Dead Shrimp Research Articles

Screening white spot syndrome virus (WSSV)-resistant molecular markers from Fenneropenaeus chinensis

White spot syndrome virus (WSSV)-resistant molecular markers were screened from the selectively bred new variety ‘Huanghai No. 2’ of Fenneropenaeus chinensis using unlabeled-probe high-resolution melting (HRM) technique. After the artificial infection with WSSV, the first 96 dead shrimps and the last 96 surviving shrimps were collected, representing WSSV-susceptible and -resistant populations, respectively. The genotypes at well-developed 39 single nucleotide polymorphisms (SNPs) loci were obtained. As revealed in the Chi-square test, 3 SNPs, genotype A/A of contig C364-89AT, genotype A/A of C2635-527CA and genotype C/T of contig C12355-592CT, were positively correlated with disease-resistance traits. Other 2 SNPs, genotype G/G of contig C283-145AG and genotype C/C of contig C12355-592CT, were negatively correlated. Moreover, analysis with BlastX program for disease-resistant SNPs indicated that 3 contigs, Contig283, Contig364 and Contig12355, matched to the functional genes of effector caspase of Penaeus monodon, peptide transporter family 1-like protein, and 40S ribosomal protein S2 of Perca flavescens with high sequence similarity. The results will be helpful to provide theoretical and technical supports for molecular marker-assisted selective breeding of F. chinensis.

Number sense and state-dependent valuation in cuttlefish.

Identifying the amount of prey available is an important part of an animal's foraging behaviour. The risk-sensitive foraging theory predicts that an organism's foraging decisions with regard to food rewards depending upon its satiation level. However, the precise interaction between optimal risk-tolerance and satiation level remains unclear. In this study, we examined, firstly, whether cuttlefish, with one of the most highly evolved nervous system among the invertebrates, have number sense, and secondly, whether their valuation of food reward is satiation state dependent. When food such as live shrimps is present, without training, cuttlefish turn toward the prey and initiate seizure behaviour. Using this visual attack behaviour as a measure, cuttlefish showed a preference for a larger quantity when faced with two-alternative forced choice tasks (1 versus 2, 2 versus 3, 3 versus 4 and 4 versus 5). However, cuttlefish preferred the small quantity when the choice was between one live and two dead shrimps. More importantly, when the choice was between one large live shrimp and two small live shrimps (a prey size and quantity trade-off), the cuttlefish chose the large single shrimp when they felt hunger, but chose the two smaller prey when they were satiated. These results demonstrate that cuttlefish are capable of number discrimination and that their choice of prey number depends on the quality of the prey and on their appetite state. The findings also suggest that cuttlefish integrate both internal and external information when making a foraging decision and that the cost of obtaining food is inversely correlated with their satiation level, a phenomenon similar to the observation that metabolic state alters economic decision making under risk among humans.

Comparing the Response of the Brown ShrimpCrangon crangon(Linnaeus, 1758) to Prolonged Deprivation of Food in Two Seasons

ABSTRACT Crustaceans often undergo periods of starvation, due to natural food shortage or physiological constraints. During these periods, several metabolic and behavioral changes can occur. This study evaluates how the brown shrimp Crangon crangon (Linnaeus, 1758) responds to prolonged deprivation of food in two seasons of the year, and how this species mobilizes its energetic reserves. Shrimps caught in June (summer) and October (autumn) 2010 in the Minho estuary (north of Portugal) were placed in individual cages in experimental aquaria and kept in starvation until the last shrimp died or was sacrificed (six shrimp per aquarium every week). The energetic content, total lipids and total protein, and the oxygen consumption rate were compared between seasons, sacrificed and naturally dead shrimp, and considering the weeks of starvation. Summer shrimp proved to be better prepared to endure starvation than those caught in autumn: they survived 2.5 times longer, had a higher Fulton condition factor, higher e...

새우의 급성간췌장괴사병과 수입 위생 조치

In 2009 outbreaks of an emerging disease in shrimp farms were first reported in China. The disease was known as early mortality syndrome (EMS), or acute hepatopancreatic necrosis disease (AHPND). Since 2009, the disease has been spread to Vietnam, Malaysia, Thailand and Mexico. The causative agent of the disease was identified as Vibrio parahaemolyticus. It is a common seawater inhabitant bacterium, and the pathogen can sometimes contaminate seafood. The disease has caused mass mortality of cultivated shrimp, and huge economic losses in the countries named above. In order to prevent the introduction and establishment of AHPND, emergency measures, such as strengthening of import conditions and even import bans, were put in place by many other countries. In Korea, on the other hand, there are large quantities of shrimp imports from countries, such as Thailand and Vietnam. Transportation of live and fresh dead shrimp is highly likely, and could be a transmission pathway if the shrimp are sourced from populations in AHPND endemic areas. It is important to recognize that importing countries may provisionally adopt sanitary or phytosanitary measures on the basis of available pertinent information, including that from the relevant international organizations, as well as from sanitary or phytosanitary measures applied by other countries based on "Agreement on Application of Sanitary and Phytosanitary Measures". It is pertinent that Korea also takes proper emergent measures to keep out diseases and provide safe seafood.

Cytotoxicity of selected medicinal plants used in Mt. Frere District, South Africa.

In South African traditional medicine, some are plants known to combat pediatric diseases and are commonly used by traditional healers. The aim was to evaluate cytotoxicity effects of plants. The ground plant material was exhaustively extracted using methanol, acetone and water separately for 72 hrs. These organic solvents were removed from filtrates using a rotavapour. Stock solutions were prepared at 40 mg/ml Dimethyl sulfoxide (DMSO) and test solutions were transferred into vials and 10 brine shrimps introduced in each. The number of dead shrimps was counted to ascertain toxicity. Ten A. salina nauplii (larva) were transferred into each sample vial and filtered brine solution was added to make 5 ml. The nauplii were counted macroscopically in the stem of the pipette against a lighted background. A drop of dry yeast suspension was added as food to each vial. Probit analysis was used to determine the concentration at which lethality to brine shrimp represents 50 % (LC50). All the tested extracts showed that the concentration is directly proportional to death of brine shrimps. Fifty percent lethality (LC50) of the tested crude extract ranged between 4.1 and 4.6 µg/ml with methanol extract of B. abyssinica being the lowest and T. acutiloba the highest. This study revealed that 100% of plant crude extracts screened for activity against Artemisia salina larvae showed strong cytotoxicity below 10 µg/ml and plant species with LC50 values < 1000 µg/ml may not make good paediatric remedies due to their inherent toxicity.

Toxicological evaluation of the plant products using Brine Shrimp (Artemia salina L.) model

Many natural products could serve as the starting point in the development of modern medicines because of their numerous biological and pharmacological activities. However, some of them are known to carry toxicological properties as well. In order to achieve a safe treatment with plant products, numerous research studies have recently been focused on both pharmacology and toxicity of medicinal plants. Moreover, these studies employed efforts for alternative biological assays. Brine Shrimp Lethality Assay is the most convenient system for monitoring biological activities of various plant species. This method is very useful for preliminary assessment of toxicity of the plant extracts. Rapidness, simplicity and low requirements are several advantages of this assay. However, several conditions need to be completed, especially in the means of standardized experimental conditions (temperature, pH of the medium, salinity, aeration and light). The toxicity of herbal extracts using this assay has been determined in a concentration range of 10, 100 and 1000 µg/ml of the examined herbal extract. Most toxicity studies which use the Brine Shrimp Lethality Assay determine the toxicity after 24 hours of exposure to the tested sample. The median lethal concentration (LC50) of the test samples is obtained by a plot of percentage of the dead shrimps against the logarithm of the sample concentration. LC50 values are estimated using a probit regression analysis and compared with either Meyer’s or Clarkson’s toxicity criteria. Furthermore, the positive correlation between Meyer’s toxicity scale for Artemia salina and Gosselin, Smith and Hodge’s toxicity scale for higher animal models confirmed that the Brine Shrimp Lethality Assay is an excellent predictive tool for the toxic potential of plant extracts in humans.

Antimicrobial Susceptibility of Vibrio Species Isolated From Brackish Water Shrimp Culture Environment

A comparative study regarding prevalence and antimicrobial susceptibility of Vibrio species in the black tiger shrimp (Penaeus monodon Fabricius) culture environment as well as market samples were analyzed. Total Vibrio counts were found to range up to 2.5 × 103 cfu/gm and 60 cfu/gm in shrimp and Gher water samples, respectively. Results revealed that the total vibrio count were found to be higher in samples taking from the market shops in Dhaka city than Gher shrimp samples and the live shrimps were not contaminated with Vibrio species but dead shrimps were found contaminated, which were collected from shrimp Gher. In the antimicrobial susceptibility test, highest 28.57% resistance among total Vibrio isolates was observed to penicillin and cephalexin. Among the seven isolates of Vibrio species from Gher water sample one isolate displayed multi-drug resistance (MDR). The main reason of antibiotic resistance could be the indiscriminate application of antibiotics in shrimp farming and release of shrimp pond effluent to estuarine ecosystems or post harvest contamination of shrimps with the antibiotic resistant bacteria through the environment and human handling. DOI: http://dx.doi.org/10.3329/jbas.v36i2.12964 Journal of Bangladesh Academy of Sciences, Vol. 36, No. 2, 213-220, 2012

Feeding in the dark: lateral-line-mediated prey detection in the peacock cichlid Aulonocara stuartgranti

The cranial lateral line canal system of teleost fishes is morphologically diverse and is characterized by four patterns. One of these, widened lateral line canals, has evolved convergently in a wide range of teleosts, including the Lake Malawi peacock cichlids (Aulonocara), and has been attributed to its role in prey detection. The ability to study Aulonocara in the laboratory provides an opportunity to test the hypothesis that their reported ability to feed on invertebrate prey living in sandy substrates in their natural habitat is the result of lateral-line-mediated prey detection. The goal of this study was to determine whether Aulonocara stuartgranti could detect hydrodynamic stimuli generated by tethered brine shrimp (visualized using digital particle image velocimetry) under light and dark conditions, with and without treatment with cobalt chloride, which is known to temporarily inactivate the lateral line system. Fish were presented with six pairs of tethered live and dead adult brine shrimp and feeding behavior was recorded with HD digital video. Results demonstrate that A. stuartgranti: (1) uses the same swimming/feeding strategy as they do in the field; (2) detects and consumes invertebrate prey in the dark using its lateral line system; (3) alters prey detection behavior when feeding on the same prey under light and dark conditions, suggesting the involvement of multiple sensory modalities; and (4) after treatment with cobalt chloride, exhibits a reduction in their ability to detect hydrodynamic stimuli produced by prey, especially in the dark, thus demonstrating the role of the lateral line system in prey detection.

Real time PCR quantification of WSSV infection in specific pathogen free (SPF) Litopenaeus vannamei (Boone, 1931) exposed to antiviral nucleotide

ObjectiveTo investigate the level of WSSV transmission from the infected tiger prawn Penaeus monodon (P. monodon) to specific pathogen free Litopenaeus vannamei (L. vannamei) in laboratory captivity condition in relation to PCR detection, histopathological observation and viral genome sequence. MethodsViral DNA was isolated from purified virions by treatment with proteinase K (0.2 mg/mL) and Sarkosyl (1%). The purity and concentration of the DNA were determined by agarose gel electrophoresis. Moribund and dead shrimp were removed and processed for indirect immunofluorescence (IIF) analysis. Histological observation of infected L. vannamei shrimps were revealed by the degenerated cells which were characterized by intranuclear inclusions in the tissues of WSSV infected mid-gut gland, lymphoid organ, gill lamellae and gut epithelium. Total DNA was extracted, from shrimp hemolymph and tissues, with a High Puree PCR template preparation kit. WSSV-DNA was detected using a commercial 2-step PCR detection kit. ResultsThe present study compares the real-time PCR results with SYBR Green I concentration ranging from 0.2 to 0.7×. The positive standard was used in the range of 102, 104 106, 108 and 1010 copies/ng of DNA in general. The PCR analysis showed the appearance of a prominent band from the PCR amplified product of WSSV-DNA at internal control band of 848 bp. Moderate and severe levels were observed as 650 bp and 910 bp (200 & 2 000 copies) in various transmission routes. The WSSV content in moribund shrimp of all the experimental species (L. vannamei) approximately ranged in nucleotide application by quantification method from 0.000 001 WSSV copies/μg of total DNA. In whole moribund infection animal, approximately 0.02 WSSV copies/μg of DNA was detected in nucleotide applied animal. ConclusionThese results indicate that wild brood stock and native culture shrimp P. monodon may be infected with WSSV and can get transferred into the SPF L. vannamei farming environment. Based on the studies, we made in captivity condition in different WSSV transmission route in dissimilar infection range with the use of nucleotide for antiviral drugs. There is an urgent need to address and develop antiviral drugs and molecular based viral genome technique for control measures to salt away the aquaculture environments.

UJI TOKSISITAS EKSTRAK ETANOL BUAH MERAH (Pandanus conoideus Lam) ASAL MANOKWARI PAPUA BARAT DENGAN METODE BRINE SHRIMP LETHALITY TEST

A research on Toxicity assay of Extract Ethanol Red Fruit (Pandanus conoideus Lam) upon Brine Shrimp Lethality Test which intended to know the toxic effect of Red Fruit and supposed to determine Lc50 of the Red fruit by method Brine Shrimp Lethality Test. The ethanol ekstrak, was made in various concentration 10, 100, 1000 µg/ml. Mean while, for negative control was seawater which made by the same concentration. In each concentration has put in 10 brine shrimp. Each concentration has put 1 drop of yeast extract as source of food. The volume is added to 10 ml, observation is done after 24 hours treatment. Observation result in dead brine shrimp data. From the counted of probit analysis method obtained Lc50 value was that 52,844 ± 25,679 µg/ml. Mean while in negative compare there were not brine shrimp killed. Key words : Pandanus conoideus Lam , Brine Shrimp Lethality Test, Toxicity assay

UJI TOKSISITAS EKSTRAK ETANOL BUAH MERAH (Pandanus conoideus Lam) ASAL MANOKWARI PAPUA BARAT DENGAN METODE BRINE SHRIMP LETHALITY TEST

A research on Toxicity assay of Extract Ethanol Red Fruit (Pandanus conoideus Lam) upon Brine Shrimp Lethality Test which intended to know the toxic effect of Red Fruit and supposed to determine Lc50 of the Red fruit by method Brine Shrimp Lethality Test. The ethanol ekstrak, was made in various concentration 10, 100, 1000 µg/ml. Mean while, for negative control was seawater which made by the same concentration. In each concentration has put in 10 brine shrimp. Each concentration has put 1 drop of yeast extract as source of food. The volume is added to 10 ml, observation is done after 24 hours treatment. Observation result in dead brine shrimp data. From the counted of probit analysis method obtained Lc50 value was that 52,844 ± 25,679 µg/ml. Mean while in negative compare there were not brine shrimp killed. Key words : Pandanus conoideus Lam , Brine Shrimp Lethality Test, Toxicity assay

Characterization of WSSV resistance in selected families of Litopenaeus vannamei

Disease outbreaks caused by the white spot syndrome virus (WSSV) have resulted in high economic losses in the shrimp-culture industry worldwide. After three generations of selective breeding for WSSV resistance, 29 families of Litopenaeus vannamei (G3) were selected and divided into three groups: relatively high-resistance, moderate-resistance, and susceptible families, which had average survival rates of 22.7±12.30, 9.08±1.46, and 0.78±1.70%, respectively, after WSSV infection. Appearance of the mortality peak in the relatively high-resistance families was postponed by 2–3days compared to the same in susceptible families and unselected shrimps. Using real-time quantitative polymerase chain reaction, the copy number of WSSV DNA in the muscles of surviving shrimps from the relatively high-resistance family 14 was significantly lower than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 36hourspostinfection (hpi) and thereafter; for example, at 48hpi, the DNA copy number was (23.87±2.56)×106copiesg−1 in the high-resistance families against (40.03±2.54)×106, (65.70±3.44)×106, and (47.87±3.95)×106copiesg−1 body weight, respectively, in the other groups. The WSSV copy number in the muscles of dead shrimps from the relatively high-resistance family 14 was significantly higher than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 48hpi and thereafter; for example, at 72hpi, the DNA copy number was (54.1±3.9)×106copiesg−1 compared to values of (31.5±2.6)×106, (11.6±2.9)×106, and (25.4±2.7)×106copiesg−1 body weight in the other groups, respectively. The results indicated that shrimps from the relatively high-resistance families could inhibit the replication of WSSV and therefore had apparent WSSV resistance. These results provide a direction for the future selective breeding of shrimps for WSSV resistance.

Effects of Limited Dissolved Oxygen Supply on the Growth and Energy Allocation of Juvenile Chinese Shrimp, Fenneropenaeus chinensis

Abstract Information about the effect of hypoxia exposure on energy allocation is helpful for better understanding how aquatic animals tolerate and adapt to a hypoxic environment. The growth, molting, and energy allocation were investigated in juvenile Fenneropenaeus chinensis exposed to five different dissolved oxygen (DO) seawaters (2.09 ± 0.15, 3.10 ± 0.29, 4.13 ± 0.25, 4.73 ± 0.12, and 5.48 ± 0.09 mg/L DO) for 30 d. When DO was below 4.13 mg/L, the growth of shrimp was depressed. Feed ingestion and feed conversion efficiency decreased with the decrease of DO. Less feed ingestion was caused by lower daily metabolic energy. Higher proportion of ingested energy lost in metabolism, exuviations, and nitrogen excretion caused lower feed conversion efficiency. More energy lost in exuviations, lower survival rate, soft carapaces of dead shrimp, and cannibalism were found in oxygen deficient groups and it implicated that hypoxia could delay the shrimp recovering from molting and cause cannibalism and higher mortality. The results indicated that hypoxia was an important factor affecting the amount and fitness of shrimp stock because it could cause a high rate of mortality and growth depression.

Virulence of white spot syndrome virus (WSSV) isolates may be correlated with the degree of replication in gills of Penaeus vannamei juveniles

A standardized inoculation model was used in 2 separate experiments to gauge the virulence of 3 white spot syndrome virus (WSSV) isolates from Thailand and Vietnam (WSSV Thai-1, WSSV Thai-2, and WSSV Viet) in Penaeus vannamei juveniles. Mortality patterns (Expt 1) were compared and WSSV-positive cells quantified (Expt 2) in tissues following intramuscular inoculation of shrimp with the most (WSSV Thai-1) and least (WSSV Viet) virulent isolates as determined by Expt 1. The results of Expt 1 demonstrated that mortalities began at 36 h post inoculation (hpi) for both Thai isolate groups and at 36 to 60 hpi for the Viet isolate group. Cumulative mortality reached 100% 96 to 240 h later in shrimp challenged with the WSSV Viet isolate compared to shrimp challenged with the Thai isolates. WSSV infection was verified in all groups by indirect immunofluorescence. In Expt 2, WSSV-infected cells were quantified by immunohistochemical analysis of both dead and time-course sampled shrimp. WSSV-positive cells were detected in tissues of Thai-1 inoculated dead and euthanized shrimp from 24 hpi onwards and from 36 hpi onwards in shrimp injected with the Viet isolate. Significantly more infected cells were found in tissues of dead shrimp inoculated with the Thai-1 than in Viet isolate-inoculated shrimp. In these experiments, substantial differences in virulence were demonstrated between the WSSV isolates. The Vietnamese isolate induced a more chronic disease and mortality pattern than was found for the Thai isolates, possibly because it infected fewer cells. This difference was most pronounced in gills.

Pathogenicity Assay of Vibrio harveyi in Tiger Shrimp Larvae Employing Rifampicin-Resistant as A Molecular Marker

&lt;p&gt;Rifampicin-resistant marker was employed as a reporter to assay pathogenicity of &lt;em&gt;Vibrio harveyi&lt;/em&gt; in shrimp larvae. &lt;em&gt;V. harveyi&lt;/em&gt; M. G&lt;sub&gt;3&lt;/sub&gt; and G&lt;sub&gt;7&lt;/sub&gt; that difference not schizotyping as shown by Pulsed-Filed Gel Electrophoresis (PFGE) used in this study. Spontaneous mutation was conducted to generate &lt;em&gt;V. harveyi&lt;/em&gt; resistant to rifampicin. Two groups of shrimp post-larvae (PL&lt;sub&gt;5&lt;/sub&gt;) were immersed for 30 min in 106 CFU/ml of mutants and wild type of &lt;em&gt;V. harveyi&lt;/em&gt;, respectively; and then placed in a 2 liter shrimp rearing tank for five days. A control group was immersed in sterile seawater. Growth curve analysis and pathogenicity assay of &lt;em&gt;V. harveyi &lt;/em&gt; showed that each of the &lt;em&gt;V. harveyi&lt;/em&gt; mutant exhibited almost identical profiles to that of the wild type parental strain and did not show alteration in their pathogenicity. Sample from dead shrimp larvae showed that the dead shrimp larvae were infected by &lt;em&gt;V. harveyi &lt;/em&gt;Rf&lt;sup&gt;R&lt;/sup&gt;, indicated that rifampicin-resistant marker effective as a reporter to assay pathogenicity of &lt;em&gt;Vibrio harveyi &lt;/em&gt;in shrimp larvae.&lt;/p&gt; &lt;p&gt;Key words: shrimp larvae, &lt;em&gt;Vibrio harveyi&lt;/em&gt;, rifampicin-resistant, molecular marker&lt;/p&gt;

Combined effect of exposure to ammonia and hypoxia on the blue shrimp Litopenaeus stylirostris survival and physiological response in relation to molt stage

The effect of ambient ammonia, hypoxia and combination of both on survival and the physiological and immunological response of the blue shrimp Litopenaeus stylirostris in relation to molt stage was studied. Shrimp were submitted to 44.0-71.5 mg l­1 total ammonia-N corresponding to 2.0 mg l­1 unionized ammonia NH3-N and/or to 1.5 mg O2 l­1 (4.3 kPa) for 24 hours. Survival was recorded and the molt stages of both dead and surviving shrimp determined. Only shrimp in intermolt and premolt stages were sampled for analysis of haemolymph. Haemolymph was assayed for osmoregulatory capacity (OC), magnesium ion (Mg2+), calcium ion (Ca2+), total proteins, oxyhaemocyanin, lactate, glucose and total haemocyte count (THC).Low mortalities were recorded for shrimp submitted independently to ammonia or hypoxia. Seventy five percent of dead shrimp were in early post molt (stage A) in ammonia treatment, while hypoxia affected mainly late premolt animals (stage D2). A synergic effect of ammonia and hypoxia combination (A+O2 treatment) on mortality was observed, affecting nearly exclusively shrimp in late premolt stage D2.Analysis of molt stage repartition at the end of the experiment suggests that ammonia treatment may have accelerated molting.The common physiological response of shrimp to the different treatments was characterized by a reduced OC and an increase in Ca2+. Increase in Mg2+ could not be validated by the statistical analysis, as well as glycaemia variations. Plasmatic lactate level increased and THC decreased in shrimp submitted to hypoxia and the combination of hypoxia and ammonia. Total proteins concentration was reduced in ammonia and A+O2 treatments. The effect was more pronounced in late premolt shrimp than in intermolt shrimp. Combination of ammonia and hypoxia led to a physiological response stronger than this observed for ammonia-alone and/or hypoxia-alone treatments, except for oxyhaemocyanin.The effects of each external factor (ammonia, hypoxia) and the combination of both, and internal one (molt stage) are discussed.

The effect of raising water temperature to 33 °C in Penaeus vannamei juveniles at different stages of infection with white spot syndrome virus (WSSV)

This study investigated the effect of high water temperature (33 °C) at different stages of infection with a highly virulent and low virulent white spot syndrome virus strain (WSSV Thai-1 and WSSV Viet) in Penaeus vannamei juveniles. Shrimp were inoculated intramuscularly with either a high dose (HD) or low dose (LD). Water temperature was kept either at continuously 27 °C or switched from 27 °C to 33 °C at 0, 12 or 24 h post inoculation (hpi) for both strains and in addition at 48 or 96 hpi for WSSV Viet. The increased temperature 33 °C was maintained till the end of the experiments (120–144 hpi with WSSV Thai-1 and 240 hpi with WSSV Viet). To determine the infection status at the moment of temperature increase, five shrimp that were kept continuously at 27 °C were euthanized at 0, 12, 24, 48 and 96 hpi with each dose of two strains. WSSV infections (viral antigen VP28) in dead and euthanized shrimp were demonstrated by indirect immunofluorescence.Shrimp inoculated with HD or LD of WSSV Thai-1 and kept continuously at 27 °C till euthanasia were 100% viral antigen positive from 12 (HD) or 24 hpi (LD). Shrimp inoculated with WSSV Viet were 100% positive from 24 (HD) and 48 hpi (LD). Shrimp kept at 27 °C, showed clinical signs from 24 (HD) or 24–36 hpi (LD) with both strains. Cumulative mortalities reached 100% with WSSV Thai-1 at 60 (HD) or 84–144 hpi (LD) and with WSSV Viet 100% at 216 hpi (HD) or 90% at 240 hpi (LD). Switch of temperature to 33 °C from 0, 12 or 24 hpi was effective in reducing mortality of shrimp inoculated with the LD of both strains and with the HD of WSSV Viet. The switch to 33 °C from 24 hpi with the Thai strain (HD) and from 48 and 96 hpi with the Viet strain (LD or HD) had no effect or even accelerated the mortality rate (80–100%). All shrimp were viral antigen positive at death and euthanasia (one shrimp LD WSSV Viet) when kept continuously at 27 °C. All dead and euthanized shrimp kept at 33 °C from 0 or 12 hpi were viral antigen negative. With 33 °C from 24, 48 or 96 hpi, all dead shrimp were viral antigen positive and euthanized shrimp were negative.This study showed that 33 °C is effective to prevent disease, reduce mortality and block WSSV replication, but only in the early stages of infection.

Effect of salinity on transmission of necrotizing hepatopancreatitis bacterium (NHPB) to Kona stock Litopenaeus vannamei

Elevated salinity and temperature have been observed prior to devastating necrotizing hepatopancreatitis (NHP) outbreaks in several geographically isolated shrimp ponds. These observations have led to the hypothesis that the NHP-bacterium (NHPB) is hindered by reduced salinity, even though the mechanism is not understood. The objective of this research was to examine the effect of salinity on transmission of NHPB. The transmission rate of NHPB was estimated through laboratory experiments whereby individuals of Kona stock Litopenaeus vannamei were orally exposed to a dead NHPB-infected shrimp. For each replicate, 12 susceptible shrimp were placed with a dead NHPB-infected shrimp in a 1 m2 bottom area cylindrical tank maintained at 30 degrees C for a period of 24 h. Four salinities of 10, 20, 30, and 40 per thousand were replicated 2 times in 2 trials, giving a total of 192 shrimp exposed per os to infective material. In each trial, a negative control group was included at each salinity, giving a total of 96 shrimp exposed orally to uninfected material. After the 24 h exposure period, susceptible shrimp were individually isolated at the same physical conditions for up to 60 d to determine NHPB transmission. The NHPB was transmissible regardless of salinity: nearly a quarter of susceptible shrimp exposed to NHPB at the lowest (10 per thousand) and highest (40 per thousand) salinity examined acquired NHPB. Transmission rates were highest at the intermediate salinities of 20 and 30 per thousand, suggesting that those salinities are optimal for NHPB transmission. The observed association between high salinity and NHP outbreak in a shrimp pond is not explained by these results because reduced transmission occurred at very low and very high salinities.

Impact of daily fluctuations of optimum (27 °C) and high water temperature (33 °C) on Penaeus vannamei juveniles infected with white spot syndrome virus (WSSV)

This study evaluated the effect of daily fluctuations between optimum (27 °C) and high water temperature (33 °C) on the clinical and virological outcome of a WSSV infection in Penaeus vannamei juveniles. Shrimp were inoculated intramuscularly with a dose of 10,000 SID 50 of either virulent WSSV Thai-1 or less virulent WSSV Viet or mock inoculated. Temperature was kept either continuously at 27 °C or switched from 27 °C to 33 °C at 12 h post inoculation (hpi) and maintained at that temperature for 6 h (6 h 33 °C), 12 h (12 h 33 °C), 18 h (18 h 33 °C) or 24 h (24 h 33 °C) per day. Temperature was then lowered and maintained at 27 °C for the remaining hours of the day. The experiments ran for 132 hpi with WSSV Thai-1 and 168–192 hpi with WSSV Viet. WSSV infections were demonstrated by indirect immunofluorescence (IIF). Shrimp kept continuously at 27 °C, started to show clinical signs at 24 hpi and mortalities started at 36 hpi with both strains. Cumulative mortalities reached 100% at 60 hpi with WSSV Thai-1 and at 168–192 hpi with WSSV Viet in two experiments. At constant 33 °C (24 h 33 °C), mortalities were 0% with WSSV Thai-1 and 5% or 10% with WSSV Viet. With exposure to 33 °C for 6 or 12 h per day (6 h 33 °C or 12 h 33 °C), cumulative mortalities reached 90–100% at 72–96 hpi with WSSV Thai-1. With WSSV Viet cumulative mortalities reached 100% at 84–96 hpi or 50–95% at 96–108 hpi with 33 °C for 6 h or 12 h per day. Mortalities were clearly reduced with WSSV Thai-1 (0–40%) and WSSV Viet (5–15%) at 33 °C for 18 h per day. At constant 27 °C and at 33 °C for 6 h or 12 h per day, dead shrimp were WSSV positive and euthanized shrimp were WSSV negative. At 33 °C for 18 h or 24 h per day, the dead and euthanized shrimp were WSSV negative except the dead shrimp at 18 h 33 °C with Thai-1 were WSSV positive. The present study showed that daily temperature fluctuations have negative (6 h 33 °C with WSSV Viet) or positive effects (6 h 33 °C with WSSV Thai-1 and 12 h or 18 h 33 °C with both strains) on disease and mortality of shrimp infected with WSSV.

Development of lateral-flow immunoassay for WSSV with polyclonal antibodies raised against recombinant VP (19+28) fusion protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using colloidal gold as an indicator. The fusion protein, VP (19+28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19+28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group) of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 were classified as negative. The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.