Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P < 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2 mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3 mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3 mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.     

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1–27) and middle region (residues 35–95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.     

Protection of Procambarus clarkii against white spot syndrome virus using recombinant oral vaccine expressed in Pichia pastoris

The potential for oral vaccination of crayfish against white spot syndrome virus was investigated. The envelope proteins VP19 and VP28 were expressed in yeast (Pichia pastoris). The expressed proteins were used as oral vaccines in different forms viz., in whole culture form, whole culture sonicated form, whole culture centrifuged supernatant form, and cell residue form. The recombinant proteins were mixed with food pellets and fed to crayfish for 25 days. The vaccinated groups were divided into two even groups and challenged on the 3rd and 21st day of post vaccination. Among different vaccine groups the relative percent survival (RPS) values of sonicated form and supernatant form vaccines were found the best and met the criterion (>RPS 60%) of effective vaccine even after 21st day of post vaccination. Development of vaccine by using recombinant proteins VP19 and VP28 in yeast as expression vector was feasible with significant effects.     

Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii

The effect of hyperthermia on the development of white spot syndrome virus (WSSV) in the crayfish Procambarus clarkii was studied by competitive PCR. Crayfish were exposed to different temperatures (24 +/- 1 and 32 +/- 1 degrees C) after WSSV injection. No mortality was observed when crayfish were held at 32 +/- 1 degrees C, but mortality reached 100% when crayfish were transferred to 24 +/- 1 degrees C. Competitive PCR showed that viral levels at 32 +/- 1 degrees C remained at 10(5) copies mg(-1) tissue, while at 24 +/- 1 degrees C levels were significantly higher, rising from 10(4) to 10(10) copies mg(-1) tissue. These results suggest that hyperthermia reduces viral replication, but does not eliminate viral particles from WSSV-infected crayfish.     

A rapid assay for simultaneous detection of Spiroplasma eriocheiris and white spot syndrome virus in Procambarus clarkii by multiplex PCR

Aims: To establish a multiplex PCR method for simultaneous and rapid detection of Spiroplasma eriocheiris and white spot syndrome virus (WSSV) in Procambarus clarkii with recommendations for application to other crustacea. Methods and Results: Three primer sets were mixed at a ratio of 1 : 3 : 1 to amplify specific fragments of the S. eriocheiris, WSSV, P. clarkii crayfish (control organism) genomes, respectively. S. eriocheiris and WSSV were used to challenge the susceptible crustacea in the experimental groups. Total DNA of the samples was purified and detected by multiplex PCR. The PCR‐amplified products produced four groups of results as follows. One fragment of 1195 bp, amplified by the primer set ITS‐crayfish/28S‐crayfish, served as an internal control, showed no pathogen detection, thus confirming the specificity of our positive tests. Two groups represented by: (i) samples challenged by S. eriocheiris alone, or (ii) challenged by WSSV alone, yielded two fragments each; i.e. those from S. eriocheiris (271 bp) plus the internal control and those from WSSV (530 bp) plus the internal control. Finally, for the fourth group, in cases of double challenged treatments, all three amplified products were detected simultaneously. Conclusions: Simultaneous and rapid detection of two pathogens in P. clarkii is important to maintain productive and healthy crayfish in aquaculture. The direct detection of S. eriocheiris and WSSV from P. clarkii is practicable with multiplex PCR. Significance and Impact of the Study: This study shows that the two pathogens are simultaneously and rapidly detected in P. clarkii by multiplex PCR, thus increasing the efficiency of pathogen detection.     

Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims: To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results: The polymerase chain reaction (PCR)‐amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro‐transformation, time‐courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7‐h immersion at different time points after vaccination. Subgroups including 20 inter‐moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28‐bv and rVP28‐bs was 51·7% and 78·3%, respectively. Conclusions: The recombinant B. subtilis strain with the ability of high‐level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study: Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV.     

Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus.

The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.     

家蚕蛹表达的重组Vp28疫苗对克氏原螯虾的抗病毒保护效应

The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.     

Histological organization of the intestine in the crayfish Procambarus clarkii

Six longitudinal ridges span the length of the intestine in the crayfish Procambarus clarkii. A simple columnar epithelium with tetralaminar cuticle lines the lumen. Folds of the epithelium overlie a dense irregular connective tissue packed with mixed acinar (alveolar) glands. Mucous secretions are probably involved with formation and lubrication of faecal strings; neither the nature nor the role of the serous secretions is immediately apparent. Aggregations of cells with large cytoplasmic vacuoles, called bladder cells, appear in the subepithelial connective tissue near the tops of the intestinal ridges. The bladder cells are suitably positioned to bolster the integrity of the ridges. Striated muscle of the intestine occurs in inner longitudinal and outer circular layers. The inner longitudinal layer consists of six strips, with one strip associated with the base of each intestinal ridge. The outer circular layer is essentially complete, but there are periodic apertures in this layer on the left and right sides of the intestine, providing nerves and haemolymph vessels with access to the interior of the gut. Based on histological features, and consistent with reports on other crayfish, we conclude that the intestine of P. clarkii has a proctodeal (ectodermal) origin.     

Acclimatable cardiac and ventilatory responses to copper in the freshwater crayfish Procambarus clarkii

Mortality and physiological tests following exposure to waterborne copper were performed in the red swamp crayfish Procambarus clarkii from a central Italian population. Mortality tests gave an estimated 96 h LC50 value (with 95% confidence limits) of 162 (132-211) mg L(-1) waterborne copper II. Variations in cardiac and ventilatory rates were simultaneously monitored using a non-invasive plethysmographic technique. In experiments with different sub-lethal copper concentrations (control, 0.5, 1 and 10 mg L(-1)) performed at different times (3, 6, 96 h), copper exposure elicited a reduction in both heart and scaphognatite rates. Following exposure to 10 mg L(-1) copper for 96 h, the heart and scaphognatite rates decreased to about 35% of the initial values. The reduction was fully reversible, since crayfish exposed to 0.5, 1 and 10 mg Cu L(-1) for 96 h resumed control rates after a 3-h residence in clean water. In crayfish pre-exposed (96 h) to sub-lethal copper concentrations (0.1 and 1 mg L(-1)) and then held in control water (3 h), the reduction of heart and scaphognatite rates after exposure to 10 mg Cu L(-1) were significantly lower than in specimens pre-exposed to control water. Therefore, copper induces a concentration and time dependent reduction of both cardiac and ventilatory activity in P. clarkii; these responses can be reduced or fully abolished by pre-exposure to sub-lethal levels of the metal.     

Morphology of descending statocyst interneurons in the crayfish Procambarus clarkii Girard

Summary The morphological features of descending interneurons that responded to the artificial bending of statolith hairs were assessed with intracellular recording and staining techniques. Seven statocyst interneurons were identified on the basis of their structure and response characteristics and designated as interneurons S1 to S7. All seven identified interneurons project to the optic lobe, where the optic nerve also projects, and to the dorsal part of the tritocerebrum, where the eyestalk motoneurons originate. All except interneuron S6 also extend their major branches to other neuropilar regions. S2 projects to the dorsal part of the deutocerebrum, where the statocyst nerve terminates, and S3 to the dorsal part of deutocerebrum and the antennal lobe. Four other interneurons (S1, S4, S5, S7) also extend their branches to the parolfactory lobe to which the statocyst nerve projects as well as to the deutocerebrum and antennal lobe. The extensive dendritic projections of S1–S7 suggest that they are complex multimodal interneurons rather than simple relay interneurons, receiving at least visual and statocyst sensory information. The function of the antennal lobe branches, however, has yet to be determined since the functional role of antennal input in equilibrium control is unknown.     

Hydrodynamic orientation of crayfish (Procambarus clarkii) to swimming fish prey

Reversibly blindfolded crayfish (Procambarus clarkii) react to small swimming fish (Astyanax fasciatus mexicanus) approaching or passing nearby with antennal and cheliped movements and body turns (Fig. 3). We studied the accuracy and dynamics of crayfish orientation responses to the previously analyzed hydrodynamic disturbances caused by the fish, mostly produced by tail flicks.Antennal and cheliped movements started slightly before the onset of turning responses (Fig. 4). Antennal sweeps were performed most rapidly. 50% of the appendage sweeps resulted in contacts with the fish (Fig. 5).Most turns were directed toward the stimulus (Fig. 6). Response amplitudes increased with increasing stimulus angle. Turns were accurate for small stimulus angles, but smaller than expected for larger ones. Sweeps of ipsilateral antennae and chelipeds were generally directed backwards, while those of contralateral appendages were smaller and directed forwards. The amplitudes of appendage sweeps first increased with increasing stimulus angle and then decreased again for more caudal stimulus directions. Lateral stimuli (60°–120°) from opposite sides were usually significantly distinguished. The amplitudes of the different elements of orientation behaviour were highly correlated with each other, indicating that they were directed by the same sensory input.     

Susceptibility of the Australian freshwater crayfish Cherax destructor albidus to white spot syndrome virus (WSSV)

Cherax destructor occurs naturally and/or is farmed in all Australian mainland states and territories and is of major cultural, economical and conservation significance. The aim of this study was to determine susceptibility of the commercially important subspecies C. destructor albidus to white spot syndrome virus (WSSV), a hazard to crustaceans and currently considered to be exotic to Australia. In challenge tests by intramuscular injection, C. destructor albidus displayed a similar level of susceptibility to white spot disease (WSD) as Penaeus monodon (i.e. 100% mortality in 3 d). In one oral challenge test where C. destructor albidus was subjected to significant temperature stress, over 50% died of severe WSD within 14 d post challenge. All dead and moribund crayfish displayed histopathological lesions typical for WSD and gave positive results for WSSV in DNA dot blot hybridization tests. Survivors to 30 d (n = 3) showed no lesions and gave negative dot blot test results. In a second oral challenge test without temperature stress, mortality was delayed but reached 75% by 30 d. However, no typical WSD lesions were observed in the dead, dying or surviving crayfish and dot blot test results were negative. The results suggested that C. destructor albidus would be less susceptible than P. monodon to WSSV exposure via natural routes of infection in farms and in the wild. This information may be useful for disease import risk analysis for WSSV.     

Micro-scale fluid and odorant transport to antennules of the crayfish, Procambarus clarkii

A numerical model was developed to determine advective-diffusive transport of odorant molecules to olfactory appendages of the crayfish, Procambarus clarkii. We tested the extent of molecule transport to the surfaces of aesthetasc sensilla during an antennule flick and the degree of odorant exchange during subsequent flicks. During the rapid downstroke of a flick, odorant molecules are advected between adjacent aesthetascs, while during the slower return stroke, these odorants are trapped between the sensilla and molecular diffusion occurs over a sufficient time period to transport odorants to aesthetasc surfaces. During subsequent flicks, up to 97.6?% of these odorants are replaced with new odorant molecules. The concentration of molecules captured along aesthetasc surfaces was found to increase with increased gap spacing between aesthetascs, flick speed, and distance from the proximal end of the aesthetasc, but these changes in morphology and flicking kinematics reduce the animal's ability to take discrete samples of the odorant-laden fluid environment with each flick. Results suggest that antennule flicking allows discrete sampling of the time- and space-varying odorant signal, and high concentration odorant filaments can be distinguished from more diffuse, low concentration filaments through changes in both the timing and the encounter rate of odorant molecules to aesthetasc surfaces.     

Discovery of four natural clones in a crayfish species Procambarus clarkii

Self-cloning is quite rare in shrimp, lobsters, crayfish and crabs. Here we report the discovery of four natural clones of red swamp crayfish (Procambarus clarkii), each containing 2-6 genetically identical individuals, during the genotyping of 120 individuals with five microsatellites. The four clones were heterozygote at most of the five microsatellite loci. Phylogenetic analysis using microsatellite genotypes suggests recent origin of the four clones. Sequencing a part of the mitochondrial gene Cox I confirmed that the four clones were from the species Procambarus clarkii.     

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

Three transketolase genes have been identified in the human genome to date: transketolase (TKT), transketolase-like 1 (TKTL1) and transketolase-like 2 (TKTL2). Altered TKT functionality is strongly implicated in the development of diabetes and various cancers, thus offering possible therapeutic utility. It will be of great value to know whether TKTL1 and TKTL2 are, similarly, potential therapeutic targets. However, it remains unclear whether TKTL1 and TKTL2 are functional transketolases. Homology modelling of TKTL1 and TKTL2 using TKT as template, revealed that both TKTL1 and TKTL2 could assume a folded structure like TKT. TKTL1/2 presented a cleft of suitable dimensions between the homodimer surfaces that could accommodate the co-factor-substrate. An appropriate cavity and a hydrophobic nodule were also present in TKTL1/2, into which the diphosphate group fitted, and that was implicated in aminopyrimidine and thiazole ring binding in TKT, respectively. The presence of several identical residues at structurally equivalent positions in TKTL1/2 and TKT identified a network of interactions between the protein and co-factor-substrate, suggesting the functional fidelity of TKTL1/2 as transketolases. Our data support the hypothesis that TKTL1 and TKTL2 are functional transketolases and represent novel therapeutic targets for diabetes and cancer.     

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8–10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.