An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.     

Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.     

Helix pomatia lectin and annexin V,two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.     

Histopathological response to turpentine in the white shrimp,Penaeus setiferus

Observations are presented on the inflammatory response to a highly irritative substance, turpentine, injected into the abdominal musculature of the white shrimp, Penaeus setiferus. Injections of the irritant were administered with a tuberculin syringe between the fifth and sixth segments. Penaeid shrimp were found to be highly sensitive to turpentine, even when administered in small dosages. When sterile petroleum jelly was mixed with the turpentine to reduce the dispersion rate, the shrimp's “internal defense mechanism” was able to combat effectively the effect of the irritant. Postinjection observations of the tissues at the site of injection, gill, heart, and hepatopancreas were made at 8, 16, 24, 32, 40, 48, 72, 96, 120, 168, and 240 hr, and at 15, 20, 30, 40, 50, 60, and 120 days. The induced cellular inflammatory response consisted of infiltrating hemocytes and fibrocytes resulting in the formation of fibrous capsules, brown melanized nodules, and fibrous scar tissue in all tissues examined. The gills and hepatopancreas showed considerable tissue destruction early, but were eventually cleared of the histopathological effects of the turpentine and later appeared normal. However, extensive tissue destruction was easily distinguishable in the heart and abdominal muscle even at 120 days postinjection.     

Clearance of bacteria injected into the hemolymph of the ridgeback prawn,Sicyonia ingentis (Crustacea: Decapoda): Role of hematopoietic tissue

In an earlier investigation, radiolabelled bacteria injected into the hemolymph of the shrimp Sicyonia ingentis were cleared rapidly from circulation. Most of the bacteria were localized in the gills, followed by other organs including the heart, abdominal musculature, and hematopoietic nodules (HPN). We determined the organ-specific effectiveness of bacterial clearance and found the HPN to be the most effective on a per gram basis. Light and electron microscopy were used to determine the mechanism of clearance in the HPN. The HPN are readily permeable to individual bacteria, which then are recognized and phagocytosed exclusively by small granule hemocytes. Within the first hour, the bacteria are degraded, leaving only whorls of membrane in the phagocytic vacuoles. Subsequently, the hemocytes that ingested bacteria lyse, as has been observed in vitro. These observations support earlier suggestions that hemocytes in the HPN are functionally mature and held in reserve until they are released into circulation. © 1996 Wiley-Liss, Inc.     

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

The symbiotic water mite Unionicola formosa (Acari: Unionicolidae) ingests mucus and tissue of its molluscan host

Unionicola formosa is a symbiotic water mite that passes most of its life cycle in the mantle cavity of freshwater mussels. Although mites of this genus are often referred to as parasitic, little is known about their nutritional biology. A few species reportedly pierce the gill of a host mussel and ingest tissue or hemolymph. The present study was undertaken to identify possible sources of nutrition for U. formosa. To determine if mites ingested particulate matter in the mucous strand produced by a mussel during feeding, mussels with resident mites were exposed to a suspension of fluorescent microspheres. There was no evidence that U. formosa ingested the beads. Histochemical staining did, however, indicate a mucous material present in the midgut of the mites. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays revealed a high molecular weight component, consistent with a mucopolysaccharide, present both in the mussel gill and the mites. Results from western blots and an immunoaffinity binding assay with antibodies against mussel gill tissue and hemolymph also indicated that mites ingested host tissue. Whereas U. formosa probably does not ingest particulate material acquired by its host's suspension feeding, it is apparent that this mite utilizes host mucus, gill tissue, or hemolymph for at least part of its nutrition.     

Histopathology of Aerococcus viridans var. homari infection (Gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.     

Cellular defense reactions of insect hemocytes in vivo: Nodule formation and development in Galleria mellonella and Pieris brassicae larvae

Galleria mellonella and Pieris brassicae larvae were injected with a standardized dose of killed Bacillus cereus and other bacteria and the reactions of hemocytes followed in the first 24 hr by dissection and histology. Nodules formed in all insects injected with nonpathogens, but a pathogen, Staphylococcus aureus, failed to provoke this reaction. Within 5 min, clumps consisting of granular hemocytes, plasmatocytes, and bacteria were found attached to the internal surfaces of the insects. In the following hours, the cells comprising the clumps broke down and merged with a melanizing acellular substance, and the necrosing masses became encapsulated by plasmatocytes to form mature nodules. The role of granular hemocytes in the formation of the initial cell/bacteria aggregates is discussed along with the possible importance of nodules to the cellular defense reactions of insects.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Nitric oxide production in blowfly hemolymph after yeast inoculation

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megacephala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48h post-injection. NO levels in SIL were comparable to those measured in NIL until 12h, which might be considered the basal production, increasing at 24 and 48h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24h. l-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca(2+)-dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule.     

Morphology of hemocyte lysis and clotting in the ridgeback prawn,Sicyonia ingentis

Summary Coagulation of hemolymph in the shrimp Sicyonia ingentis was studied using light and electron microscopy. Differential counts of unclotted hemolymph show that 54% of the hemocytes are deposit cells characterized by a high nucleocytoplasmic ratio, a few granules, and cytoplasm filled with distinctive deposits. The remaining hemocytes have numerous large or small granules filling the cytoplasm. Examination of clotted hemolymph to which trypan blue had been added shows that deposit cells lyse, whereas the granulocytes exclude the dye, attach to slides, and extend filopodia. This suggests that deposit cells, not granulocytes, initiate coagulation. Ultrastructural changes in deposit cells were studied at specific times after mixing hemolymph and seawater. Deposit cells fixed immediately after removal from shrimp were shaped like elliptical discs and contained abundant, 50 nm diameter cytoplasmic deposits. After 30 s in seawater, deposit cells displayed several cytoplasmic blebs, and had aggregated the deposits. Cytolysis occurred by 45 s. Linear arrays of deposit appeared to extend through breaks in the plasma membrane, forming filamentous strands that hydrated to produce the clot. At 1 min after withdrawal, spheres of clotted hemolymph were seen, each surrounding a lysed deposit cell. Granulocytes remained relatively unchanged and trapped between adjacent expanding clots. Coagulation via hemocyte lysis is compared with other clotting mechanisms observed in various crustaceans and arthropods.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Observations on the process of wound repair in penaeid shrimp

The Petersen disk tag is a standard mark for penaeid shrimp, and attachment of the tag involves the insertion of a stainless steel pin through the shrimp's abdomen, resulting in a relatively large puncture wound. The wound healing process first observed at 24 hr post-tagging showed a pronounced hemocytic infiltration of the wound area. Hemocytes in contact with the pin became fusiform, began adhering to one another, and formed several concentric layers around the pin. Scattered foci of bacteria or nercrotic tissue in the vicinity of the would also became encapsulated by concentric layers of fusiform hemocytes, thereby forming nodules. Melanin appeared in association with the layers of hemocytes nearest the pin and in the nodules. Hemocytic infiltration was lollowed by the appearance of fibrocytes and the deposition of collagenlike fibers along the would channel 48 hr after wounding. Involution of epidermis and consequential cuticular involution into the would channel began at 96 hr after wounding. Complete epidermal and cuticular formation along the wound occurrd by 384 hr post-tagging.     

In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae

The Manduca sexta larva-specific immune protein, scolexin, was isolated and (14)CH(3)-labelled by reductive alkylation. The influence of the bacterium Streptococcus faecalis on the hemocoelic distribution of the labelled scolexin was then analyzed. During bacterial challenge, most of the scolexin signal was detected in association with the hemocyte aggregations and nodules which formed; in this respect the protein sometimes appeared to be associated with hemocytes which had phagocytized bacteria, while at other times it was most concentrated in the nodule-associated, and free, coagulum. Areas of high scolexin activity were sometimes detected at various sites on the surface of the fat body. The scolexin did not appear to bind directly to bacterial cells. Up to 24 hr following the injection of S. faecalis, the larvae were still carrying out the formation of nodules; unlike the nodules of the 3 and 6 hr intervals, the nodules observed at 21-24 hr were covered with an apparently humorally derived, coagular capsule.     

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.     

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1_His hormone or 2?×?10^?6 mol L^?1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1_His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.