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Acute toxicity and acetylcholinesterase inhibition in grass shrimp (Palaemonetes pugio) and oysters (Crassostrea virginica) exposed to the organophosphate dichlorvos: laboratory and field studies.

The use of various organophosphates to control mosquito populations is a common practice across the globe. We review the literature (LC50s) on dichlorvos, the primary breakdown product of Dibrom, and use laboratory and field experiments to determine the lethal and sublethal (bioassays) effects of dichlorvos on two widely distributed and ecologically important estuarine invertebrate species, the marsh grass shrimp, Palaemonetes pugio and the Eastern oyster, Crassostrea virginica. Laboratory results based on LC50s and sublethal acetylcholinesterase (AChE) inhibition activity bioassays indicate that adult grass shrimp are more sensitive (approximately 500 x ) to dichlorvos than juvenile oysters. Although potentially an important factor for intertidal or shallow-dwelling estuarine organisms, the toxicity of dichlorvos was not enhanced in the presence of simulated sunlight for adult P. pugio. The most notable decreases in AChE activity were for grass shrimp and oysters exposed to dichlorvos concentrations above those considered ecologically relevant. In field experiments, both species were deployed in cages in unsprayed (n = 2) and sprayed (n = 3) sites and water samples collected pre- and post-spraying. Quantifiable dichlorvos levels were measured at the two narrowest creek treatment sites following mosquito spraying, suggesting that overspray can occur and there was evidence of a sublethal AChE response at these same sites. However, experiments at the widest creek revealed no measurable dichlorvos or sublethal responses. Results from this research suggest that adult grass shrimp are more sensitive to dichlorvos than juvenile oysters. Spraying near small tidal creeks may have measurable impacts on resident species, while larger (wider) creeks appear to be capable of buffering organisms from transient fluxes of mosquito control agents that may enter the system.

Age Factors↗

Economic valuation of a mangrove ecosystem threatened by shrimp aquaculture in Sri Lanka.

Mangrove ecosystems in Sri Lanka are increasingly under threat from development projects, especially aquaculture. An economic assessment is presented for a relatively large (42 ha) shrimp culture development proposed for the Rekawa Lagoon system in the south of Sri Lanka, which involved an extended cost-benefit analysis of the proposal and an estimate of the "total economic value" (TEV) of a mangrove ecosystem. The analysis revealed that the internal benefits of developing the shrimp farm are higher than the internal costs in the ratio of 1.5:1. However, when the wider environmental impacts are more comprehensively evaluated, the external benefits are much lower than the external costs in a ratio that ranges between 1:6 and 1:11. In areas like Rekawa, where agriculture and fisheries are widely practiced at subsistence levels, shrimp aquaculture developments have disproportionately large impacts on traditional livelihoods and social welfare. Thus, although the analysis retains considerable uncertainties, more explicit costing of the environmental services provided by mangrove ecosystems demonstrates that low intensity, but sustainable, harvesting has far greater long-term value to local stakeholders and the wider community than large shrimp aquaculture developments.

Animals↗

Isolation of differentially expressed genes from white spot virus (WSV) infected Pacific blue shrimp (Penaeus stylirostris).

To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fingerprints of healthy and WSV infected blue shrimp ( Penaeus stylirostris) were compared using the mRNA differential display technique. Thirty-two unique differentially expressed, and one constitutively expressed, cDNA sequences were retrieved. Six of 32 cDNAs showed similarities with the database entries: cDNA 10G32-142 to a shrimp arginine kinase, 22C48-201 to shrimp mitochondrial ATPase gene; 22C47-197, 21G49-203 and 20A55-268 to shrimp ESTs and 20G50-206 to a WSV gene, ORF 116. The constitutively expressed gene showed significant similarity to a yeast elongation factor 1-alpha gene. The expression of a subset of differentially expressed genes (13 of 32) was further evaluated by real-time RT-PCR. Ten of 13 genes showed statistically significant changes in expression between healthy and WSV infected animals suggesting that these genes may play an important role in WSV pathogenesis.

Amino Acid Sequence↗

Assessing the health of grass shrimp (Palaeomonetes pugio) exposed to natural and anthropogenic stressors: a molecular biomarker system.

We developed a molecular biomarker system (MBS) to assess the physiological status of Palaomenetes pugio (grass shrimp) challenged with exposure to heat stress, cadmium, atrazine, and the water-accommodating fraction of either diesel fuel or bunker fuel No. 2. The MBS assayed 9 specific cellular parameters of shrimp that are indicative of a nonstressed or stressed condition: heat-shock protein 60, heat-shock protein 70, alpha B-crystallin homologue, lipid peroxide, total glutathione level, ubiquitin, mitochondrial manganese superoxide dismutase, metallothionein, and cytochrome P-450 2E homologue. Using these 9 parameters, the MBS can distinguish between the responses to each stressor, and to the nonstressed control conditions. The MBS was able to determine the structural integrity of the cell as defined by protein turnover, protein chaperoning, and lipid composition via lipid peroxide levels, and the status of key metabolic processes such as cytoskeletal integrity and glutathione redox potential. This technology aids in the accurate diagnosis of the health of shrimp because the physiological significance of changes of each parameter is well known. This technology is particularly relevant for environmental monitoring because grass shrimp are used as key indicator species in many estuarine ecosystems. Finally, this system is easy to implement, precise, and can be quickly adapted to an automated high-throughput system for mass sample analysis.

Journal Article↗

Toxic effects of juncusol, a marsh plant phenolic extract, on estuarine fish and shrimp.

Juncusol, phenolic compound, was isolated from the black needlerush, Juncus roemerianus. Different concentrations of this extract were tested for toxicity against selected species of estuarine fish and grass shrimp. Juncusol was ten times more toxic to the various species of fish than to grass shrimp. At 2.0 ppm concentration all fishes tested were killed within 36 hr. One hundred per cent mortality in the grass shrimp occurred at 20 ppm during 95 hr exposure. The LC10, LC50, and LC90 concentration for juncusol were calculated for grass shrimp and for juvenile sailfin molly.

Animals↗

Growth of the penaeid shrimp virus infectious hypodermal and hematopoietic necrosis virus in a fish cell line.

An established fish cell line, epithelioma papillosum cyprini (EPC), originating from the carp (Cyprinus carpio) was found to be susceptible to the penaeid shrimp virus, infectious hypodermal and hematopoietic necrosis virus (IHHNV). This economically important pathogen of penaeid shrimp raised in mariculture has been previously limited to growth only in young post-larval shrimps. This is, to our knowledge, the first reported successful growth of the penaeid virus in a fish cell line; it promises to provide a sensitive and convenient cell culture system for the primary isolation and detection of IHHNV which will aid immensely the production of IHHNV-free penaeid shrimp stocks for mariculture. Of added significance is the replication of a crustacean virus in a fish cell line which has never before been reported. The use of this sensitive cell line will also greatly facilitate the characterization of the IHHN virus and other crustacean viruses.

Animals↗

Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA.

Yellow head virus (YHV) is an invertebrate nidovirus which causes a severe mortality in cultured Penaeus monodon. The mortality may be prevented by prior treatment of shrimps with YHV-protease dsRNA. Whether the YHV infected shrimp might be cured by the dsRNA remains to be investigated. P. monodon injected with 10(-6) YHV showed a high virus replication and mortality within 2 days. Injection of 25 microg YHV-protease dsRNA at 3, 6, 12 or 24 h post YHV infection showed a strong inhibition of YHV replication up to 12 h. Unrelated dsRNA-GFP showed no inhibition, indicating that the inhibition was nucleic acid sequence specific through RNAi pathway. Shrimp mortality could be prevented at 3h post YHV infection by the dsRNA, but not at 24 h. These results demonstrate that YHV-protease dsRNA gives therapeutic effect and pave the way to develop a cure for YHV-infected shrimps.

Animals↗

Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA.

RNA interference (RNAi) has been shown to inhibit viral replication in some animals and plants. Whether the RNAi is functional in shrimp remains to be demonstrated. In vitro transcribed dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 were transfected into shrimp primary cell culture and found to inhibit YHV replication. dsRNA targeted to nonstructural genes (protease, polymerase, and helicase) effectively inhibited YHV replication. Those targeted structural genes (gp116 and gp64) were the least effective. These findings are the first evidence that RNAi-mediated gene silencing is operative in shrimp cells. This could be a powerful tool for studying gene function and to develop effective control of viral infection in shrimp.

Animals↗

Effect of hypo- and hypersaline conditions on osmolality and Na+/K+-ATPase activity in juvenile shrimp (Litopenaeus vannamei) fed low- and high-HUFA diets.

Fatty acid composition of cellular membranes can modify permeability and can modulate the activity of Na(+)/K(+)-ATPase. Although highly unsaturated fatty acids (HUFA) improve survival and osmoregulatory capacity to low salinities in penaeid shrimp, the possible mechanisms have not been established. For this purpose the influence of HUFA supplementation in diet (2.9 vs. 34% HUFA proportion to total fatty acids) on osmoregulatory responses of juvenile Litopenaeus vannamei submitted to an acute (15 h) or chronic exposure (21 days), to low (5 g L(-1)) and high salinities (50 g L(-1)) was analyzed. Shrimp fed the high-HUFA diet, had higher concentration of main HUFA (20:5n-3 and 22:6n-3) in polar lipids of gills. Osmotic pressure in hemolymph was significantly affected by salinity in acute (640, 751, 847 mOsm/kg for 5, 30 and 50 g L(-1), respectively), and chronic exposure (645, 713, 814 mOsm/kg), but variations between them were small compared to environmental salinity (206, 832, 1547 mOsm/kg), indicating that osmoregulation was achieved in a matter of hours. An increase in Na(+)/K(+)-ATPase activity was observed only after a chronic exposure to low salinity. Free amino acids (FAA), mainly alanine and arginine, were higher at 30 (control) and 50 g L(-1) in accordance to their role as organic osmolites. Neither osmotic pressure, Na(+)/K(+)-ATPase activity, nor FAA was affected by HUFA supplementation. However, higher water content in gills of shrimp exposed to low salinities was counteracted by increased HUFA content, which could be a result of changes in water permeability of gills. The osmoregulatory capacity of penaeid shrimp to low and high salinities was achieved within 15 h of acclimation and did not depend on HUFA supplementation in the diet.

Amino Acids↗

Starvation and diet composition affect mRNA levels of the high density-lipoprotein-beta glucan binding protein in the shrimp Litopenaeus vannamei.

A high density lipoprotein-beta glucan binding protein (HDL-BGBP) is synthesized in the hepatopancreas of the white shrimp Litopenaeus vannamei and secreted to the hemolymph. Recently, we reported the HDL-BGBP full length cDNA sequence and found that the predicted polypeptide is larger than the mature protein and also, that it contains a long 5'- and 3'-UTRs that may be involved in transcript level regulation. To test whether starvation and feeding may play a role in regulating HDL-BGBP mRNA levels, two different stimuli were evaluated: starvation and composition of diets. After 24 h, the steady state HDL-BGBP mRNA levels of starved shrimp decreased, suggesting that synthesis of the lipoprotein is less required in the absence of food. When shrimp were fed with diets containing different concentrations of protein and lipids, changes in HDL-BGBP mRNA levels were also detected. Shrimp fed the lower concentration of protein and lipid feed accumulated higher levels of HDL-BGBP mRNA. These results indicate that feeding influences HDL-BGBP transcript levels in the hepatopancreas.

Animal Feed↗

Superoxide radical production in response to environmental hypoxia in cultured shrimp.

Markers of oxidative stress in response to hypoxia and reoxygenation were assessed in Pacific white shrimp (Litopenaeus vannamei). Adult shrimp were either exposed to hypoxia (1 mg O(2)/L) for 6, 12, or 24 h followed by 1-h reoxygenation, or exposed to hypoxia for 24 h followed by 1- to 6-h reoxygenation. In all cases, shrimp maintained at constant normoxia were used as controls. Spectrophotometric techniques were applied to analyze lactate concentration, superoxide radical (O(2)(*-)) production, lipid peroxidation (TBARS), and antioxidant capacity status in muscle, hepatopancreas, and gill samples. Results indicate differences among tissues, even under control conditions. O(2)(*-) production and TBARS levels were higher in hepatopancreas than in gill or muscle. No effect of exposure to hypoxia was found. However, reoxygenation following exposure to hypoxia was found to affect the oxidative metabolism of muscle and hepatopancreas from cultured shrimp. Lactate concentration and O(2)(*-) production increased while antioxidant capacity decreased in hepatopancreas and muscle in the first hours of reoxygenation. This could translate into tissue damage, which may significantly jeopardize the commercial aquaculture product.

Animals↗

Purification, characterization and cDNA cloning of a novel lipopolysaccharide-binding lectin from the shrimp Penaeus monodon.

In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

Animals↗

Update on viral accommodation, a model for host-viral interaction in shrimp and other arthropods.

Comparatively little published information is available on the mechanistic response of shrimp and other arthropods to viral pathogens. Much of the literature has been focused on the use of viruses for biological control of insect pests or disease vectors and on the use of baculoviruses as a means of heterologous protein expression in insect cell lines. The situation changed dramatically with the rapid global increase in cultivation of penaeid shrimp and the massive farm losses that have occurred due to viral pathogens. Urgency to solve these problems has led to a closer examination of the shrimp response to viral pathogens in the hope of finding new methods of disease control. Field observations and results of laboratory experiments in the past decade indicate that shrimp may be capable of a specific, adaptive response to viral pathogens that cannot be explained by current knowledge and understanding of their cellular and humoral defenses. Hallmarks of this response are specific tolerance to single and multiple viral infections without gross or histological signs of disease, a phenomenon common to crustaceans and insects. The concept of viral accommodation was introduced in 1998 as a simple testable hypothesis to explain these phenomena. Key elements of the hypothesis were an unknown mechanism for specific memory of pathogens and the role of this memory in dampening viral triggered apoptosis. Recent field and research results have supported predictions of the viral accommodation hypothesis and suggest that memory may be provided by the viral pathogens themselves in persistent infections that result in reduced severity of disease. The well-known phenomenon of defective interfering viral particles may play an important role in this process, but it cannot explain cross protection that has recently been described for heterologous viral infections. The major conclusion is that homologous and heterologous reduction in disease severity resulting from persistent viral infections (i.e., accommodated viral infections) may be a key process that has evolved from host viral interaction in the arthropod line.

Animals↗

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g. the Interferon response) have long been thought to be restricted to vertebrates. Recent work in an underappreciated experimental model, the penaeid shrimp, is challenging these traditional distinctions, by demonstrating the existence of both innate (non sequence-specific) and RNAi-related (sequence-specific) antiviral phenomena in crustacea. Here we discuss the evidence for this bivalent role of dsRNA in the initiation of antiviral responses in shrimp, and present new data that suggest that the antiviral functions of the shrimp RNAi machinery have imposed selective pressures on an evolving viral pathogen. These findings open the door for the discovery of novel mechanisms of innate immunity, and provide a basis for the future development of strategies to control viral diseases in the commercially important penaeid shrimp.

Animals↗

Toxicity of three pesticides individually and in mixture to larval grass shrimp (Palaemonetes pugio).

This study examined the toxicity of three pesticides, singly and in mixture, to grass shrimp (Palaemonetes pugio) larvae. The pesticides included atrazine, an herbicide used on turf grass and field crops; fipronil, a persistent insecticide used against termites and fire ants; and imidacloprid, a systemic insecticide used in agricultural and home products. Fipronil was the most toxic to shrimp larvae with a 96-h LC50 of 0.68microg/L (95% CI 0.57-0.79microg/L). Shrimp larvae were less sensitive to imidacloprid with a 96-h LC50 of 308.8microg/L (95% CI 273.6-348.6microg/L). Atrazine was non-toxic to shrimp larvae at concentrations up to 10,000microg/L. In mixtures, fipronil plus atrazine and imidacloprid plus atrazine had no change in toxicity compared to fipronil and imidacloprid tested singly. Similarly, a fipronil/imidacloprid mixture did not show greater than additive toxicity. However, when atrazine was added to the fipronil/imidacloprid mix, greater than additive toxicity occurred.

Animals↗

Elimination of chloramphenicol, sulphamethoxazole and oxytetracycline in shrimp, Penaeus chinensis following medicated-feed treatment.

Muscle residue depletion of chloramphenicol (CAP), sulphamethoxazole (SMZ) and oxytetracycline (OTC) following oral administration was evaluated in shrimp, Penaeus chinensis under field conditions. Three groups of shrimps were cultured in tanks filled with seawater and fed a commercial medicated diet containing 2000 mg kg(-1) CAP, SMZ and OTC, respectively, twice daily for 3 days. Sampling was conducted at different intervals (0, 0.5, 1, 1.5, 2, 3, 4, 8, 10, 12, 16, 24, 36, 48, 72, 96, 120, 144, 168 h) after the cessation of medication. Drug analysis was carried out by HPLC. The elimination half-life (t(1/2)) of CAP was 10.04 h, the elimination half-life (t(1/2)) of SMZ was 5.68 h and that of OTC was 16.12 h. If the EU MRPL value of 0.3 ng/g for CAP and MRL value of 0.1 microg/g for SMZ and OTC quoted for muscle from finfish is extended to shrimp muscle, extrapolation of the data indicates that it would be passed after a 139.7 h (95% CI=132.0-144.4 h), 30.6 h (95% CI=27.2-33.1 h), 90.3 h (95% CI=87.9-92.5 h) withdrawal period for CAP, SMZ and OTC in shrimp muscle, respectively.

Administration, Oral↗

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress.

The white shrimp Litopenaeus vannamei were challenged with tryptic soy broth (TSB)-grown Vibrio alginolyticus at a dose of 1 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of nitrite-N at 0 (control), 1.12, 5.15, 11.06 and 21.40 mg l(-1). Mortality of shrimp in 5.15, 11.06 and 21.40 mg l(-1) was significantly higher than those in the control solution after 48-168 h. L. vannamei that had been exposed to control, 0.98, 4.94, 9.87 and 19.99 mg l(-1) nitrite-N for 96 h were examined for THC (total haemocyte count), phenoloxidase activity, and respiratory burst (release of superoxide anion). The THC and phenoloxidase activity decreased when the shrimp were exposed to 4.94, 9.87 and 19.99 mg l(-1) nitrite-N, whereas, the respiratory burst increased significantly at 9.87 and 19.99 mg l(-1) nitrite-N after 96 h. It is therefore suggested that nitrite in water caused a depression in the immune ability of L. vannamei and an increased susceptibility to V. alginolyticus infection, together with an increase of superoxide anion production, possibly to cytotoxic levels for the host.

Animals↗

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei.

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to V. alginolyticus infection.

Animals↗