Cannibalism in juvenile blue-swimmer crabs Portunus pelagicus (Linnaeus, 1766): effects of body size, moult stage and refuge availability

Factors influencing the rate of cannibalism in juvenile blue-swimmer crabs Portunus pelagicus were investigated under controlled conditions using time-lapse video recordings. This study was undertaken to improve blue-swimmer crab culture and experimentally addressed (1) prey vulnerability (2) cannibal-victim interactions, and (3) activity patterns of juveniles in varying degrees of refuge. Crabs used in the study were aged 15 weeks and sorted into two size classes; small (less than or equal to 60 mm carapace width (CW)) and large (greater than or equal to65 mm CW) of a similar sex ratio. Vulnerability and thus survival was influenced by body size variation, moult stage and refuge availability. Crabs with carapace width less than or equal to 60 mm were more vulnerable than larger individuals, as indicated by significant differences in survival rates. As predicted, juveniles in transition stages associated with ecdysis were especially vulnerable. Premoult (redliner) crabs appeared to be in a high state of agitation as evidenced by the frequency of agonistic encounters and this may be a contributing factor to the high mortality observed at this critical premoult stag. increases in refuge density increased survival of juveniles proportionally, indicating that the quantity of shelter is important for reducing cannibalism in this species. Cannibal-victim interactions were frequently asymmetrical in terms of size and moult stage. Cannibals were significantly heavier than victims, and were predominantly at intermoult stage. Sexual biases among cannibals and victims were not found in this study. Activity patterns of juveniles were influenced by the experimental conditions. Crabs provided with high refuge showed reduced aggressive activity and increased time spent resting, but unchanged locomotion or feeding activity. Regular grading as well as the presence of suitable shelter for newly moulted crabs is recommended for improving culture of P. pelagicus. Research into inducing synchronous moulting may also yield promising results. (C) 2004 Elsevier B.V. All rights reserved.

Molecular tools for studying puberty in the grey mullet, Mugil cephalus: cloning of cDNAs that regulate reproductive function

The cDNAs coding for the brain GnRHs (AY373449-51), pituitary GH, SL and PRL, and liver IGFs (AY427954-5) were isolated. Partial cDNA sequences of the brain (Cyp19b) and gonadal (Cyp19a) aromatases have also been obtained. These tools would be utilized to study the endocrine regulation of puberty in the grey mullet.

Mutation scanning analysis of Marteilia sydneyi populations from different geographical locations in eastern Australia

Marteilia sydneyi (Paramyxea) is the causative agent of QX disease in oysters. In spite of the economic impact of this disease, its origin and the precise reason(s) for its apparent spread in Australian waters are not yet known. Given such knowledge gaps, investigating the population genetic structure(s) of M. sydneyi populations could provide insights into the epidemiology and ecology of the parasite and could assist in its prevention and control. In this study, single strand conformation polymorphism (SSCP)-based analysis of a region (195 bp) of the first internal transcribed spacer (ITS-1) of ribosomal DNA was employed to investigate genetic variation within and among five populations of M. sydneyi from oysters from five different locations in eastern Australia. The analysis showed the existence of a genetic variant of M. sydneyi common to the Great Sandy Strait, and the Richmond and Georges Rivers, as distinct from variants at the Pimpama and Clarence Rivers. Together with historical and other information relating to the QX disease outbreaks in eastern Australia, the molecular findings support the proposal that the parasite originated in the Great Sandy Strait and/or Richmond River and then extended southward along the coast. From a technical perspective, the study demonstrated the usefulness of SSCP as a tool to study the population genetics and epidemiology of M. sydneyi. (C) 2003 Elsevier Ltd. All rights reserved.

Correlating gene expression with larval competence and the effect of age and parentage on metamorphosis in the tropical abalone Haliotis asinina

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P < 0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.

Developmental expression of two Haliotis asinina hemocyanin isoforms

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

Esterified astaxanthin levels in lobster epithelia correlate with shell colour intensity: Potential role in crustacean shell colour formation

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton. (c) 2005 Elsevier Inc. All rights reserved.

Digenean trematodes infecting the tropical abalone Haliotis asinina have species-specific cercarial emergence patterns that follow daily or semilunar spawning cycles

Approximately 1-2% of the tropical abalone Haliotis asinina inhabiting Heron Island Reef are infected with opecoelid digeneans. These largely inhabit the haemocoel surrounding the cerebral ganglia and digestive gland-gonad complex, and infected abalone typically have significantly reduced or ablated gonads. Observations of infected abalone reveal two distinct cercarial emergence patterns, one which correlates tightly with the abalone's highly regular and synchronous fortnightly spawning cycle, and the other which occurs in a circadian pattern. The former appears to be a novel emergence strategy not previously observed in digeneans. While the cercariae in all abalone are morphologically indistinguishable, comparison of sequences from the internal transcribed spacer 2 (ITS 2) region of the ribosomal DNA reveals a 5.7% difference between cercariae displaying different emergence patterns, indicating these are two distinct species that probably belong to the same genus. The ITS 2 sequences of the species with the daily emergence pattern are identical to that of an undescribed adult opecoelid from the gut of the barramundi cod, Cromileptes altivelis. Combined molecular, morphological and emergence data suggest that while these opecoelid cercariae use the same first intermediate host and are closely related species-members of the genus Allopodocotyle-they fill different ecological niches that are likely to include different definitive hosts.

Heritability estimates for growth in the tropical abalone Haliotis asinina using microsatellites to assign parentage

The tropical abalone Haliotis asinina is a wild-caught and cultured species throughout the Indo-Pacific as well as being an emerging model species for the study of haliotids. H. asinina has the fastest recorded natural growth rate of any abalone and reaches sexual maturity within one year. As such, it is a suitable abalone species for selective breeding for commercially important traits such as rapid growth. Estimating the amount of variation in size that is attributable to heritable genetic differences can assist the development of such a selective breeding program. Here we estimated heritability for growth-related traits at 12 months of age by creating a single cohort of 84 families in a full-factorial mating design consisting of 14 sires and 6 dams. Of 500 progeny sampled, 465 were successfully assigned to their parents based on shared alleles at 5 polymorphic microsatellite loci. Using an animal model, heritability estimates were 0.48 +/- 0.15 for shell length, 0.38 +/- 0.13 for shell width and 0.36 +/- 0.13 for weight. Genetic correlations were > 0.98 between shell parameters and weight, indicating that breeding for weight gains could be successfully achieved by selecting for shell length. (c) 2006 Elsevier B.V. All rights reserved.

Expressed sequence tag analysis of genes expressed during development of the tropical abalone Haliotis asinina

The tropical abalone. Haliotis asinina. is,in ideal species to investigate the molecular mechanisms that control development. growth, reproduction and shell formation in all cultured haliotids. Here we describe the analysis of 232 expressed sequence tags (EST) obtained front a developmental H. asinina cDNA library intended for future microarray studies. From this data set we identified 183 unique gene Clusters. Of these, 90 clusters showed significant homology with sequences lodged in GenBank, ranging in function from general housekeeping to signal transduction, gene regulation and cell-cell communication. Seventy-one clusters possessed completely novel ORFs greater than 50 codons in length, highlighting the paucity of sequence data from molluscs and other lophotrochozoans. This study of developmental gene expression in H. asinina provides the foundation for further detailed analyses of abalone growth, development and reproduction.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.

Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine

This study investigated the chromosome ploidy level of Marsupenaeus (Penaeus) japonicus (Bate) non-viable (unhatched) embryos and nauplii after exposure to 6-dimethylaminopurine (6-DMAP), timed to stop either polar body (PB) I, or PBI and II extrusion. Embryos from eight separate families or spawnings were exposed to 150 or 200 mu M 6-DMAP from 1- to 3-min post-spawning detection (psd) for a 4- to 5-min duration (timed to stop PBI extrusion). Separate aliquots of embryos from five of the same spawnings were also exposed to 200 mu M of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). For one spawning, a third aliquot of embryos was exposed to 400 p M of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). At 18-h psd, non-viable embryo and nauplii samples were taken separately for fluorescent activated cell sorting (FACS). FACS revealed that there were diploids and triploids among all treated non-viable embryos and nauplii. All control non-viable embryos and nauplii were diploid. Percentages of triploid induction for the 4- to 5-min and 16-min durations were not significantly different (P > 0.05). Additionally, no difference was found in the triploidy level of nonviable embryos compared to nauplii in these treatments. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 4- to 5-min duration ranged from 29.57% to 99.23% (average 55.28 +/- 5.45%) and from 5.60% to 98.85% (average 46.70 +/- 7.20%), respectively. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 +/- 11.00%) and from 47.5% to 99.24% (average 79.38 +/- 5.24%), respectively. To our knowledge, this is the first documentation of successful PBI or PBI and II inhibition in shrimp. This study conclusively shows that treatment of M. japonicus embryos with 6-DMAP at 1- to 3-min pscl for either a 4- to 5-min duration (timed to stop PBl extrusion) or 16-min duration (timed to stop both PBI and II extrusion) results in viable triploid nauplii. (c) 2006 Elsevier B.V. All rights reserved.

Short-term hyperthermic treatment of Penaeus monodon increases expression of heat shock protein 70 (HSP70) and reduces replication of gill associated virus (GAV)

Disease is the result of interactions amongst pathogens, the environment and host organisms. To investigate the effect of stress on Penaeus monodon, juvenile shrimp were given short term exposure to hypoxic, hyperthermic and osmotic stress twice over a 1-week period and estimates of total haemocyte count (THC), heat shock protein (HSP) 70 expression and load of gill associated virus (GAV) were determined at different time points. While no significant differences were observed in survival and THC between stressed and control shrimp (P>0.05), HSP 70 expression and GAV load changed significantly (P

The effectiveness of heat, cold and 6-dimethylaminopurine shocks for inducing tetraploidy in the Kuruma shrimp, Marsupenaeus japonicus (Bate)

In this study tetraploid Marsupenaeus japonicus (Bate) embryos were produced by preventing the first division in mitosis. The effectiveness of temperature and chemical shocks for producing tetraploid M. japonicus were assessed when applied at different times postspawning and for different durations. Tetraploid M. japonicus embryos (spawned at 27 degrees C) were produced by heat shocks at 35 degrees C and 36 degrees C in three and eight spawning samples respectively, and a cold shock at 5 degrees C in a single spawning sample. All temperature shocks inducing tetraploidy were applied 18-23 min postspawning for a 5-10 min duration. The percentage of spawnings successfully inducing tetraploid embryos (i.e., frequency of induction) ranged from 33.33% to 66.67% for the 21, 22 and 23 min postspawning heat shock treatment regimes. The percentage of tetraploid embryos within an induction (i.e., induction rate), as determined by flow cytometry, ranged from 8.82% to 98.12% (ave. S.E.) (34.4 +/- 21.4%) for the 35 degrees C shock treatments, from 13.12% to 61.02% (35.0 +/- 5.0%) for the 36 degrees C shock treatments and was 15% for the 5 degrees C cold shock treatment. No tetraploids were produced for spawnings that received heat shocks above 36 degrees C or below 35 degrees C, or for cold shocks above 5 degrees C for any of the tested postspawning treatment and duration times. Chemical shock with 150 mu M 6-dimethylaminopurine did not result in tetraploid M. japonicus embryos at any of the tested postspawning treatment times and durations. Tetraploid M. japonicus embryos were nonviable, with no tetraploid larvae being detected by flow cytometry. Based on our results heat shocking of M. japonicus embryos at 36 degrees C, 23 min postspawning for a 5-10 min duration is the most effective means to produce tetraploids through inhibition of the first mitotic division (taking into consideration the importance of frequency and induction rate equally).