Genome-wide identification and expression profile of the sox gene family in different tissues and during embryogenesis in the Pacific white shrimp (Litopenaeus vannamei).

Sox transcription factors play essential roles in a variety of critical physiological processes. Still, members of the sox gene family have not yet been genome-wide identified in shrimps. In this study, a total of five members of the sox gene family were identified from the genome of Pacific white shrimp Litopenaeus vannamei and classified into three subgroups based on the conserved HMG-box domain. Among them, three belong to the SoxB subgroup (one in B1 and two in B2), one in the SoxC subgroup, and one in the SoxE subgroup. The five sox genes had different sex-biased expression in some tissues. Sox21, soxB1, and sox14 had a higher expression in ovary than in testis. In comparison, sox4 had a male-biased specific expression in the gonad, hepatopancreas, gill, and eyestalk. There was no difference in soxE gene expression between testis and ovary. During embryonic development, the expression level of three sox genes (soxB1, sox21, and soxE) was higher in gastrulation stage compared to previous stages, declined in limb bud stage and then increased in intramembrane nauplius stage; the expression of sox4 was detected in blastula stage and continued to increase in the following two stages and then surged in intramembrane nauplius stage; the highest expression of sox14 was in the fertilized egg stage, and the expression level decreased with the development of the embryo. These results suggest that the shrimp sox gene family may be involved in gametogenesis, tridermogenesis, and neurogenesis.

Isolation of the sex-determining gene Sex-lethal (Sxl) in Penaeus (Litopenaeus) vannamei (Boone, 1931) and characterization of its embryogenic, gametogenic, and tissue-specific expression.

The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes.

Differential regulation of the lipoxygenase pathway in shrimp hepatopancreases and ovaries during ovarian development in the black tiger shrimp Penaeus monodon.

Dietary polyunsaturated fatty acids (PUFAs) are critical to the success of ovarian development in marine crustaceans, especially for domesticated species such as the black tiger shrimp Penaeus monodon. These fatty acids are stored in a midgut gland called the hepatopancreas and subsequently serve as an energy source or are incorporated in yolk during ovarian development. PUFAs are known precursors of hydroxy fatty acids, including hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid (HEPE), which are catalyzed by lipoxygenases (LOX). In previous studies, 8-HEPE has been shown to regulate female reproduction and adipogenesis in marine crustaceans. However, whether the biosynthesis of 8-HEPE in these species is the result of LOX activity has yet to be investigated. In this study, 8-HEPE was identified exclusively in P. monodon hepatopancreases using liquid chromatography-mass spectrometry. Treatment with nordihydroguaiaretic acid resulted in the reduction of 8-HEPE, suggesting the enzyme-dependent catalysis of 8-HEPE in hepatopancreases. Additionally, a full-length P. monodon LOX (PmLOX) was amplified from shrimp ovary cDNA. Sequence analysis revealed that the putative PmLOX contains domains and catalytic residues required for LOX catalytic function. Furthermore, PmLOX expression increased steadily as shrimp ovary maturation progressed, while PmLOX expression and the amount of 8-HEPE decreased in shrimp hepatopancreases. These findings not only suggest differential requirements for hydroxy fatty acid biosynthesis in shrimp ovaries and hepatopancreases during the P. monodon ovarian development, but also provide insights into the LOX pathway in marine crustaceans.

Behavioural and biochemical alterations in Penaeus monodon post-larvae diet-exposed to inorganic mercury.

Mercury is a metal naturally present in the environment with concentrations in aquatic systems increasing annually due to human activities. This represents a great concern mainly due to its high toxicity to organisms and consequences for human health. Most studies regarding the toxic effect of mercury have focussed on freshwater species using water as the exposure and uptake pathway. In contrast, the present study investigated the effects of dietary exposure of mercury to the marine crustacean Penaeus monodon post-larvae during 96 h to evaluate changes in behaviour (swimming activity and risk taken) and in biochemical biomarkers [acetylcholinesterase (AChE) and glutathione S-transferase (GST)]. Results showed a decrease in swimming activity with an increase in mercury exposure, but no changes were observed regarding the behavioural response 'risk taken'. Prawns from medium (0.56 µg g-1) and high (1.18 µg g-1) treatments had their GST activity reduced in relation to the beginning of experiment (time 0), while AChE activity was increased in the low (0.15 µg g-1) treatment in relation to time 0. In the present study, behaviour analysis were clearer than biochemical biomarkers and results might indicate P. monodon populations from a mercury contaminated environment might be at risk, since the behavioural alterations observed increases the risk of predation.

Transcriptome profiles of Penaeus (Marsupenaeus) japonicus animal and vegetal half-embryos: identification of sex determination, germ line, mesoderm, and other developmental genes.

There is virtually no knowledge of the molecular events controlling early embryogenesis in Penaeid shrimp. A combination of controlled spawning environment, shrimp embryo micro-dissection techniques, and next-generation sequencing was used to produce transcriptome EST datasets of Penaeus japonicus animal and vegetal half-embryos. Embryos were collected immediately after spawning, and then blastomeres were separated at the two-cell stage and allowed to develop to late gastrulation, then pooled for RNA isolation and cDNA synthesis. Ion Torrent sequencing of cDNA from approximately 500 pooled animal and vegetal half-embryos from multiple spawnings resulted in 560,516 and 493,703 reads, respectively. Reads from each library were assembled and Gene Ontogeny analysis produced 3479 annotated animal contigs and 4173 annotated vegetal contigs, with 159/139 hits for developmental processes in the animal/vegetal contigs, respectively. Contigs were subject to BLAST for selected developmental toolbox genes. Some of the genes found included the sex determination genes sex-lethal and transformer; the germ line genes argonaute 1, boule, germ cell-less, gustavus, maelstrom, mex-3, par-1, pumilio, SmB, staufen, and tudor; the mesoderm genes brachyury, mef2, snail, and twist; the axis determination/segmentation genes ß-catenin, deformed, distal-less, engrailed, giant, hairy, hunchback, kruppel, orthodenticle, patched, tailless, and wingless/wnt-8c; and a number of cell-cycle regulators. Animal and vegetal contigs were computationally subtracted from each other to produce sets unique to either half-embryo library. Genes expressed only in the animal half included bmp1, kruppel, maelstrom, and orthodenticle. Genes expressed only in the vegetal half included boule, brachyury, deformed, dorsal, engrailed, hunchback, spalt, twist, and wingless/wnt-8c.

Ultrastructure of putative germ granules in the penaeid shrimp Marsupenaeus japonicus.

Knowledge about the specification of the germ line in penaeid shrimp would allow development of techniques to control germ cell formation and/or fate to produce reproductively sterile shrimp for genetic copyright purposes. Recent studies have traced the localization of an RNA-enriched intracellular body (ICB) in the putative germ line of four penaeid shrimp species. It is hypothesized that the ICB may serve as a putative germ granule and marker of germ line fate. In this study semi-thin and ultra-thin sections of Marsupenaeus japonicus embryos were prepared, and the dimensions and ultrastructure of the ICB was examined at different stages of embryogenesis. The ICB was an aggregation of electron dense granules, small vesicles and multi-vesicular bodies (MVBs), similar to germ granules from other species. Lamellar membranes and mitochondria were localized at the periphery of the ICB. Using fluorescence microscopy, microtubules were also observed between the centrosome and the ICB. The localization of the ICB in the D lineage and putative germ cell line, the enrichment of RNA in the ICB, and the ultrastructural similarities to other germ granules characterized in this study support the hypothesis that the ICB contains germ granules.

Triploidy induction in the Pacific white shrimp Litopenaeus vannamei: an assessment of induction agents and parameters, embryo viability, and early larval survival.

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-µm 6-DMAP final concentration at 1 min post-spawning detection for a 6 to 8 min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7 min 30 s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8 min post-spawning detection for a 4 to 10 min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.

Molecular cloning and expression analysis of the Mitogen-activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Ploidy manipulation and polyploid detection in the white shrimp Litopenaeus vannamei (Boone 1931) (Decapoda, Penaeidae).

Ploidy manipulation has been rarely used in the genetic improvement of cultured marine shrimps. Although polyploid induction has been proven to be successful in Penaeids, including the species Litopenaeus vannamei, the methodology still requires some improvements. In the present work, different thermal shock treatments on ploidy manipulation were tested and a protocol for detecting polyploid individuals was also established. Fertilized eggs were treated by cold (10°C) and heat (38°C) thermal shocks for 8, 12, 15, 18, 20, and 22 min to induce polyploidy. Nuclear measurements within distinct treatments revealed a significant deviation in relation to the mean diameter of nuclei in the control individuals. Triploid and tetraploid metaphases were observed within treated individuals, confirming the increase of interphasic nuclear diameter. The cold thermal shock was more efficient than the hot ones, besides leading to a higher and more homogeneous hatchery rate. A mean number of three nucleoli per nucleus were observed in diploid individuals, while treated samples usually presented up to five nucleoli per nucleus. The standardization of protocols to obtain and detect polyploid products allows further utilization of such methods on a commercial scale in order to evaluate the performance of polyploid individuals in the genetic improvement of L. vannamei.

Cytological defects during embryogenesis in heat-induced tetraploid Kuruma shrimp Penaeus japonicus.

Tetraploid shrimp embryos have been induced; however, in all cases no postlarvae were produced. This study determined when tetraploid Penaeus japonicus became non-viable and identified unique abnormalities to aid in elucidating the causes of lethality. Embryonic development was analyzed using flow cytometry to determine ploidy and laser scanning confocal microscopy for cytological examination of embryogenesis. Abnormalities exclusive to tetraploids were identified from the 1-cell stage: an off-centre pronucleus, polypolar spindles, delayed time to first mitosis and polypolar cleavage. Following first mitosis in the tetraploids, 50% of the cells did not contain DNA. This unique abnormality was not resolved later in development and is therefore believed to be a lethal trait. Causes of this phenomenon likely stemmed from abnormal mitotic spindle regeneration following the mitotic heat shock. Consequently, the findings of this study indicate that current methods of tetraploidy induction using heat shock appear unsuitable for viable tetraploid shrimp production.

Effects of cryoprotectant on the embryos of banded coral shrimp (Stenopus hispidus); preliminary studies to establish freezing protocols.

The addition of cryoprotectants is a necessary step in cryopreservation procedures because they can minimize cellular injury during cryopreservation. Toxicity of cryoprotectant depends on the type, concentration, temperature and exposure period. The aim of this study was to investigate the toxicity of cryoprotectant to embryos of banded coral shrimp (Stenopus hispidus) in order to inform the development of a cryopreservation protocol. Three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) embryos were selected and exposed to different concentration of cryoprotectants (0.25M-5M) for an equilibration period of 10, 20 or 30 min at room temperature. Hatching percentage indicated that the toxicity of tested cryoprotectants increased in the order of methanol, ethylene glycol (EG), dimethyl sulphoxide (DMSO), glycerol and dimethylacetamide (DMA). The No Observed Effect Concentrations (NOECs) for eye-formation stage embryos were 1M, 0.25M, 0.25M, 0.25M and 0.25M respectively after 10 min incubation whilst the NOECs for heart beat and pre-hatch stage embryos were 1M, 0.5M, 0.5M, 0.5M and 0.25M respectively. Pre-hatch stage embryos appeared to be more tolerant to cryoprotectant toxicity than eye-formation and heart beat stage embryos.

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea, Malacostraca, Decapoda).

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.

Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp Marsupenaeus japonicus.

In order to identify genes involved in oogenesis and spermatogenesis in penaeid shrimp Marsupenaeus japonicus, a modified annealing control primer (ACP) system was adapted to identify genes differentially expressed in ovary and testis at different developmental stages. By using 20 pairs of ACP primers, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r). Bioinformatics analyses show that this gene encodes a protein of 241 amino acids with a predicted molecular mass of 27.4 kDa. Real time PCR analyses demonstrated that the expression level changed significantly in the developing testis and ovary. In the stage 2 of testis, it reached its highest expression level, the lowest expression level present in the stage 1 of ovary. The significantly different expression levels in developing testis and ovary suggest that UBE2r has an important role in oogenesis and spermatogenesis. This article is the first report of UBE2r in crustaceans and also is the first report showing that UBE2r is differentially expressed at different stages of the developing ovary and testis in an animal.

Expression of clottable protein of tiger shrimp (Penaeus monodon) in gonads and its possible role as nutrient source for the embryo.

We have investigated the expression of clottable protein (CP) in gonad of tiger shrimp (Penaeus monodon) and extent of its phosphorylation. Polyclonal antibodies against purified CP were prepared from rabbit serum. Using this anti-CP antiserum, the temporal expression of CP in gonads of tiger shrimp was analyzed. It was found that the CP occurs only in mature ovaries but not in immature ovaries and testes. Results of RT-PCR confirmed that these tissues expressed low levels of CP mRNA transcripts. Upon eyestalk-ablation, the ovaries in female shrimps were induced to develop, and the CP expression levels in ovaries were traced chronically by RT-PCR analyses. The expression level peaked on day 3 with an increase of about 40 folds relative to the basal level and returned to normal level (as the control shrimp) at day 12. The shrimp embryos at different intervals from spawning to 16h post-spawning were also collected, and it was found that CP contents were gradually decreased in the embryos until the nauplii were hatched. In addition, purified CP was shown to react with specific anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine antibodies suggesting that CP is a phosphoprotein with all types of phosphorylations. Taken together the results suggest that expression of CP in shrimp ovaries is coupled to ovarian development and CP possibly supply nutrition for shrimp embryo.

Temperature effect on behaviour, oxygen consumption, ammonia excretion and tolerance limit of the post larvae of shrimp Penaeus indicus.

The present study has been carried out to know the effect of temperature on behaviour, equilibrium loss and tolerance limit of the post larvae of shrimp Penaeus indicus. The experimental temperatures were selected based on the thermal tolerance limit. The experiments were conducted at a specific temperature for duration of 48 hr. The thermal tolerance experiments were conducted in two ways: in direct exposure and in gradually increasing temperature. The upper and lower lethal temperatures for the post larvae of shrimp P. indicus were 43.5 degrees C and 8 degrees C respectively. During tolerance experiment, no mortality was observed at 33 degrees C and 35 degrees C. But at 38 degrees C with gradual increase in temperature, 30% loss of equilibrium and mortality were recorded in 24.31 hrs and 25.07 hrs, and the remaining 70% were alive. On the contrary, when the post larvae of shrimps were directly exposed to 38 degrees C, almost 80% loss of equilibrium and mortality were recorded in 30.22 hrs and 30.40 hrs, remaining 20% were alive. At 40 degrees C with gradual increase in temperature, 100% loss of equilibrium and mortality were recorded in 25.32 hrs and 25.56 hrs. On the other hand, when the post larvae of shrimps were directly exposed to 40 degrees C, 100% loss of equilibrium was observed in 0.37 hrs and mortality in 1.40 hrs. These behavioral responses include an elevated temperature of 12 degrees C, surfacing, dashing against glass wall, jumping out of the water, etc. In general, the rate of oxygen consumption and ammonia excretion was found to enhance with increasing temperature. In the present study, it was found that gradual increase in temperature favours the shellfish population to escape from the thermal exposure as compared to direct exposure.

Ontogenesis of haemocytes in shrimp (Fenneropenaeus chinensis) studied with probes of monoclonal antibody.

Monoclonal antibodies (Mabs) specifically against haemocytes of shrimp (Fenneropenaeus chinensis) were employed to study the ontogenesis of haemocytes by immunofluorescence assay technique (IFAT) with the aids of avidin-biotin complex method. Different developmental stages were identified by means of histochemical staining. Stages including blastula, gastrula, limb bud stage, embryonic-nauplius and nauplius were examined. With Mab 2A3 against both hyline, semi-granular and granular haemocytes, the first positive cells were detected at the limb bud stage of the embryos. Distinguishable positive haemocytes with a diameter of about 7 microm appeared in embryonic-nauplius. Whereas, with Mab 1H11 specific to cytoplasmic granulars in haemocytes, the first positive granular haemocytes were observed at the stage of embryonic-nauplius. Increased number of both total and granular haemocytes at the stage of nauplius impressed a possible enhancement of the immune system as the embryogenesis progressed.

A PL10 vasa-like gene in the kuruma shrimp, Marsupenaeus japonicus, expressed during development and in adult gonad.

A PL10 vasa-like gene was isolated from the Kuruma shrimp Marsupenaeus japonicus and therefore called Mjpl10. It is differentially expressed during embryonic, larval, and postlarval development, and in female and male gonads. Using absolute real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrate that Mjpl10 transcripts are present in the two-cell embryo, suggesting it is maternally expressed, and continually at low levels throughout embryogenesis. Mjpl10 expression increases significantly in the first 25 h after hatching (nauplii IV) and then decreases in a linear fashion by 316-fold over the next 52-day period. Its continued expression throughout embryonic and larval development is compatible with a conserved role in early germ cell specification. Transcript levels of Mjpl10 are also detected in the ovary and testes of mature adults.

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.

Muscle development in dendrobranchiate shrimp, with comparison with Artemia.

The muscle pattern of malacostracan and entomostracan crustacean nauplius larvae was compared using fluorescent phallotoxins. In the dendrobranchiate malacostracan Sicyonia ingentis, F-actin staining was first detected in limb setae at 12 h, likely within sensory nerves. Staining of F-actin was detected in the trunk at 15 h and grew into the naupliar limbs. Sarcomeres were detected at 19 h, identifying the structures as extrinsic limb muscles. The extrinsic limb muscles enlarged but retained their general pattern during the later nauplius stages. Longitudinal trunk muscles and circumferential visceral muscle (VM) developed in the post-naupliar region during nauplius instars 4 and 5, at the time when the gut also formed. In the anostracan branchiopod Artemia salina, the newly hatched nauplius contained an extensive system of extrinsic and intrinsic limb muscles. The gut was almost complete at hatching, along with its associated circumferential VM. Muscles similar in position and structure could be identified in nauplii from the two taxa, but different anatomical origins of extrinsic muscles were evident. Whether the naupliar limb muscles are homologous in malacostracans and branchiopods remains an open question. The strong musculature of the dendrobranchiate naupliar limbs correlates with the use of all three pairs of limbs for swimming.

Purification and characterization of hatching enzyme from shrimp Penaeus chinensis.

By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular weight of Penaeus HE is about 43.0 kDa in SDS-PAGE. The Penaeus HE had obvious choriolytic activity, which was optimal at pH 6.0 and temperature of 40 degrees C, respectively. The Km value of the HE for casein was 7.47 mg ml(-1). The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin, and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin, and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI, and leupeptin. These results indicate that the HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, Zn2+, Ca2+, Mg2+, and Cu2+. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn2+, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.