The influence of seasonal temperature variation on embryonic development in two species of spider crabs (Majoidea).

The aim of this study was to analyze the ecological influence of seasonal temperature variations on embryonic development in two species of Majoidea. Leurocyclus tuberculosus and Libinia spinosa. These species inhabit stable populations in the San José Gulf (42°25'41″S, 64°08'27″O, Patagonia-Argentina) in a wide depth range and seasonal temperature variations from 8 °C to 18 °C. The influence of seasonal temperatures on embryonic development was examined in ovigerous females of both species, through a bioassay in laboratory conditions at 8 °C, 14 °C, and 18 °C from gastrula to hatching. Additionally, monthly female samplings were conducted for one year, characterizing their spatial depth distribution associated with the seawater temperature in the study area and their ovigerous/non ovigerous condition. Laboratory experimental results showed that duration of incubation increased 67 % in L. tuberculosus and 55% in L. spinosa by a decline in temperature from 18 °C to 8 °C with one diapause period. The embryonic development for the 3 assayed temperatures was divided in five periods. The assayed temperatures did not affect the morphology of the embryo, and its growth during each period did not present significant differences among the different temperatures. Field analysis showed significant differences in the spatial distribution of ovigerous females which were associated to the assessed temperatures along the year. This distribution might modulate the development of embryos leading to two spawnings synchronized with the phyto- and zooplankton peaks in the San José gulf. Finally, ecological and potential impact of ocean warming in these species was also addressed.

Molecular cloning, characterization, and expression analysis of a sex-biased transcriptional factor sox9 gene of mud crab Scylla paramamosain.

Sox9 gene, a crucial member of the Sox gene family, is present in various organisms and involved in many physiological processes, especially in sex determination and gonad development. In this study, we reported a sox9 gene (designated as Spsox9) from Scylla paramamosain through analyzing published gonad transcriptome data. Meanwhile, the accuracy was validated by PCR technology, and the 3' sequences were cloned with 3' RACE technology. The full-length cDNA of Spsox9 is 2843 bp, consisting of a 243 bp 5' UTR, an 1124 bp 3' UTR, and a 1476 bp ORF encoding 491 amino acids. Furthermore, to better understand its conservation among crustacean species, the sox9 gene ortholog was identified in several other crustaceans species with their published transcriptome data, respectively. All of the Sox9 proteins identified in the current study had the common feature of Sox proteins (HMG domain) and were highly conserved among analyzed crustacean species. In all examined tissues, the Spsox9 was mainly expressed in the gonad (testis and ovary), eyestalk, and cerebral ganglion. During embryo development, Spsox9 was highly expressed in 5 pairs of appendages, 7 pairs of appendages, and eye-pigment formation stage. During ovary development, the expression level of Spsox9 remained stable in the first 4 stages (O1-O4) and decreased in the tertiary vitellogenesis (O5) stage. During testis development, the expression level of Spsox9 was highest in the spermatid stage (T2) and was significantly different from that in the spermatocyte stage (T1) and mature sperm stage (T3) (p < 0.05). In addition, Spsox9 exhibited a sex-biased expression pattern in T1 and O1. These present results indicated that the Spsox9 gene might play crucial roles in the gonad and embryo development of mud crab.

Cyclin B protein undergoes increased expression and nuclear relocation during oocyte meiotic maturation of the freshwater prawn Macrobrachium rosenbergii and the Chinese mitten crab Eriocheir sinensis.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Characterization of gonad differentially expressed SoxB2 genes in mud crab Scylla paramamosain.

Members of sox gene family play critical roles in development, and some of them have crucial functions in sexual dimorphism. To understand the role of two SoxB2 genes, Sox14b and Sox21 of mud crab Scylla paramamosain, the full-length 1939 bp SpSox14b cDNA sequence and 861 bp SpSox21 cDNA sequence were obtained from the crab's transcriptome database, which encode 397 and 259 amino acids respectively. The results of sq-PCR showed that SpSox14b was expressed in all tissues, while SpSox21 was only expressed in the testis and brain. qRT-PCR showed that the expression level of SpSox14b in ovary was significantly higher than that of testis, and during the gonad development its expression was the highest in O2 (previtellogenesis) stage. The expression level of SpSox21 in testis was much higher than in brain, and was significantly higher in T3 (the mature sperm stage) than in other stages of testis development. Meanwhile, in different stages of larval development, SpSox21 was low expressed in zoea, then increased significantly in megalopa. Therefore we speculated that SpSox14b and SpSox21 may play different roles in the gonad development of mud crab, especially SpSox21 may be involved in the development and maintenance of testis. The expression level of SpSox14b and SpSox21 during the eye-pigment formation was significantly higher than that in other embryonic development stages, the results of whole-mount in situ hybridization showed that SpSox14b and SpSox21 were mainly located near the head and the compound eyes in eye-pigment formation stage and hatching. It suggested that they may be involved in the formation of brain nerves and are related to the regulation of body segments, and play different roles in sexual dimorphism.

Discovery of sex-related genes from embryonic development stage based on transcriptome analysis in Eriocheir sinensis.

Investigation of sex determination system in Eriocheir sinensis is important because of sex-dimorphism in its growth traits. However, little information about the sex-related genes in embryonic development stages were exposed. To obtain more information of sex determination in Chinese mitten crab, we performed the transcriptome analysis in embryonic development stage (fertilized egg stage, cleavage stage, blastula stage, gastrula stage and heartbeat stage) of Chinese mitten crab using nextgeneration sequencing technology. Thirty-one of 32,088 annotated unigenes were identified as sex-related genes including 16 genes involved in primary sex determination in model organisms and 8 genes of SOX family and 7 genes of DMRT gene family. Heatmap based on the RPKM value in five embryonic development stages indicated that these genes were clustered into two branches. Analysis of the differentially expressed 12 genes, including 3 genes of SOX family, 3 genes of DMRT gene family and 6 genes involved in primary sex determination in model organisms, showed significantly difference between the first three periods (fertilized egg stage-cleavage stage-blastula stage) and the last two periods (gastrula stage-heartbeat stage) and all 12 genes were up-regulated after blastula stage. In conclusion, we inferred that sex determination might be initiated after blastula stage in E. sinensis. Transcriptome analysis from embryonic development stage could provide a background information for further investigation in sex determination of Eriocheir sinensis.

Identification and Characterization of a Luteinizing Hormone Receptor (LHR) Homolog from the Chinese Mitten Crab Eriocheir sinensis.

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain.

The early developmental stages of the mud crab Scylla paramamosain suffer from high mortality caused by pathogen infections; however, few immune associated factors are known. Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) functions as a typical pathogen recognition receptor and plays an important role in the innate immune system of invertebrates. In this study we characterized a LGBP gene (SpLGBP) which was highly expressed in the late embryonic, zoea I larval stage and hepatopancreas of S. paramamosain.. It encodes 364 amino acids, composed of several conserved domains like the bacterial glucanase motif. The recombinant SpLGBP protein (rSpLGBP) was obtained through the E.coli expression system, in which two 6◊His-tags were added to both C and N terminals during vector construction for the improvement of purification efficiency. In vivo the study showed that the SpLGBP mRNA was significantly up-regulated under Vibrio parahaemolyticus and a lipopolysaccharide (LPS) challenge in the hemocytes and hepatopancreas. The ELISA binding assay in vitro indicated that the rSpLGBP was capable of binding to LPSs and peptidoglycan (PGN). The rSpLGBP could agglutinate both G+ and G- bacteria in the presence of Ca2+. Our results suggest that SpLGBP may play an immunological role against pathogenic infection in the early developmental stages of S. paramamosain.

Re-description of Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Brachyura: Grapsoidea: Varunidae) zoeal development using confocal laser scanning microscopy.

A number of authors have described the zoeal development of the Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, 1853, while some of these studies only recognised 5 zoeal stages, two of them described six. The present study re-examined the zoeal stages of E. sinensis from laboratory-reared material using confocal laser scanning microscopy and visualised the images using the open-source software programmes ImageJ and Drishti. From these images 6 zoeal stages were re-described and compared with all previous larval descriptions of the Chinese mitten crab. Comments on the variation of some setal characters are also described in zoeal stages IV-VI.

Hepatopancreatic cells of a stone crab Menippe frontalis from Perú: separation, viability study, and evaluation of lipoperoxidation against cadmium contamination.

Crustaceans are frequently used as bioindicators, and changes in their metabolism at the hepatopancreas (HP) level are often followed in these studies. The HP is the site of digestion, absorption, nutrient storage, and toxic metal detoxification, enabling crabs to survive in metal contaminated regions. Cellular damage and high lipid peroxidation (LPO) levels have been found in crab populations under high cadmium (Cd) concentrations. The aim of this work was to separate and characterize the HP cells of the stone crab Menippe frontalis from the Pacific Ocean, Perú (5° 5' 21″ S-81° 6' 51″ W) and to measure the cellular viability and LPO after exposure to the non-essential metal Cd. The HP cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at concentrations of 10, 20, 30, and 40%. We found the same cell types that were described for other species (e.g., Ucides cordatus, Atlantic Ocean, Brazil). High cellular viability against 1 mmol L-1 of Cd was observed for resorptive (R) cells in 20% sucrose layer (88 ± 8%, *P < 0.05, ANOVA), and blister (B) cells in the 40% sucrose layers (92 ± 7%, *P < 0.05, ANOVA). Cd (1 mmol L-1) caused an increase in LPO levels, suggesting that crabs from polluted areas can be affected by toxic metals, generating a physiological stress. The gradient sucrose methodology can be used for different species and results in a similar separation, viability, and cellular identification. The results are a starting point for toxic metal studies for species distributed across different geographic coordinates.

Entrainment of the Circadian Rhythm in Egg Hatching of the Crab Dyspanopeus sayi by Chemical Cues from Ovigerous Females.

The subtidal crab Dyspanopeus sayi has a circadian rhythm in larval release with a free-running period of 24.1 h. Under constant conditions, eggs hatch primarily in the 4-h interval after the time of sunset. The study tested the new model for entrainment in subtidal crabs, which proposes that the female perceives the environmental cycles and entrains the endogenous rhythm in the embryos. Results verified the model for D. sayi. Hatching by embryos collected from the field when they had not yet developed eye pigments, and were kept in constant conditions attached to their mother, exhibited the circadian hatching rhythm. Attached embryos could also be entrained to a new photoperiod in the laboratory before they developed eye pigments. Further, mature embryos removed from the female hatched rhythmically, indicating that a circadian rhythm resides in the embryos. However, if mature embryos with eye pigments were removed from the female and exposed to a new light-dark cycle, they could not be entrained to the new cycle; rather, they hatched according to the timing of the original light-dark cycle. Nevertheless, detached, mature embryos would entrain to a new light-dark cycle if they were in chemical, but not physical, contact with the female. Thus, the female perceives the light-dark cycle, and uses chemical cues to entrain the circadian rhythm of hatching by the embryos.

Unravelling polar lipids dynamics during embryonic development of two sympatric brachyuran crabs (Carcinus maenas and Necora puber) using lipidomics.

Embryogenesis is an important stage of marine invertebrates with bi-phasic life cycles, as it conditions their larval and adult life. Throughout embryogenesis, phospholipids (PL) play a key role as an energy source, as well as constituents of biological membranes. However, the dynamics of PL during embryogenesis in marine invertebrates is still poorly studied. The present work used a lipidomic approach to determine how polar lipid profiles shift during embryogenesis in two sympatric estuarine crabs, Carcinus maenas and Necora puber. The combination of thin layer chromatography, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry allowed us to achieve an unprecedented resolution on PL classes and molecular species present on newly extruded embryos (stage 1) and those near hatching (stage 3). Embryogenesis proved to be a dynamic process, with four PL classes being recorded in stage 1 embryos (68 molecular species in total) and seven PL classes at stage 3 embryos (98 molecular species in total). The low interspecific difference recorded in the lipidomic profiles of stage 1 embryos appears to indicate the existence of similar maternal investment. The same pattern was recorded for stage 3 embryos revealing a similar catabolism of embryonic resources during incubation for both crab species.

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in Scylla paramamosain, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based on the evident results that it was highly expressed in maternal ovaries and in the early developmental stages of the zygote and early embryo stage whereas it gradually decreased in zoea 1. SpFLT-1 positively responded to the challenge of Vibrio alginolyticus with a significantly increased level of mRNA expression in the hemocytes and gills at 3 hours post infection (hpi). The SpFLT-1 protein was detected densely in the same fraction layer where the Vibrio protein was most present in the hemocytes and gills at 3 hpi. Furthermore, it was found that the expression of SpFLT-1 decreased to the base level following disappearance of the Vibrio protein at 6 hpi in the gills. Silencing SpFLT-1 inhibited the endocytosis rate of V. alginolyticus but overexpression of the gene could facilitate bacterial entry into the epithelioma papulosum cyprinid cells. Our study indicated that SpFLT-1 may act as a key protein involved in the process of bacterial infection and this sheds light on clarifying the pathogenesis of pathogens infecting S. paramamosain.

Pesticide effects on crabs: how environmental concentrations of endosulfan and chlorpyrifos affect embryos.

The objectives of this work were to evaluate the effects of environmentally relevant chlorpyrifos and endosulfan, concentrations in the incubation period, effective hatching and survival of embryos and neonates of the freshwater burrowing crab, Zilchiopsis collastinensis (Decapoda, Trichodactylidae). Both pesticides were prepared from commercial and technical grade products. The exposure to about 100, 200, and 400 ng endosulfan L(-1), and 48, 240, and 1,200 ng chlorpyrifos L(-1) did not cause differences in the incubation period or in effective hatching but decreased survival of neonates, especially in the concentrations prepared from the technical grade product. Even if these concentrations are below the median lethal concentration (LC50) values for embryos, these caused a significant decrease in the survival of neonates, i.e. when crabs are outside the egg and not protected by chorion. The decrease in the neonate population caused by these concentrations, which could be found in the environment, might impact aquatic communities.

[Fertility and Embryonic Mortality Level of the Japanese Mitten Crab Eriocheir japonica (Crustacea: Decapoda: Varunidae) in Primorye].

Reproductive biology of the Japanese mitten crab Eriocheir japonica (de Haan, 1835) was studied in the northern part of its habitat. It was revealed that E. japonica from the water bodies of Primorye, in contrast to crabs in the central part of the habitat on the Japanese Islands, lays fewer eggs in the first clutch, does not have direct dependence between the size-weight characteristics of females and the number of eggs in the third clutch, and shows higher level of embryonic mortality. The loss of eggs during the embryonic period in each of three sequentially laid clutches was 16, 34, and 52%, respectively (28% in total during the period of reproduction). Fecundity was approximately 43%. It was registered that the specifics of reproductive biology of E. japonica (monocyclicity, prolonged period of reproduction, portion spawning, indirect embryonic development, high fertility, relatively small and yolk enriched eggs, pelagic planktotrophic larva, high level of energy costs on reproduction, egg bearing on the pleopods, high level of embryonic mortality), an intermediate strategist, is in a good agreement with its life strategy, thereby enabling this species to realize its reproductive potential in the northern part of the habitat.

Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus.

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus.

Circadian rhythm in larval release by the crab Rhithropanopeus harrisii: entrainment model.

The subtidal crab Rhithropanopeus harrisii has a circadian rhythm in larval release; under constant conditions eggs hatch in the 2-3-h interval after the time of sunset in nontidal estuaries. Eggs that are removed from the female hatch rhythmically, indicating that the circadian rhythm resides in the embryos. The model for entrainment is that mature embryos have functional sensory systems that detect and entrain to environmental cycles. This model was reexamined by confirming that the visual system of advanced embryos responds to light and thus could mediate entrainment to the light/dark cycle. We then determined whether the hatching rhythm of mature embryos that are removed from the female can be entrained to new light/dark cycles. Contrary to expectations, these embryos did not entrain to new cycles. Instead, they remained entrained to the light/dark cycle to which they were exposed when still attached to the female, suggesting that the female entrains the rhythm. Indeed, hatching by embryos collected from the field when they had not yet developed eye pigments, kept in constant conditions attached to their mother, exhibited the circadian hatching rhythm. They could also be entrained to a new photoperiod in the laboratory. The role of the female is further supported by experiments showing that the hatching rhythm in embryos carried by females lacking one but not both eyes can be entrained to a new cycle in the laboratory. Thus, the revised model is that the female perceives the light/dark cycle and entrains the circadian rhythm in the embryos.

Sex-lethal gene of the Chinese mitten crab Eriocheir sinensis: cDNA cloning, induction by eyestalk ablation, and expression of two splice variants in males and females.

Sex-lethal (Sxl) plays an important role in sex determination in insects. In this study, the full-length complementary DNA (cDNA) of Sxl of the Chinese mitten crab Eriocheir sinensis (EsSxl) was cloned. EsSxl is expressed during different developmental stages of embryos, and its expression level in the cleavage stage is lower than that in other stages of embryonic development, such as the original zoea stage when it reaches the highest level. The expression level of EsSxl in eight different tissues was investigated. EsSxl was expressed in seven examined tissues, excluding eyestalk, and the highest expression levels were observed in testis and hepatopancreas. EsSxl expression in testis was 13-fold higher than that in ovary. After induction by eyestalk ablation, changes in EsSxl expression were also tissue-specific, with EsSxl expression increasing by 2.6-fold and 11.5-fold in ovary and muscle, respectively, between 0 and 7 days after eyestalk ablation and decreasing by 2.0-fold in testis between 0 and 3 days after eyestalk ablation and by 265-fold in hepatopancreas between 0 and 7 days after eyestalk ablation. Two splice variants of EsSxl were identified, and the only difference between them was a 77-bp alternatively spliced intron that is transcripted in EsSxl1 and skipped in EsSxl2. Both variants were expressed in males and females, demonstrating the lack of a sex-specific expression pattern for Sxl in Chinese mitten crab.

Changes in biochemical composition and digestive enzyme activity during the embryonic development of the marine crab, Charybdis japonica (Crustacea: Decapoda).

In this study, we investigated the biochemical composition and digestive enzyme activity during embryonic development in the marine crab, Charybdis japonica. Water, protein, and ash content increased, while levels of lipids and carbohydrates decreased significantly during embryonic development, and a marked correlation between water content and egg volume (r = 0.97; P < 0.01) was recorded. The utilization percentages of lipids and carbohydrates were 32.72% and 91.56%, respectively. The predominant essential amino acids (EAA) were lysine, leucine, arginine and valine, and the major nonessential amino acids (NEAA) were glutamic acid and aspartic acid. From the fertilized egg stage to the protozoea stage, total amino acid (TAA) concentration increased from 52.18% to 55.11% on a dry weight basis, but the ratio of EAA/TAA decreased from 52.57% to 48.90%. The quantitatively more important fatty acids were C16:0, C18:1n-9c, C16:1, C22:6n-3 (docosahexaenoic acid, DHA), and C22:2. Polyunsaturates (PUFA) and monounsaturated (MUFA) were consumed at similar rates (34.20% and 36.70%, respectively); both were consumed at higher rates than saturates (SFA) (26.56%). In particuler, n-3 fatty acids decreased significantly, with a high consumption rate of 43.74%. Activities of trypsin and pepsin increased during both the early and later embryonic stages, but decreased during the middle stages. Lipase activity increased gradually during embryonic development, except in the protozoea stage with a significant decrease, while activities of amylase and cellulase showed an ascending trend after an initial decline. The activity of all digestive enzymes increased, except for that of lipase, from the heartbeat stage to the protozoea stage.

Embryology of the spider crabs Leurocyclus tuberculosus (H. Milne-Edwards & Lucas 1842) and Libinia spinosa (H. Milne-Edwards 1834) (Brachyura, Majoidea).

The embryonic development of the spider crabs Leurocyclus tuberculosus and Libinia spinosa was divided into five periods based on the differentiation of: (I) cleavage, (II) embryonic primordium, III) optic lobes, (IV) optic lobes pigmented and (V) chromatophores presence. Different traits such as spines, setae and telson morphology distinguish the two species from period III until hatching. Egg volume was greater in Leurocyclus tuberculosus than in Libinia spinosa. The duration of each period was different during development. Whereas in Leurocyclus tuberculosus period II (morphogenesis) is the longest, in Libinia spinosa the period IV is the longest. Complete embryonic development at 14'C lasted 36.7 +/- 3.1 days in Leurocyclus tuberculosus and 57.4 +/- 4.4 days in Libinia spinosa.

Fecundity, embryonic and ovarian development of blue swimming crab, Portunus pelagicus (Linnaeus, 1758) in coastal water of Johor, Malaysia.

Blue swimming crab, Portunus pelagicus is widely study and research throughout the Indo-West Pacific, but little is known of its reproductive biology in Malaysia. The present study describes the fecundity, embryonic development and ovarian development stages of the P. pelagicus from Johor coastal water, Malaysia. Carapace width range of berried crabs sampled was from 9.64 to 13.32 cm, while the body weight range was from 75 to 235 g. The mean number of egg produced by females in different sizes ranged from 105443.333 +/- 35448.075 per eggs batch. Mean egg size during embryonic development at stage 1 was 0.307 +/- 0.037, while 0.386 +/- 0.039 and 0.396 +/- 0.033 for stage 2 and stage 3, respectively. Study showed that there was significant (p < 0.05) relationship between the number of eggs and carapace width/body weight. Mean diameter oocyte during ovarian development at stage 1 was 97.732 +/- 12.391 while for stage 2 was 149.516 +/- 23.287. Stage 3 showed increasingly of size with mean diameter was 158.506 +/- 27.616 and 181.013 +/- 24.339 for stage 4.