A parvo-like virus in cultured redclaw crayfish Cherax quadricarinatus from Queensland,Australia

In the summer of 1999/2000, an epizootic occurred in cultured juvenile redclaw crayfish Cherax quadricarinatus on one commercial crayfish farm in northern Queensland, Australia. Mortalities occurred over 4 wk, with up to 96% cumulative mortalities in 2 earthen ponds stocked with juveniles. The crayfish were weak, anorexic and lethargic. A transmission trial was conducted, using filtered, cell-free extract prepared from infected crayfish as inoculum. The disease was reproduced, with on-going mortalities occurring in inoculated crayfish over 55 d. Experimentally inoculated crayfish showed gross signs of malaise, anorexia and disorientation before dying. Two types of intranuclear inclusion bodies (INIBs) were seen in tissues of endodermal, ectodermal and mesodermal origin by light microscopy with haematoxylin and eosin (H&E) stained sections. 'Early'-stage INIBs were eosinophilic, rounded and located centrally within slightly enlarged nuclei while 'late'-stage INIBs were well-rounded and deeply basophilic. The gills, cuticular epithelium and epithelial cells of the foregut, midgut and hindgut were the most heavily infected tissues. By transmission electron microscopy, virions with an average diameter of 19.5 nm were seen within electron-dense granular inclusion bodies within enlarged nuclei of both naturally and experimentally infected crayfish. The size of the virions and cytopathology are consistent with characteristics of viruses in the Family Parvoviridae. This is the first reported case of mass mortality caused by a parvo-like virus infection in C. quadricarinatus.     

Predation on invasive redclaw crayfish Cherax quadricarinatus by native fishes in the Kafue River,Zambia

The stomach contents of eight species of predatory or omnivorous fish caught in gillnets in the Kafue River in May and June 2010 were examined to determine the relative importance, expressed as ‘prominence value’ (PV), of crayfish Cherax quadricarinatus in their diet. Four species, Clarias gariepinus, Schilbe intermedius, Serranochromis robustus and Serranochromis angusticeps, had fed on C. quadricarinatus. The PV of C. quadricarinatus was highest in C. gariepinus, in which the quantity and size of crayfish eaten was significantly correlated with standard length (SL) of fish >200 mm SL. Predated C. quadricarinatus were significantly smaller (mean carapace length [CL] 30 mm; SE 1.2) than those caught in gillnets (mean CL 76 mm; SE 0.9). No C. quadricarinatus remains were found in the stomachs of Oreochromis andersonii, Synodontis macrostigma, Serranochromis macrocephalus or Mormyrus lacerda.     

Ultrastructure and cytopathology of a rickettsia-like organism causing systemic infection in the redclaw crayfish, Cherax quadricarinatus (Crustacea: decapoda), in Ecuador

A study of the ultrastructural characteristics of an intracellular bacterium infecting the redclaw crayfish, Cherax quadricarinatus, a pathogen referred to previously as a rickettsia-like organism (RLO), revealed the presence of different developmental stages. These included a rod-shaped and uniformly electron-dense elementary body (EB) and an intermediate body (IB). The length of the EB varied between 0.48 and 0.6 microm, and the diameter was 0.3 microm. The IB was 0.75 to 1.1 microm long by 0.36 to 0.44 microm in diameter. Although the EB of this bacterium has ultrastructural characteristics similar to those of Rickettsiella, no information is available regarding its genetic relationship to this genus, and the intracellular bacterium should continue to be referred to as a rickettsia-like organism. The hemocytes had different levels of infection, and the RLO proliferated inside these cells. The EB appeared to be free in the cytoplasm of infected hemocytes and other cells; however, this might be a fixation artifact. The EB was also contained in membrane-bound vacuoles along with the IB. RLO colonies were observed inside small granular cells. No large granular cells were observed in the sections examined; therefore, no data were obtained regarding infection of this type of hemocyte. The fixed phagocytes on the external side of the terminal hepatic arterioles had an activated interrupted layer containing RLO bacteria. Stem cells in the hematopoietic tissue were also infected, and some cells were apparently being released into circulation.     

Description of a presumptive hepatopancreatic reovirus, and a putative gill parvovirus, in the freshwater crayfish Cherax quadricarinatus

The redclaw freshwater crayfish Cherax quadricarinatus has a reputation for being hardy and resistant to handling stress. However, in recent years, possibly since 1996, C. quadricarinatus farmers in northern Queensland have noted a decrease in stress resistance in their stock. A presumptive reovirus in the hepatopancreas, and a putative parvovirus in the gills, were associated with chronic mortalities in C. quadricarinatus at one northern Queensland farm. Hypertrophic nuclei with marginated chromatin were observed in gill epithelium in moribund crayfish which had recently been relocated to a laboratory from the holding tank facility on the farm. Affected nuclei appeared to be vacant or contained a faint granular basophilia in H&E stained sections. However, toluidine blue staining revealed a homogeneously granular appearance of the nuclei. Transmission electron microscopy revealed approximately 20 nm diameter virus-like particles within the nucleus. Eosinophilic, Feulgen-negative, cytoplasmic inclusions were observed in distal hepatopancreatocytes in 1 moribund C. quadricarinatus collected from the same on-farm holding tank approximately 6 mo later. This crayfish did not display the gill lesions. Transmission electron microscopy showed that the inclusions contained icosahedral virus particles 35 to 40 nm in diameter. The histopathology and preliminary virus morphology of the presumptive hepatopancreatic reovirus, and the histopathology, ultrastructural pathology and preliminary virus morphology of the putative gill parvovirus, are reported herein.     

Temnocephalan symbionts of the freshwater crayfish Cherax quadricarinatus from northern Australia

Four species of turbellarian temnocephalan symbionts (Platyhelminthes: Temnocephalida) are reported for the first time from the external surfaces of Cherax quadricarinatus, a freshwater crayfish from northern Australia. Three of these species — Temnocephala rouxii Merton, 1913, Notodactylus handschini (Baer, 1945), and Diceratocephala boschmai Baer, 1953 — are known previously from related crayfish in New Guinea. The newly discovered fourth species, Decadidymus gulosus n. gen., n. sp., has an unusual combination of characters linking it with both the Temnocephalidae and the Scutariellidae. Together the four species possess an array of characters that challenges current concepts of families in the order. D. boschmai has an almost completely ciliated epidermis, a feature otherwise unknown in the order.     

The life history and biology of Diceratocephala boschmai (Platyhelminthes; Temnocephalida), an ectosymbiont on the redclaw crayfish Cherax quadricarinatus

Adults of the temnocephalan Diceratocephala boschmai, an ectocommensal on the crayfish Cherax quadricarinatus, deposited eggs on the host carapace. After 15 to 20 days at 28 °C, the eggs hatched into ciliated miniature adults with undeveloped reproductive organs, that remained attached to the host alongside the eggs. They grew and became gravid within 53 to 70 days if they were not dislodged. Most oviposited on the host on which they had hatched. Although larval temnocephalans have been described from in vitro work, this is the first temnocephalan life history to be described in vivo.Juvenile worms frequently changed their position on the carapace. Adult worms were sedentary just prior to, and during oviposition, and were frequently found at the base of the large chelae, the ventral surface of the antennae and antennules, and at the base of the walking legs. The ventral margins of the abdomen, the mouthparts, and the interorbital-rostral area were secondarily inhabited.It was calculated using an indirect regression that adult Diceratocephala boschmai survived on average 91 days when removed from the host but they did not produce eggs in vitro. On the host, adult worms lived an average of 48 days after the commencement of oviposition.Abbreviations C cilia - E eyespot - Es ejaculatory sac - Ex excretory canal - G gut - Gl glands - I intertentacular flange - M muscular peduncle - O ovary - P penis - Pg pharyngeal glands - Ph pharynx - R rhabdites - S seminal vesicle - T tentacles - Te testes - U uterus - Vd vas deferens - Vit vitelline glands     

High-density lipoprotein associated with secondary vitellogenesis in the hemolymph of the crayfish Cherax quadricarinatus

The high-density lipoproteins LPI and LPII were isolated from the hemolymph of the crayfish Cherax quadricarinatus by gradient ultracentrifugation and high-performance liquid chromatography (HPLC). Both lipoproteins contained a carotenoid moiety. LPI is comprised of a single polypeptide with an approximate molecular mass of 96 kDa. LPII was composed of two similar native components, LPIIa and LPIIb, both having polypeptides of 80 and 177 kDa. Both under natural conditions and after endocrine manipulations, LPI was present in males and in females, regardless of the female reproductive stage. LPII was present only in secondary-vitellogenic females, but not during the winter reproductive arrest period. LPII was also absent from young females that had received androgenic gland implants. LPII also appeared in the hemolymph of intersex individuals from which the androgenic gland had been removed. It is therefore suggested that LPII serves as a marker indicating the onset of secondary vitellogenesis in C. quad'iariicarintus females.     

Methods to enhance the intensity of intranuclear bacilliform virus infection in Cherax quadricarinatus

Many studies have examined the morphology, pathology and epizootiology of the intranuclear bacilliform virus (IBV) of Cherax quadricarinatus, but little research has been conducted to acquire specific knowledge of the virus. This is partly due to difficulties in detecting the virus and in obtaining sufficient material for viral isolation and purification. As quantified by light microscopy, we significantly (p < 0.01) enhanced IBV intensities from 10.56 to 16.67% in C. quadricarinatus by using salinity stress (12 ppt) and ingestion of infected hepatopancreatic tissue, which increased intensities from 4.33 to 10.77%. It was also found that phosphotungstic acid-eosin stain was superior to standard haematoxylin and eosin stain in visualizing IBV inclusion bodies. It is expected that these new techniques will enhance the detectability of the virus and provide sufficient viral material for viral purification, characterization and development of molecular tools for detection and phylogenetic analysis.     

A scanning electron microscope study of Craspedella sp. from the branchial chamber of redclaw crayfish,Cherax quadricarinatus,from Queensland,Australia

Epidermal topography was examined, including papillate ridges, grooves and ciliated sensory papillae of Craspedella sp. from the branchial chamber of redclaw crayfish, Cherax quadricarinatus, from Queensland, Australia. Rhandites were observed to discharge from ducts opening mainly in a small distal region of the ventral epidermis of the three central (of five) tentacles. These regions, devoid of ciliated sensory papillae, serve to adhere the anterior end of the worms during locomotion. Secretions from glands associated with the posterior attachment organ were observed to discharge from pores on the outside region of the ventral surface of the disc.A comparison of various scanning electron microscopy (SEM) fixation techniques showed that (1) hot fixatives at 90 °C provide most information on the largest number of epidermal structures and (2) different fixation regimes highlight different epidermal features.     

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus

The RPCH and β-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C. quadricarinatus RPCH and β-actin mRNAs, as cDNAs, were quantified every 3 h by RT-PCR. Single cosinor analysis supports the notion of β-actin and RPCH mRNA circadian behavior in animals subjected to 12 h:12 h light/dark regimes. In constant darkness RPCH mRNA concentration changes to ultradian cycles.     

Patterns of molecular diversity in wild stocks of the redclaw crayfish (Cherax quadricarinatus) from northern Australia and Papua New Guinea: impacts of Plio-Pleistocene landscape evolution

1. Analysis of mitochondrial and nuclear DNA (microsatellites) in 379 individuals, collected from 15 localities in northern Australia and Papua New Guinea (PNG), demonstrated that wild redclaw crayfish ( Cherax quadricarinatus ) populations consist of two highly divergent Australian lineages and two PNG lineages.
2. The disjunction between the two Australian lineages occurs over a distance of approximately 200 km in the south-western corner of the Gulf of Carpenteria. These data conflict with an earlier study that detected no significant differentiation in 23 variable allozyme loci in redclaw sampled from northern Australia, but concur broadly with the previous recognition of two morphologically distinct species ( C. quadricarinatus and C. bicarinatus ) across northern Australia, and a third species in PNG ( C. albertsii ).
3. The inferred timing and patterns of divergence evident in the molecular data presented here closely align with a similar pattern reported in a co-distributed freshwater decapod crustacean, and broadly reflect patterns in some vertebrate taxa with similar distributions across northern Australia and PNG.
4. These congruent patterns most probably reflect periodic Plio-Pleistocene land and freshwater connections between Australia and New Guinea.     

Molecular cloning and expression analysis of chymotrypsin-like serine protease from the redclaw crayfish (Cherax quadricarinatus): A possible role in the junior and adult innate immune systems

A novel chymotrypsin-like serine protease (CLSP) was isolated from the hepatopancreas of the redclaw crayfish Cherax quadricarinatus (Cq-chy). The full-length cDNA of Cq-chy contains 951 nucleotides encodes a peptide of 270 amino acids. The mature peptide comprising 223 amino acids contains the conserved catalytic triad (H, D, and S). Similarity analysis showed that Cq-chy shares high identity with chymotrypsins from the fiddler crab; Uca pugilator. Cq-chy mRNA expression in C. quadricarinatus was shown to be: (a) tissue-related with the highest expression in the hepatotpancreas and widely distributed, (b) highly responsive in the hepatopancreas to White Spot Syndrome Virus (WSSV) challenge, and (c) differently regulated in immature and adult crayfish. In this study we successfully isolated Cq-chy. Our observations indicate that Cq-chy is differently involved in the immature and adult innate immune reactions, thus suggesting a role for CLSPs in the invertebrate innate immune system.     

Invasive Australian crayfish Cherax quadricarinatus in the Sanyati Basin of Lake Kariba: a preliminary survey

The invasion of Cherax quadricarinatus, the Australian redclaw crayfish, in the Sanyati Basin of Lake Kariba, Zimbabwe, is reported. A total of 79 crayfish were caught at 10 out of 12 sampling sites in the Sanyati Basin in November–December 2012. The average catch per unit effort (CPUE) varied from 1.1 to 4 crayfish per trap per night, carapace length ranged from 29 to 93.5 mm, and weight ranged from 4 to 196.2 g. Most crayfish were between 50 and 59.9 mm carapace length. Males (average 82.6 g) were significantly heavier than females (37.2 g) and males were larger in carapace length, carapace width, chela length and chela width. A feral population of C. quadricarinatus is now established in the Sanyati Basin. Possible modes of dispersal within the basin are discussed.     

Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus

In this study, we explored the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish, Cherax quadricarinatus, by investigating activities of enzymes related to innate immune function during infection. After 6-12 h of exposure to WSSV, the activities of four enzymes, phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD) and lysozyme (LSZ), increased in the gills of C. quadricarinatus but then sharply decreased during longer infection times. Except for PO, the activities of other enzymes in the WSSV-infected crayfish (Group II) were significantly lower than those of the controls at 72 h post-exposure (P < 0.01). Interestingly, the enzyme activities in the group treated with polysaccharides before challenge with WSSV (Group III) were higher than those in Group II. This phenomenon demonstrated that the polysaccharides could improve the immuno-enzyme activities and enhance the organism's antiviral defenses. Morphological examination by transmission electron microscopy revealed abundant WSSV particles and significant damage in the gills of infected crayfish. WSSV infection caused parts of the gill epithelium and microvilli to be reduced in number and size or damaged; meanwhile, the mitochondria morphology changed, with parts of the cristae diminished leaving large vacuoles. Moreover, electron dense deposits appeared and heterochromatinized nuclei could be seen in blood cells with ruptured nuclear membranes and outflow of nucleoplasm. The findings of this study furthers our understanding of the biochemical alterations induced by viral infections, including changes in the antioxidant status, oxidative stress and lysozyme activity, which could help to advance strategies for control of WSSV in crayfish.     

Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus)

Vitellins from ovaries and eggs at different stages of development in freshwater crayfish (Cherax quadricarinatus) were examined by chromatography, PAGE and SDS-PAGE. With these methods, two forms of vitellin (Vt1 and Vt2) were observed in ovaries and eggs (stages I and V). In ovaries in secondary vitellogenesis, native molecular mass was 470 (Vt1) and 440 (Vt2) kDa. The electrophoretic pattern of the eggs proved to be more complex. The protein molecular mass depend on the development stage of the egg: stage I, 650 kDa (Vt1) and 440 kDa (Vt2); stage V, 390 kDa (Vt1) and 340 kDa (Vt2). The identified vitellins appear to be lipo-glycocarotenoprotein. A similar vitellin polypeptide composition was observed in the two forms of vitellin from ovaries and eggs in stage V. In ovaries the SDS-PAGE analysis showed four subunits with molecular weights of approximately 180, 120, 95 and 80 kDa (Vt1 and Vt2). The polypeptide composition in the two forms of vitellins in stage I and stage III eggs were different at 195, 190, 130 and 110 kDa (Vt1) and 116 and 107 kDa (Vt2). On the other hand, in stage V eggs, 110, 95, 87 and 75 kDa (Vt1 and Vt2) were identified. Two antibodies (Ab1 and Ab2) were prepared against the purified proteins of stage V eggs and their specificity was demonstrated by radial immunoprecipitation, and Western blotting analysis. Two forms of vitellins were also found in stage V eggs after chromatography on Sepharose CL-2B column and hydroxylapatite and polyacrylamide gel electrophoresis.     

OIE white spot syndrome virus PCR gives false-positive results in Cherax quadricarinatus

White spot syndrome virus (WSSV) is an intranuclear bacilliform virus (IBV) that is a serious, notifiable crustacean pathogen. The Office International des Epizooties (OIE) PCR protocol for WSSV uses primer sets initially developed by Lo et al. (1996). It yields a first-step PCR amplicon of 1441 bp and a nested PCR amplicon of 941 bp. An amplicon (941 bp) purported to specifically detect WSSV was obtained when using template DNA extracted from Cherax quadricarinatus in a WSSV PCR detection protocol recommended by the OIE. Sequencing and analysis of the 941 bp amplicon and an occasional 550 bp amplicon from C. quadricarinatus revealed no phylogenetic relationship with WSSV, and suggested a possible lack of sufficient primer specificity for WSSV in the OIE test. This suggestion was supported by the fact that the OIE outer primer sequence (146F1) was present in both the forward and reverse position of the 941 bp and the forward position of the 550 bp nested amplicons from C. quadricarinatus. As WSSV is a notifiable pathogen, the consequences of false-positive results are harsh in WSSV-free zones and can lead to incorrect quarantine and unnecessary destruction of animals. Therefore, urgent attention and revision is necessary for the current OIE PCR protocol for WSSV detection.