Presence and distribution of FMRFamide-like immunoreactivity in the sea cucumber Holothuria scabra (Jaeger, 1833)

Since the discovery of FMRFamide, a cardioactive neuropeptide in the mollusk Macrocallista nimbosa (Lightfoot, 1786) by Price and Greenberg (Science 197:670–671, 1977), there has been a steady increase in our understanding of the distribution and diversity of FMRFamide and FMRFamide-related peptides (FaRPs) across phyla. The sea cucumber Holothuria scabra (Jaeger, 1833) (Echinodermata: Holothuroidea) is a high-premium tropical economic species that is overexploited globally and may be in imminent danger of extinction in some areas. Conservation of this species and its amenability to culture is however hampered by the limited knowledge about its biology, including the neurohormonal system. Using indirect immunofluorescence technique with antisera raised against the tetrapeptide FMRFamide as well as reverse-phase HPLC, we have demonstrated the widespread distribution of FMRFamide-like immunoreactivity in the tissues of H. scabra, including the coelomic fluid, suggesting that FaRPs might have neurohormonal activities in this species.     

Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand

In 2001-2002 throughout Thailand, black tiger shrimp Penaeus monodon farmers reported very unusual retarded growth. We have called this problem monodon slow growth syndrome (MSGS). Based on decreased national production, estimated losses due to this phenomenon were in the range of 13 000 million baht (approximately 300 million US dollars) in 2002. Since rearing practices had not changed, it was considered possible that the MSGS problem may have arisen from a new or existing pathogen. To examine this possibility, cultivated shrimp were sampled from 32 commercial rearing ponds that reported abnormally slow growth from eastern, central and southern regions of Thailand. Shrimp were randomly sampled from each pond and grouped into normal and small shrimp. Normal shrimp were defined as those with body weights (BW) of 24 g or more while small shrimp were defined as those that weighed 16.8 g or less. Pleopods were used for detection of monodon baculovirus (MBV), heptopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) using specific polymerase chain reaction (PCR) assays. In addition, some shrimp were processed for normal histopathology and transmission electron microscopy (TEM). Most of the shrimp specimens were infected by at least 1 of these viruses but many had dual or multiple infections. Prevalence of HPV and combined HPV/MBV infections in the small shrimp was significantly higher than in the normal shrimp. In addition to the viruses, a new microsporidian species, gregarines and bacteria were also observed but were not significantly associated with the MSGS problem. Some of the small shrimp gave negative results for all these pathogens by PCR and histology and no new and unique histopathology was recognized in any of the samples. The findings suggested that HPV infection was a contributing factor but not the overriding factor responsible for MSGS. It is possible that MSGS is caused by an unknown pathogen or by some other presently unknown, non-pathogenic factor.     

High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand

White spot syndrome virus (WSSV) presently causes the most serious losses to shrimp farmers worldwide. Earlier reports of high DNA sequence homology among isolates from widely separated geographical regions suggested that a single virus was the cause. However, we have found surprisingly high variation in the number of 54 bp DNA repeats in ORF94 (GenBank AF369029) from 55 shrimp ponds (65 shrimp samples) experiencing WSSV outbreaks in Thailand in 2000 and 2002. These were detected by PCR amplification using primers ORF94-F and ORF94-R flanking the repeat region. Altogether, 12 different repeat groups were found (from 6 to 20 repeats) with 8 repeats being most frequent (about 32%). Extracts prepared from individual shrimp in the same outbreak pond belonged to the same repeat group while those collected at the same time from separate WSSV outbreak ponds, or from the same ponds at different times, usually belonged to different repeat groups. This suggested that different outbreaks were caused by different WSSV isolates. In contrast to the highly variable numbers of repeats, sequence variation within the repeat region was confined to either T or G at Position 36. These variations may be useful for epidemiological studies on the local and global movement of WSSV, since there is high variation in the number of repeats (good for local studies) but little sequence change (good for global studies).     

Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is widespread in cultured Penaeus monodon and P. vannamei in Thailand. It causes runt-deformity syndrome that is characterized by physical abnormalities and stunted growth in P. vannamei, but causes no apparent disease in P. monodon. In both species, the virus may produce Cowdry Type A inclusions in tissues of ectodermal and mesodermal origin, but these are common in P. vannamei and rare in P. monodon. The virus can be more easily detected in both species by IHHNV-specific PCR primers. By in situ hybridization (ISH) using specific IHHNV probes, fixed phagocytes associated with myocardial cells tended to show strong positive reactions in both shrimp species. Ovarian and neural tissue (neurons in the nerve ganglia and glial cells in the nerve cord) were ISH positive for IHHNV only in P. vannamei. By transmission electron microscopy, necrotic cells were found in the gills of IHHNV-infected P. vannamei, while paracrystalline arrays of virions and apoptotic cells rather than necrotic cells were found in the lymphoid organ of IHHNV-infected P. monodon. Thus, it is possible that apoptosis in P. monodon contributes to the absence of clinical disease from IHHNV. These findings reveal different responses to IHHNV infection by the 2 shrimp species. A curious feature of IHHNV infection in P. monodon was inconsistency in the comparative viral load amongst tissues of different specimens, as detected by both ISH and real-time PCR. This inconsistency in apparent tissue preference and the reasons for different cellular responses between the 2 shrimp species remain unexplained.     

Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon

No controlled studies on the effect of infectous hypodermal and necrosis virus (IHHNV) on Penaeus monodon have been previously reported. Here we describe domesticated P. monodon that became positive for IHHNV and other viruses at variable levels of prevalence during cultivation in 16 open-air, earthen ponds. These were stocked with domesticated postlarvae (PL) that tested negative for 7 shrimp viruses including IHHNV at 6% prevalence in 3 checks using polymerase chain reaction (PCR) methods. These PL were derived from domesticated female broodstock that individually tested negative for the same viruses. At 4 mo of culture, the shrimp in some ponds without obvious mortality tested positive by PCR methods for IHHNV and 3 other viruses at variable levels of maximum estimated prevalence (MEP). Stained tissue sections showed no lesions typical of IHHNV, but in situ hybridization tests with an IHHNV-specific DNA probe were positive. There was no significant difference in mean body weight (i.e. ca. 25 g) between shrimp groups positive or negative for IHHNV. Similar results were obtained with IHHNV negative and positive adults at 1 yr. Adults that individually tested negative for all 7 viruses and some that tested lightly positive for IHHNV were bred for the next generation. There were no significant differences in the number of eggs (> 600 000) and nauplii (ca. 300,000) produced by females negative and positive for IHHNV. From these females, 11/49 (22%) IHHNV PCR-positive PL batches were obtained from PCR-negative spawners, while 8/11 (73%) were obtained from IHHNV PCR-positive spawners. The results suggested that IHHNV infection can be transmitted vertically but does not seriously retard growth of P. monodon or affect fecundity of lightly infected broodstock.     

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.     

L-(3H) glutamate binding to a membrane preparation from the optic lobe of the giant freshwater prawn Macrobrachium rosenbergii de Man.

Membrane preparation from the optic lobe of the giant freshwater prawn, Macrobrachium rosenbergii de Man, was examined for the presence of specific L-(3H) glutamate binding. The optic lobes were isolated from live animals. The tissue was homogenized and the membrane fraction isolated by differential centrifugation. The membrane suspension was incubated with 10-1,000 nM of L-(3H) glutamate at 37 degrees C for 60 min. Nonspecific binding was determined by incubating the mixture with 100 microM L-glutamate. L-(3H) glutamate specifically bound to the membrane fraction with a dissociation equilibrium constant (Kd) of 205 nM and maximum number of binding sites (Bmax) of 2.04 n mol/mg protein. By using LIGAND computerized program, the saturation isotherm binding pattern indicates a single type of binding. To determine the type of glutamate receptors, competitive inhibition and IC50 of several glutamate agonists and antagonists were determined. The study reveals a metabotropic type of binding site.     

Retention of radioactive substances in the hypothalamus, anterior pituitary, and reproductive organs of male rats after 3H-melatonin administration

Radioactive concentrations were determined in serum, lung, hypothalamus, anterior pituitary, testis, and accessory sex organs of adult male rats at 2, 20, 30 and 60 min after intravenous 3H-melatonin administrations. The retention patterns of 3H-melatonin and other radioactive substance (3H-non-melatonin) in these tissues were compared. The anterior pituitary demonstrated best tissue retention with highest concentration of 3H-melatonin. The testis and prostate gland accumulated 3H-non-melatonin in an increasing manner from 2 to 60 min. The radioactive substances were also preferentially and progressively located in the nuclear fraction of the anterior pituitary, hypothalamus, testis, and prostate gland. This study leads to the following suggestions: the anterior pituitary is another target organ of melatonin; melatonin is converted into other active material which exerts its action in the testis and prostate gland; melatonin and its active derivative exert their action through the nuclei of their respective cells.     

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.