A multigene and morphological analysis expands the diversity of the seabod shrimp Xiphopenaeus Smith, 1869 (Decapoda: Penaeidae), with descriptions of two new species.

After being stable for nearly a century, the taxonomic history of the genus Xiphopenaeus has been marked by many changes in the last three decades. The taxonomic status of the Atlantic species has a low resolution, and many species are still undefined and grouped as cryptic species. Here we employed an integrative approach to define the species of Xiphopenaeus and the morphological characters needed to differentiate them. We combined the analyses of two molecular markers (COI and 16 S rDNA), scanning electron microscopy and light microscopy. Based on specimens from 17 localities from the Atlantic and Pacific oceans, we detected five divergent genetic groups, three in the Atlantic (A1, A2, A3) and two in the Pacific (P1, P2). Male secondary sexual characters were able to differentiate four out of the five genetic groups. Group A1 corresponds to X. kroyeri, and A2 and A3 correspond to new species. We redescribed the genus and two new species are described and illustrated: Xiphopenaeus dincao nov. sp. (A2) and Xiphopenaeus baueri nov. sp. (A3). Since the holotype of X. riveti was missing and the specimen analysed from group P2 was a female, the status of the species of Xiphopenaeus from the Pacific remains unresolved.

Imaging aquatic animal cells and associated pathogens by atomic force microscopy in air.

Atomic force microscopy (AFM) is a sophisticated imaging tool with nanoscale resolution that is widely used in structural biology, cell biology, and material science, among other fields. However, to date it has rarely been applied to the study of aquatic animals, especially on one of the main cultured species, shrimp. One reason for this is that no shrimp cell line established until now, primary cell is fragile and difficult to be studied under AFM. In this study, we used AFM to image three different types of biological material from shrimp (Litopenaeus vannamei) in air, including hemocytes and two associated pathogens. Without obvious deformations when the cells were imaged in air and in the case for the haemocytes and the cells were fixed as well. The result suggests hydrophobic glass coverslips are a suitable substrate for adhesion of these samples. The method described here can be applied to the preparation of other fragile biological samples from aquatic animals for high-resolution analyses of host-pathogen interactions and other basic physiological processes.

The gastric sieve of penaeid shrimp species is a sub-micrometer nutrient filter.

Unlike that of vertebrates, the penaeid shrimp stomach is of ectodermic origin and is thus covered by a cuticle that is sloughed upon molting. It is composed of two chambers, here called the anterior and posterior stomach chambers, ASC and PSC, respectively. The PSC contains a filtration structure variously called a pyloric filter, filter press, gastric filter or gastric sieve (GS), and the last of these will be used here. The GS resembles an elongated, inverted-V, dome-like, chitinous structure with a midline ridge that is integral to the ventral base of the PSC. The dome surface is covered with a carpet-like layer of minute, comb-like setae bearing laterally branching setulae. This carpet serves as a selective filter that excludes large partially digested food particles but allows smaller particles and soluble materials to enter hepatopancreatic ducts that conduct them into the shrimp hepatopancreas (HP), where further digestion and absorption of nutrients takes place. Although the GS function is well known, its exclusion limit for particulate material has not been clearly defined. Using histological and ultra-structure analysis, we show that the GS sieve pore diameter is approximately 0.2-0.7 µm in size, indicating a size exclusion limit of substantially less than 1 µm. Using fluorescent microbeads, we show that particles of 1 µm diameter could not pass through the GS but that particles of 0.1 µm diameter did pass through to accumulate in longitudinal grooves and move on to the HP, where some were internalized by tubule epithelial cells. We found no significant difference in these sizes between the species Penaeus monodon and Penaeus vannamei or between juveniles and adults in P. vannamei This information will be of value for the design of particulate feed ingredients such as nutrients, therapeutic drugs and toxin-absorbing materials that may selectively target the stomach, intestine or HP of cultivated shrimp.

A morphological study of the male reproductive tract, post-testicular acrosome maturation and spermatophore formation in the black tiger prawn (Penaeus monodon).

Inferior larval production rates of domesticated Penaeus monodon broodstock has been a major hurdle to the expansion of its aquaculture, so that a better understanding of basic male reproductive biology is critical to improve the reproductive performance of this commercially important penaeid species. Following our previous study of spermatogenesis in the species, this study explored the mechanism of spermatophore formation with regards to the contribution of the reproductive tract epithelium by light and transmission electron microscopy. Four types of epithelial secretions (S1-4) were observed contributing to the three layers of the spermatophore. The primary layer of spermatophore was composed of S1 and S2, which were released from the secretory epithelial cells of the proximal vas deferens (PVD) and the secretory epithelial cells of the sperm-bearing lumen of the mid vas deferens, respectively. The secondary layer of the spermatophore was composed of S3, the secretory product of epithelial cells in the accessory tubule lumen of the mid vas deferens. The outer layer of the spermatophore was formed from S4 which was secreted by the epithelial cells in the posterior mid vas deferens and the terminal ampulla. Unique folds of the vas deferens epithelium appeared to play an important role in the formation of the spermatophore, particularly in the formation of the laminated structure of the spermatophore appendage. With respect to acrosome maturation along the reproductive tract, most spermatozoa did not have a fully developed anterior spike and a subacrosomal region when in the PVD, whereas both structures were fully formed by the time the spermatozoa reached the mid vas deferens and increased electron density when in the terminal ampulla; this observation represents the first morphological evidence of post-testicular acrosome maturation in this taxon.

The functional anatomy of external genitalia in the black tiger prawn (Penaeus monodon) studied by micro-computed tomography and scanning electron microscopy.

Although artificial insemination has been used for decades in Penaeus monodon aquaculture, the interaction of male and female external genitalia during spermatophore transfer has not been fully documented. As a result, studying the functional anatomy of this process may help to better refine the insemination technique. The sexual act in penaeoid prawns is virtually impossible to observe directly; as a result, this study aimed to describe the functional anatomy and interaction of external genitalia, such as the petasma, appendices masculinae and genital papillae of the male, with that of the thelycum and genital lobes of the female, using a combination of micro-computed tomography and scanning electron microscopy. We hypothesise that the spermatophores are ejaculated into the ventromedial groove of the petasma and squeezed by abdominal flexure into the spermathecae of female's thelycum. During this process, the 'spike-like' setae observed on the petasma and appendices masculinae are speculated to control the transferring direction of the spermatophores. The approach of three dimensional remodelling and animation reported in this study may prove useful in the examination of further hypotheses related to the functional anatomy of external genitalia and/or appendages for other crustacean species.

The oxidative stress and antioxidant responses of Litopenaeus vannamei to low temperature and air exposure.

Low temperature and air exposure were the key attributes for waterless transportation of fish and shrimp. In order to investigate the oxidative stress and antioxidant responses of the live shrimp Litopenaeus vannamei in the mimic waterless transportation, live shrimp were cooled at 13 °C for 3 min, stored in oxygen at 15 °C for 12 h, and then revived in water at 25 °C. The survival rate of shrimp under this waterless transportation system was over 86.67%. The ultrastructure of hepatopancreas cells were observed while activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-Px), antisuperoxide anion free radicals (ASAFR), total antioxidant capacity (TAOC), reactive oxygen species (ROS) production, content of malondialdehyde (MDA) and relative mRNA expressions of CAT and GSH-Px in the hemolymph and hepatopancreas were determined. Slight distortions of some organelles in hepatopancreas cells was reversible upon the shrimp revived from the cold shock. The activities of SOD, POD, CAT, GSH-Px, TAOC, ROS production and relative mRNA expressions of CAT and GSH-Px increased following the cold shock and reached peak levels after 3 or 6 h of storage, and then decreased gradually. There was no significant difference between the fresh and the revived shrimp in SOD, POD, GSH-Px, TAOC, ROS, MDA and relative mRNA expressions of CAT and GSH-Px. The oxidative stress and antioxidant responses were tissue-specific because hepatopancreas seemed to have a greater ability to defend against organelle damage and was more sensitive to stress than hemolymph based on the results of SOD activity, MDA content and GSH-Px mRNA expression. These results revealed that low temperature and air exposure caused significant oxidative and antioxidant responses, but did not lead to irreversible damages in this waterless system.

Prevalence and distribution of covert mortality nodavirus (CMNV) in cultured crustacean.

An emerging covert mortality nodavirus (CMNV) was proved to be the infectious agent of shrimp viral covert mortality disease (VCMD). Prevalence and distribution of CMNV were investigated by using the methods of reverse transcription loop-mediated isothermal amplification (RT-LAMP), nested reverse transcription PCR, gene sequencing, histopathology, in situ RNA hybridization (ISH) and transmission electron microscope (TEM) in this study. RT-LAMP results showed that CMNV positive samples appeared in the cultured crustaceans including Litopenaeus vannamei, Fenneropenaeus chinensis, Marsupenaeus japonicus, Penaeus monodon, and Macrobrachium rosenbergii, and mostly distributed the coastal provinces in China. The prevalence rates of CMNV among the collected samples in 2013, 2014 and 2015 were 45.93% (130/283), 27.91% (84/301) and 20.85% (54/259), respectively. CMNV infection in M. japonicas and P. monodon was verified by ISH. The presence of CMNV particles were confirmed by TEM analysis in the CMNV positive samples diagnosed by RT-LAMP. The high prevalence and wide epidemic distribution of CMNV in this investigation revealed that it was necessary to pay close attention to the high risk of CMNV transmission in farmed crustaceans.

Sperm ultrastructure of shrimps from the family Penaeidae (Crustacea: Dendrobranchiata) in a phylogenetic context.

We describe the sperm ultrastructure of six penaeid species, including at least one member of each tribe (Penaeini, Parapenaeini and Trachypenaeini). Fragments of the vas deferens of the Penaeidae Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Litopenaeus schmitti, Parapenaeus americanus, Rimapenaeus constrictus and Xiphopenaeus kroyeri were fixed and processed according to the routine for transmission electron microscopy. The morphological results were contextualized in an evolutionary perspective using molecular markers for the phylogenetic reconstruction of this group. A phylogram was proposed by Bayesian inference based on 1007 bp of 33 sequences of the combined genes (16S rDNA and COI mtDNA) from 27 dendrobranchiate specimens. Our findings show that morphological differences in the sperm ultrastructures of members among the tribes of Penaeidae can be used as a baseline to understand their evolutionary relationships. Individuals from the Penaeini tribe show plesiomorphic characteristics in the sperm ultrastructure compared to the Trachypenaeini tribe from which they were derived, such as shrimp from family Sicyoniidae. The morphological complexity of the sperm of the different penaeid members corroborated with the genetic phylogeny, which showed different clades for each tribe and the close relationship with Sicyoniidae. The sperm features of the selected species studied here reflected their evolutionary history. These features confirm the previous phylogenetic hypothesis and question the monophyly of Penaeidae, which should be verified in the future with a more complete set of representative members of each tribe.

Effects of cryopreservation on ultrastructural morphology of white shrimp (Litopenaeus vannamei) sperm.

BACKGROUND: The structural integrity of a spermatozoon is very important for the processes of fertilization and embryo development. OBJECTIVE: To provide valuable data for developing better cryopreservation techniques for shrimp sperm. MATERIALS AND METHODS: Using scanning electron microscopy and transmission electron microscopy, we examined the morphological alteration of Litopenaeus vannamei sperm after cryopreservation with different concentrations of dimethylsulfoxide (DMSO). RESULTS: We found that the damaged post-thaw sperm presented either vesiculated acrosomal contents, wrinkled membranes, perforated membranes, and loss of the acrosomal spike. The seriously damaged sperm showed missing acrosomal spikes, deformed nuclei, burst nuclear membranes, and vacuolated nuclei. In addition, we found that the post-thaw sperm stored with 5% DMSO had the highest viability rate and lowest DNA damage coefficient by eosin-nigrosin staining and comet assay. CONCLUSION: Our results suggested that cryopreservation has deleterious effects on ultrastructural morphology of L. vannamei sperm, especially on acrosomal spikes and membranes.

Three-dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block-face scanning electron microscopy.

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF-SEM provided a large dataset of sequential electron-microscopic-level images that facilitated comprehensive ultrastructural observations and three-dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane-less dense granules. A large electron-lucent vesicle and "arch-like" structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF-SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. RESULTS: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. CONCLUSIONS: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.

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.

Changes in oviduct structure in the black tiger shrimp, Penaeus monodon, during ovarian maturation.

OBJECTIVE: To examine the structure of the oviduct of the shrimp Penaeus monodon. METHODS: The oviducts of P. monodon with three different major groups of ovarian development (Group (Gr.) 1: Stages I & V; Gr. 2: Stages II & III; and Gr. 3: Stage IV) were examined by light, transmission electron, and scanning electron microscopies, respectively. RESULTS: The epithelium of the oviduct in Gr. 1 was composed of tall simple columnar cells with their basal nuclei located on the basement membrane and its thick collagen fibers. In Gr. 2, the oviduct seemed to produce some substances and their epithelial cells became transitional with centrally located nuclei and formed some vacuoles. Obviously, the epithelial cells in Gr. 3 (at Stage IV) were disorganized, disrupted, and shed accumulated spherical secretory substances including some cellular contents into the lumen. CONCLUSIONS: The structural changes of the P. monodon oviduct were related to ovarian maturation stages (Grs. 1-3). Prior to spawning, only the oviduct epithelium at ovary Stage IV produced and secreted a number of spherical secretion substances into the lumen. These substances may act as the oviductal lubricants to facilitate the spawning process.

Protein hydrolysate of salted duck egg white as a substitute of phosphate and its effect on quality of Pacific white shrimp (Litopenaeus vannamei).

Protein hydrolysate from salted egg white (PHSEW) with different degrees of hydrolysis (DH) (3%, 6%, and 9%) was produced using pepsin. Disappearance of proteins with molecular weight (MW) of 108 and 85 kDa with the concomitant formation of proteins with MW of 23, 20, 13, and 5 kDa was observed in PHSEW. The use of PHSEW for quality improvement of Pacific white shrimp (Litopenaeus vannamei) was investigated. Shrimp soaked in 4% NaCl containing 7% PHSEW and 2.5% mixed phosphates (0.625% sodium acid pyrophosphate [SAPP] and 1.875% tetrasodium pyrophosphate [TSPP]) had the highest cooking yield with the lowest cooking loss (P < 0.05), compared with shrimps with other treatments. Nevertheless, no difference in weight gain was obtained in comparison with those treated with 4% NaCl containing 3.5% mixed phosphate (P > 0.05). Cooked shrimp treated with 4% NaCl containing 7% PHSEW and 2.5% mixed phosphate or those treated with 4% NaCl containing 3.5% mixed phosphate had the higher score of appearance, texture, and overall likeness but less shear force, in comparison with the control (no treatment) (P < 0.05). Microstructure study revealed that muscle fibers of cooked shrimp from both treatments had the swollen fibrils and gaps, while the control had the swollen compact structure. Therefore, use of PHSEW could reduce phosphate residue in shrimps without an adverse effect on sensory properties.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis.

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Ran GTPase regulates hemocytic phagocytosis of shrimp by interaction with myosin.

Ran GTPases, one family of small G protein superfamily, have been widely demonstrated to be involved in the transport system between cytoplasm and the nucleus. However, the function of Ran GTPase in immunity remains unclear. In our study, it was found that the Ran GTPase (designated as PjRan) was up-regulated in virus-resistant shrimp, indicating that the PjRan might be implicated in the innate immune system against virus infection. On the basis of protein interactions, it was found that the PjRan interacted with myosin, a crucial protein in the process of phagocytosis to form a protein complex. The RNAi and mRNA assays showed that the PjRan could regulate shrimp hemocytic phagocytosis. Further data evidenced that the depletion of PjRan by RNAi caused a significant increase of virus copies, and the overexpression of PjRan resulted in a significant decrease of virus copies, suggesting that the PjRan participated in the antiviral immunity by regulating phagocytosis. Therefore, our study revealed a completely novel aspect of Ran GTPase in phagocytosis by the direct interaction with the cytoskeleton protein and presented a novel pathway concerning to antiviral immunity, which will help to better understand the molecular events in immune response against virus infection in invertebrates.

Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion.

RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank EU170438) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank AA083987) and YHV-2 (GenBank AF227196), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank EU123854) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence (EU853170) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.

Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon.

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.

Properties, translucence, and microstructure of Pacific white shrimp treated with mixed phosphates as affected by freshness and deveining.

Effects of freshness and deveining on some properties, translucence, and microstructure of Pacific white shrimp (Litopenaeus vannamei) soaked in 2.5% NaCl containing different phosphates were studied. Shrimp soaked in all solutions had increases in weight gain and cooking yield with lowered cooking loss, compared with the control (P < 0.05). However, efficacy of mixed phosphates in quality improvement of ice-stored shrimp was lower than fresh shrimp. Deveining resulted in increased weight gain and yield (P < 0.05). Nevertheless, samples treated with phosphates became more translucent. Shrimp stored in ice for 7 d and treated with mixed phosphates were generally more translucent than fresh counterparts (P < 0.05). Shrimp soaked in 2.5% NaCl containing 0.875% sodium acid pyrophosphate (SAPP) and 2.625% tetrasodium pyrophosphate (TSPP) were generally less translucent and had high weight gain and cooking yield along with low cooking loss. The microstructure study revealed that the muscle fibers were less attached with the loss of Z-disks after being treated with mixed phosphates. Cooked meats of fresh shrimp and ice-stored shrimp had more compact fiber arrangement with the shrinkage of sarcomere compared with raw samples. Disintegration was observed at the M-line in ice-stored shrimp treated with mixed phosphates after cooking, while such a phenomenon was not found in the cooked fresh sample treated with phosphates. T(max) and enthalpy of both myosin and actin peaks shifted to lower values when shrimp were treated with mixed phosphates (P < 0.05). Those changes were generally more pronounced in ice-stored shrimp. Therefore, freshness and deveining process had an impact on the quality of Pacific white shrimp treated with phosphates.

Food digestion by cathepsin L and digestion-related rapid cell differentiation in shrimp hepatopancreas.

Cathepsin L (CatL) has been readily localized in the large vacuole and in the apical complex of the digestive B-cell of the shrimp hepatopancreas. Immunogold technique revealed the occurrence of CatL in zymogen granule, digestive body and digestive vacuole of the B-cell in the hepatopancreas of Metapenaeus ensis. Coalescences of zymogen granule with sub-apical vacuole, and of two small digestive bodies were observed. This progressive coalescence of CatL vesicles is direct evidence of involvement of CatL in intracellular digestion. Released CatL vesicles and free CatL were found in the lumen of hepatopancreatic tubule. CatL mRNA existed in F-cell, but not in the mature B-cell. This finding supports the previous suggestion that F-cell is the precursor of B-cell. F-cell is a transient form. Transition from F-cell to B-cell is fast. We define F-cell as the transcribing cell, F/B-cell as the enzyme-synthesizing cell and B-cell as the enzyme-secreting cell. For the first time, we suggest that R-cell is the replacing cell for the leaving B-cell. CatL degrades nutrient intracellularly and extracellularly. The most interesting finding is that CatL is transcribed in one type of cell, and the very cell evolves quickly to a morphologically different cell where the enzyme functions.