female T. gigas in 2009–2010 (Phase-1) was higher (38 crabs) than in 2010–2011 (Phase-2: 7 crabs) and 2012–2013 (Phase-3: 9 crabs) for which both increased overexploitation (for edible and fishmeal preparations) as well as anthropogenic disturbances in the vicinity (sand mining since 2009, land reclamation for wave breaker/parking lot constructions in 2011 and fishing jetty construction in 2013) are responsible. In this context, the physical infrastructure developments have altered the sediment close to nesting sites to be dominated by fine sand (2.5Xφ ) with moderately-well sorted (0.6–0.7σφ), very-coarse skewed (−2.4SKφ), and extremely leptokurtic (12.6Kφ) properties. Also, increased concentrations of Cadmium (from 4.2 to 13.6 mg kg−1) and Selenium (from 11.5 to 23.3 mg kg−1) in the sediment, and Sulphide (from 21 to 28 µg l−1) in the water were observed. In relation to the monsoonal changes affecting sheltered beach topography and sediment flux, the spawning crabs have shown a seasonal nest shifting behaviour in-between Sites I-III during 2009–2011. However, in 2012–2013, the crabs were mostly restricted to the areas (i.e., Sites I and II) with high oxygen (5.5–8.0 mg l−1) and moisture depth (6.2–10.2 cm). In view of the sustained anthropogenic pressure on the coastal habitats on one hand and decreasing horseshoe crabs population on the other, it is crucial to implement both conservation and management measures for T. gigas at Pantai Balok. Failing that may lead to the loss of this final spawning ground on the east coast of P. Malaysia.

Thermal tolerance and locomotor activity of blue swimmer crab Portunus pelagicus instar reared at different temperatures

Owing to its potential market value, the blue swimmer crab Portunus pelagicus is of great economic importance. The temperature of water significantly affects the physiological function and production efficiency of these crabs. The aim of the present study was therefore to examine the critical thermal minimum (CTMin), critical thermal maximum (CTMax), acclimation response ratio (ARR), escaping temperature (Tesc), and locomotor behavior of P. pelagicus instars at 20 °C, 24 °C, 28 °C, 32 °C, and 36 °C. The CTMax ranged from 39.05 °C to 44.38 °C, while the CTMin ranged from 13.05 °C to 19.30 °C, and both increased directly with temperature. The ARR ranged from 0.25 to 0.51. The movement of crabs (walking before molting) correlated positively with the acclimation temperature. These results indicate that the parameters evaluated varied with temperature. Furthermore, the high CTMax indicates the potential of this species to adapt to a wide range of temperatures. In addition, the implications of these findings for portunid crabs behavior and distribution in their natural habitat are also discussed.

Gut Lactobacillus sp. bacteria as probiotics for Portunus pelagicus (Linnaeus, 1758) larviculture: effects on survival, digestive enzyme activities and water quality

In this study, the effects of indigenous probiotic, Lactobacillus rhamnosus, as an additive to water in Portunus pelagicus larviculture was investigated on survival rate, digestive enzyme activities and water quality. All experiments were triplicated and designed with three different administrations of probiotic 1.0 × 106 cfu mL−1 (T-1), 5.0 × 106 cfu mL−1 (T-2) and 1.0 × 107 cfu mL−1 (T-3) added daily and one control without any probiotic. The supplementation of the probiotic significantly (p < 0.05) increased survival rate for all treatments over the controls. Highest survival rate (10.0 ± 0.9%) of larvae was achieved in T-2. Addition of probiotic bacteria affected the bacterial load (cfu mL−1) in rearing water. The specific activities of protease and amylase enzymes in probiotic treatments were significantly higher (p < 0.05). The highest specific activities of protease (0.17 ± 0.01 U mg−1 protein) and amylase (0.36 ± 0.02 U mg−1 protein) were determined in treated group T-2, respectively. Addition of probiotic affected the pH and nitrogen content of rearing water. The results of this study suggest that the indigenous L. rhamnosus added to rearing water at a certain concentration significantly enhanced survival rate, measured digestive enzyme activities and improved the water quality of P. pelagicus larvae.