Distribution of neuropeptide F in the ventral nerve cord and its possible role on testicular development and germ cell proliferation in the giant freshwater prawn, Macrobrachium rosenbergii.

Neuropeptide F in invertebrates is a homolog of neuropeptide Y in mammals and it is a member of FMRFamide-related peptides. In arthropods, such as insects, there are two types of neuropeptide F comprising long neuropeptide F (NPF) and short neuropeptide F (sNPF). Both NPFs are known to play a crucial role in the regulations of foraging, feeding-related behaviors, circadian rhythm, stress responses, aggression and reproduction in invertebrates. We have earlier found that in the giant freshwater prawn, Macrobrachium rosenbergii, there are three isoforms of NPF and four isoforms of sNPF and that NPFs are expressed in the eyestalks and brain. In the present study, we investigate further the tissue distribution of NPF-I in the ventral nerve cord (VNC) and its role in the development of testes in small male (SM) Macrobrachium rosenbergii. By immunolocalization, using the rabbit polyclonal antibody against NPF-I as a probe, we could detect NPF-I immunoreactivity in the neuropils and neuronal clusters of the subesophageal ganglia (SEG), thoracic ganglia (TG) and abdominal ganglia (AG) of the SM prawns. In functional assays, the administrations of synthetic NPF-I (KPDPTQLAAMADALKYLQELDKYYSQVSRPRFamide) and sNPF (APALRLRFamide) peptides significantly increased the growth rates of SM prawns and significantly increased the gonadosomatic index (GSI) and proliferations of early germ cells in the seminiferous tubules of their testes. It is, therefore, suggestive that NPFs may play critical roles in energy homeostasis towards promoting growth as well as testicular development in prawns that could be applied in the aquaculture of this species.

Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii.

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, -II, -III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and -II precursor proteins, which show high similarity (91-95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and -II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Variation of prostaglandin E2 concentrations in ovaries and its effects on ovarian maturation and oocyte proliferation in the giant fresh water prawn, Macrobrachium rosenbergii.

Prostaglandins (PGs) are important bioactive mediators for many physiological functions. In some decapod crustaceans, prostaglandin E2 (PGE2) has been detected in reproductive organs, and may play a role in the control of ovarian maturation. However, in the freshwater prawn, Macrobrachium rosenbergii, the presences of PGE2 and key enzymes for PGE2 biosynthesis, as well as its effects on ovarian maturation have not yet been investigated. In this study we reported the presence of PGE2, cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) in the ovarian tissues of M. rosenbergii, using immunohistochemistry. Intense immunoreactivities of PGE2 (PGE2-ir), COX1 (Cox1-ir) and PGES (PGES-ir) were detected in previtellogenic oocytes (Oc1 and Oc2), while the immunoreactivities were absent in the late vitellogenic oocytes (Oc4). This finding supports the hypothesis that the PGE2 biosynthesis occurs in the ovary of this prawn. To ascertain this finding we used LC-MS/MS to quantitate PGE2 concentrations during ovarian developmental cycle. The levels of PGE2 were significantly higher in the early ovarian stages (St I and II) than in the late stages (St III and IV). Moreover, we found that administration of PGE2 stimulated the ovarian maturation in this species by shortening the length of the ovarian cycle, increasing ovarian-somatic index, oocyte proliferation, and vitellogenin (Vg) level in the hemolymph.

Changes in plasma angiotensin II, aldosterone, arginine vasotocin, corticosterone, and electrolyte concentrations during acclimation to dry condition and seawater in the crab-eating frog.

The crab-eating frog Fejervarya cancrivora inhabits mangrove swamps and marshes in Southeast Asia. In the present study, circulating angiotensin II (Ang II), aldosterone (Aldo), arginine vasotocin (AVT), and corticosterone (Cort) concentrations as well as various blood parameters were studied under osmotically stressful conditions. Following acclimation to hyperosmotic seawater and dry condition for 5days, body weight was significantly decreased. Under both conditions, plasma Na(+), Cl(-), and urea concentrations, hematocrit values (Ht; blood volume indicator), and osmolality were significantly increased. Dehydration associated with hypovolemic and hyperosmotic states of body fluids was induced during acclimation to hyperosmotic seawater and dry condition in the crab-eating frogs. Ang II, Aldo, AVT, and Cort were maintained within relatively narrow concentration ranges in the control frogs; however, in frogs under dry and hyperosmotic seawater conditions, large variations were observed among individuals in each group. Mean plasma Ang II and Aldo concentrations significantly increased in hyperosmotic seawater-acclimated and desiccated frogs. Although mean plasma AVT concentrations in dehydrated frogs of both the groups were approximately 2.0-3.5 times higher than those in the control frogs, the differences were not significant because of the variation. There was a significant correlation between plasma osmolality and AVT as well as Ang II but not Aldo. A significant correlation was also observed between Ht and AVT as well as Ang II. Plasma Ang II was significantly correlated with plasma Aldo. These results indicate that the crab-eating frogs may exhibit similar physiological responses to both seawater-acclimated and dry conditions. It appears that under dehydrated conditions, osmoregulatory mechanisms participate in stabilization of the situation. The renin-angiotensin system may have pivotal roles in body fluid regulation under volemic and osmotic stress in the Fejervarya species with unique osmoregulation.

The effects of serotonin, dopamine, gonadotropin-releasing hormones, and corazonin, on the androgenic gland of the giant freshwater prawn, Macrobrachium rosenbergii.

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research.

The anthelmintic effect of plumbagin on Schistosoma mansoni.

The anthelmintic effects of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) and praziquantel (PZQ) against adult Schistosoma mansoni in vitro were compared by estimating the relative motility (RM) values, survival indices (SI) and alterations of the tegument of flukes incubated in M-199 medium containing 1, 10 and 100 µg/ml of the drugs, at 1, 3, 6, 12 and 24 h. For the parasites incubated in 10 µg/ml, the RM values of the PB-treated group decreased at a more rapid rate than the PZQ-treated group. When incubated in 100 µg/ml all PB-treated flukes appeared immobile from 1 to 24 h when 91-100% died, while in the PZQ-treated group RM values were higher than that of PB and most flukes were still alive at 1-12 h, and at 24 h only 21% of flukes were killed. Furthermore, male parasites were more affected by PZQ than females as their RM values were significantly less than that of females at all doses. While in PB treatment, males and females showed less difference in response to the drug as their RM values were closer than those treated with PZQ. When observed by SEM, the tegument of untreated S. mansoni displayed no alteration, while in PB treated parasites it exhibited swelling, blebbing, loss of spines, disruption of tubercles and ridges, leading to erosion and lesion, exposure of the basal lamina, and sloughing of the tegument. PZQ induced similar tegumental changes as those observed in PB treatment but at longer incubation time and higher doses. These data indicated that PB had more anthelmintic effect on both sexes of adult S. mansoni than PZQ.

Paramphistomum cervi: the in vitro effect of plumbagin on motility, survival and tegument structure.

Paramphistomiasis causes enteritis and anemia in livestocks and result in substantial production and economic losses. It is considered a neglected tropical disease, with no effective trematodicidal compound for treatment. Plumbagin (PB), a compound founds to be rich in the roots of Plumbago indica, is a naphthoquinone derivatives which can induce oxidative stress in parasites. In this study we have evaluated the anthelmintic activity of PB against adult Paramphistomum cervi by incubating the parasites in M-199 medium containing 0.1, 1.0, 10 and 100 µg/ml of the PB, and albendazole (ABZ) at the concentration of 100 µg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observed by scanning electron microscopy (SEM). After 12 h exposure with 100 µg/ml ABZ, flukes showed decreased contraction and motility. At 24 h incubation they showed only active movement of some part of the body. The PB-treated flukes at all concentrations showed rapid decrease of motility at 3 h incubation. In 0.1, 1.0 and 10 µg/ml of PB, the RM values were decreased sharply from 3 to 12 h, and then they were killed since 12 h in the incubation with 10 µg/ml of PB. The highest parasite mortality was found as early as 3h when they were incubated with 100 µg/ml of PB. The morphological changes on the tegumental surface were similar in both flukes treated with ABZ and PB, which sequentially comprised of swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesions were formed which exposed the basal lamina. The damage appeared more severe on the ventral than the dorsal surface, and earlier on the anterior part and lateral margins of middle third when compared to the posterior part of the parasites's bodies. The severity and rapidity of the damages were enhanced with increasing concentration of PB, which showed stronger activity than ABZ. Hence, PB has a potential to be an anthelmintic drug against adult P.cervi.

Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals.

Characterization and expression of cathepsin B2 in Fasciola gigantica.

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites' antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.

Fasciola gigantica: histology of the digestive tract and the expression of cathepsin L.

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite's body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.

Effect of gonadotropin releasing hormone on the expression of luteinizing hormone and estrogen in the nerve ganglia and ovary of a tropical abalone, Haliotis asinina Linnaeus.

Gonadotropin releasing hormone (GnRH) is a peptide brain hormone that is involved in the regulation of reproduction in vertebrates via stimulation of the secretion of the pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in their turn stimulate sexual development and sex steroid hormone secretion by the gonads. The tropical abalone, Haliotis asinina, in common with many other invertebrates contains a peptide with a similar structure to GnRH. This study looks at its possible involvement in reproduction by injecting groups of one-year-old female abalone at the mature phase by injecting them with synthetic H. asinina (Has) GnRH at doses of 0, 250 and 500 ng/g and then measuring the amount of material in nerve ganglia, ovary and hemolymph that cross-reacted with enzyme-linked immunosorbent assays (ELISA) for vertebrate LH and steroid, estradiol. Immunohistochemistry, using antibodies for the same two compounds, was also carried out to examine the location of immunoactivity in the tissues of the animals. There were slight (in some cases statistically significant) increases in LH-immunoactivity and estradiol in the hemolymph and tissues. However, this applied to the lower dose only (i.e the dose-response relationship was non-monotonic). Using immunohistochemistry, LH-immunoreactive cells were observed in types 1 and 2 neurosecretory (NS1 and NS2) cells within the cerebral and pleuropedal ganglia of H. asinina. In addition, LH-immunoreactive nerve fiber bundles were strongly detected in both ganglia. The immunoactivity against the estrogen appeared to be localized in the granulated cells within the connective tissue and trabeculae of the mature ovary. There was no positive staining in the cytoplasm of any stage of the germ cells. The interpretation of these findings is presently hindered by the fact that the homologous gene for vertebrate LH has not yet been identified in the genomes of any mollusks (so the cause of the immunostaining is as yet unknown) and also by the fact that mollusks are known to readily absorb steroids from the environment and store them long-term in the form of fatty acid esters. More work, involving identification of the protein that cross-reacts with the LH antiserum and also exclusion of the possibility that the estradiol is of exogenous origin, will have to be carried out before these findings can be used to manipulate reproduction in this species.

Fasciola gigantica: immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke.

Specific monoclonal antibody (MoAb) to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in the tissues of metacercariae, newly excysted juvenile (NEJ), 1, 3, 5, and 7-week-old juveniles of Fasciola gigantica by using indirect immunofluorescence, immunoperoxidase and immunogold techniques. Both indirect immunofluorescence and immunoperoxidase detections showed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes as well as epithelial linings of the oral sucker. Unlike adult F. gigantica, it was not detected in spermatogenic cells in the testes, cells of Mehlis'gland, oocytes within the ovary, and ovum within the egg of parasites. At the ultrastructural level, the immunogold labeling showed deposit of gold particles specifically in G2 tegumental granules and on the surface membrane. Thus, this 28.5 kDa antigen is expressed in the tegument and associated structures of juvenile parasites, and it could be a major component of the G2 granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is the replenishment and turnover of the surface membrane to prevent the attachment of the host immune effector cells.

Fasciola gigantica: immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen.

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.

Fasciola gigantica: anthelmintic effect of the aqueous extract of Artocarpus lakoocha.

The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,3',5'- tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 microg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 microg/ml as the positive control, for 3, 6, 12 and 24h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3h incubation with TCZ at the concentration 80 and 175 microg/ml. TCZ markedly reduced the parasite's motility at the concentration of 175 microg/ml at 6h, and killed the worms after 12h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite's motility similar to TCZ at 3h incubation. In 250 and 500 microg/ml of the crude extract, the values were decreased from 3 to 12h, then they were stable between 12 and 24h and reduced to the level approximately 30-40% of the control. At 750 and 1000 microg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12h and killed the parasites between 12 and 24h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250-500 and 750-1000 microg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage.

Cloning, characterization, and expression of vitelline protein BI and its encoding gene in the liver fluke, Fasciola gigantica.

A cDNA containing a 813 bp open reading frame encoding vitelline protein BI (FgVPBI) of Fasciola gigantica was cloned. FgVPBI has 96% sequence identity with VPBI of Fasciola hepatica and 84% identity with VPBII F. hepatica. It is far less similar to eggshell precursor proteins of other trematode species, for example, 29% identity with C. sinensis. Northern blot hybridization of total RNA from adult parasites demonstrated a FgVPBI transcript with a size of 1,000 nucleotides. FgVPBI mRNA is localized in the vitelline cells in both vitelline glands and intrauterine eggs. Recombinant FgVPBI was expressed as a 31.5 kDa protein in Escherichia coli and used for production of a polyclonal antiserum in rabbits. The FgVPBI antiserum detected immunoblotted rFgVPBI and native eggshell precursor protein at molecular weights of 31.5 kDa and 31 kDa, respectively. Immunolocalization showed strong staining in the cytoplasm of vitelline cells, in eggshell globules and the shells of eggs.

Starvation Promotes Autophagy-Associated Maturation of the Testis in the Giant Freshwater Prawn, Macrobrachium rosenbergii.

Autophagy is a degradative process of cellular components accomplished through an autophagosomal-lysosomal pathway. It is an evolutionary conserved mechanism present in all eukaryotic cells, and it plays a fundamental role in maintaining tissue homeostasis both in vertebrates and invertebrates. Autophagy accompanies tissue remodeling during organ differentiation. Several autophagy-related genes and proteins show significant upregulations following nutrient shortage (i.e., starvation). In our previous study, we found that in female giant freshwater prawns subjected to a short period of starvation autophagy was up-regulated in consonant with ovarian maturation and oocyte differentiation. Whether and how starvation-induced autophagy impacts on testicular maturation and spermatogenesis of the male prawns remained to be investigated. In this study, we analyzed the effects of starvation on histological and cellular changes in the testis of the giant freshwater prawn Macrobrachium rosenbergii that paralleled the induction of autophagy. Under short starvation condition, the male prawns showed increased gonado-somatic index, increased size, and late stage of maturation of seminiferous tubules, which contained increased number of spermatozoa. Concurrently, the number of autophagy vacuoles and autophagy flux, as monitored by transmission electron microscopy and the autophagic marker LC3, increased in the testicular cells, indicating that a short period of starvation could induce testicular maturation and spermatogenesis in male M. rosenbergii along with modulation of autophagy.

Existence of an egg-laying hormone-like peptide in male reproductive system of the giant freshwater prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species. A better understanding of the molecular components of reproduction in this species would help to advance the prawn production. In the present study, we demonstrated the presence of an egg laying hormone (ELH)-like peptide in the male reproductive system. First, an antibody to the abalone (a)ELH was generated, and by Western blot it was shown to specifically bound to a protein from the male M. rosenbergii reproductive tissues with a similar size to molluscan ELH. This aELH-like peptide was localized in spermatogonia in the testes of all three male morphotypes: blue claw, orange claw and small males. Moreover, the aELH-like peptide was detected in the epithelium of the spermatic duct and its associated smooth muscle cell layers and on the outer surface of spermatozoa. As well, the aELH-like peptide was detected in the spermatophore located in the female thelycum at 4-6 h post-mating, indicating that it was transferred to the female during copulation. Taken together, we suggest that this aELH-like peptide could be as a male inducing factor that helped to accelerate female spawning. Liquid chromatography of crude extracts and immunoblot analysis suggested that the aELH-like peptide could be further purified for ultimate characterization.

Immunohistochemical expression of type II collagen in the lingual mucosa of rats during organogenesis of the tongue.

OBJECTIVES: We examined the timing of the appearance and distribution of type II collagen as a possible component of the extracellular matrix that is involved in the morphogenesis of the rat tongue. METHODS: We examined the immunofluorescence of type II collagen, differential interference contrast (DIC) images, and images recorded in transmission mode after toluidine blue staining by laser-scanning microscopy (LSM) during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples. RESULTS: Immunoreactivity specific for type II collagen was scattered on cells over a wide area of the mesenchymal connective tissue of the fetal tongue on day 15 after conception (E15), when the lingual epithelium was composed of one or two layers of cuboidal cells. Immunoreactivity specific for type II collagen was recognisable on cells of the lamina propria of the lingual mucosa and around the developing lingual muscle of fetuses at E17 and E19. On E19, the epithelium was clearly of the stratified squamous type. At postnatal stages after birth (P0), immunoreactivity became more and more significant in the connective tissue of the lamina propria with the advancing of morphogenesis of the filiform papillae. In addition, immunoreactivity was widely distributed in the connective tissue around the lingual muscle, as myogenesis in the tongue advanced. The lingual epithelium was composed of stratified squamous cells, and keratinization of the lingual epithelium proceeded gradually as morphogenesis of filiform papillae continued during postnatal development. CONCLUSION: Type II collagen appeared not only in the connective tissue of the lamina propria as the morphogenesis of filiform papillae occurred and the lingual epithelium became keratinized but also in the endomysium and perimysium around the lingual muscle after myogenesis of the tongue is complete at P0.

Newly developed technique for dual localization of keratins 13 and 14 by fluorescence immunohistochemistry.

It is difficult to visualize histological details on semi-ultrathin sections by light microscopy after immunohistochemical labeling because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize the immunoreactivity of keratins 13 (K13) and 14 (K14), we used a newly developed technique for dual localization of antigens by fluorescence immunohistochemistry and confocal laser-scanning microscopy in transmission mode, after staining specimens with toluidine blue. Using this approach, we examined the immunolocalization of K13 and K14 on the lingual epithelium of fetal and juvenile rats by immunofluorescence while monitoring morphological changes in the filiform papillae by laser-scanning microscopy, in transmission mode, of the same sections. No K13 and K14 immunoreactivity was detected on the lingual epithelium of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of a few layers of cuboidal cells. K14 immunoreactivity was first detected on the lingual epithelium of fetuses on E17 and K13 immunoreactivity on E19. The number of layers of cuboidal cells in the lingual epithelium also increased from E17 to E19. K13 and K14 immunoreactivity was distinct at all postnatal stages examined. Although the respective patterns of K13 and K14 immunoreactivity differed as the filiform papillae developed, K13 immunoreactivity was generally evident in the suprabasal cells of the interpapillary cell columns and K14 immunoreactivity was detected in the basal and suprabasal cells of the papillary and interpapillary cell columns. Our newly developed technique for dual localization of antigens should be useful for investigations of very small specimens, such as fetal tissues and organs.

Autophagy-Associated Shrinkage of the Hepatopancreas in Fasting Male Macrobrachium rosenbergii Is Rescued by Neuropeptide F.

Invertebrate neuropeptide F-I (NPF-I), much alike its mammalian homolog neuropeptide Y, influences several physiological processes, including circadian rhythms, cortical excitability, stress response, and food intake behavior. Given the role of autophagy in the metabolic stress response, we investigated the effect of NPF-1 on autophagy during fasting and feeding conditions in the hepatopancreas and muscle tissues of the male giant freshwater prawn Macrobrachium rosenbergii. Starvation up-regulated the expression of the autophagy marker LC3 in both tissues. Yet, based on the relative levels of the autophagosome-associated LC3-II isoform and of its precursor LC3-I, the hepatopancreas was more responsive than the muscle to starvation-induced autophagy. Injection of NPF-I inhibited the autophagosome formation in the hepatopancreas of fasting prawns. Relative to the body weight, the muscle weight was not affected, while that of the hepatopancreas decreased upon starvation and NPF-1 treatment could largely prevent such weight loss. Thus, the hepatopancreas is the reserve organ for the nutrient homeostasis during starvation and NPF-I plays a crucial role in the balancing of energy expenditure and energy intake during starvation by modulating autophagy.