Penaeus Monodon Prawns Research Articles

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

Mourilyan virus (MoV) is a newly identified virus of Penaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a approximately 0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (approximately 85 nm diameter) to ovoid MoV particles (approximately 85 x 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi.

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in ∼10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.

The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses.

Summary. We report here a 5596 nt sequence comprising the 3′-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3′-poly(A) tail of the 26235 nt genome of GAV. The putative 1640 amino acid (aa) ORF3 protein (MW = 182049 Da, pI = 6.62) contains 15 potential N-linked glycosylation sites, 15 potential O-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (TM) domains. Three of the predicted TM domains occur in the amino-terminal 228 aa, two in the central portion, and one near the carboxy-terminus of ORF3. Only one short (83 aa) open reading frame (ORF4) was identified between ORF3 and the 3′-poly(A) tail. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids.

Gill-associated nidovirus of Penaeus monodon prawns transcribes 3'-coterminal subgenomic mRNAs that do not possess 5'-leader sequences.

Sequence analysis of the approximately 20 kb 5'-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a-1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining approximately 6.2 kb of the GAV genome downstream of ORF1a-1b to a 3'-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3'-coterminal subgenomic (sg) mRNAs of approximately 6 kb and approximately 5.5 kb. Primer extension and 5'-RACE analyses showed that the sgmRNAs initiate at the same 5'-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5'-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26.2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5'-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.

Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.

A 20089 nucleotide (nt) sequence was determined for the 5' end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a approximately 22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU 'slippery' sequence associated with -1 ribosomal frameshifting. A 131 nt stem-loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and 'H-type' pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the 'SDD' polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a-1 frameshift suggests that GAV represents the first invertebrate member of the Order NIDOVIRALES:

Primary cell cultures isolated from Penaeus monodon prawns.

We have devised a cell culture system for Penaeus monodon prawn cells that uses a defined synthetic medium. Organs were removed from adult prawns ranging in size from 13--19cm rostrum-to-telson length. Cultures consisted of either a blend of hematopoietic and lymphoid cells or ovarian cells. The cells divide rapidly in culture, doubling on average once per week for 5 to 6 weeks. These cultures continue to survive for at least 5 months but the rates of cell division are low after the first 5--6 weeks. In the literature, unicellular eukaryotic marine organisms such as chytrids may contaminate marine cell cultures. In some cases these eukaryotic contaminants may be difficult to distinguish from prawn cells unless detailed ultrastructure or characteristic developmental stages such as zoospores can be observed. Alternatively, we prepared molecular probes from repeated DNA sequences 100--400 bp in length in the P. monodon genome. These species-specific probes were hybridised to genomic DNA from cell culture to confirm proliferation of P. monodon cells in our cultures.

The Effects of Different Types of Stress on Blood Glucose in the Giant Tiger Prawn Penaeus monodon

Abstract— Juvenile Penaeus monodon prawns were exposed to a variety of putative stressom and blood glucose concentrations monitored as an index of metabolic stress. Prawns were held under controlled conditions and food deprived for 12–18 h before blood sampling. Juvenile prawns maintained in tanks at densities of 9, 23, 36 and 50 prawns/m2 for 4 wk did not have significantly different baseline levels of blood glucose, with mean concentrations varying from 1.1 to 1.3 mmol/L. In contrast, as an index of overall condition, antennal length and rostral spine length were significantly degraded in the 36 and 50 prawns/m2 groups compared to the 9 and 23 prawns/m2 groups (P < 0.05). Blood glucose levels were significantly elevated after prawns were exposed to air for 10 min and also increased Significantly when dissolved oxygen was decreased from 6.5 mg/L to 2.1 mg/L. (P < 0.05). Gassing of water from pH 8.5 to 5.9 with carbon dioxide, with a corresponding decrease of dissolved oxygen from 6.6 to 5.9 mg/ L, induced a rapid and significant rise in blood glucose levels from 1.1 to 2.3 mmol/L. In contrast, a decrease of water pH from 8.3 to 5.9 with sulfuric acid, did not result in a significant change in blood glucose concentrations. These findings are discussed in relation to pond management for maintaining prawns in a low stress environment.

The Effects of Prophylactic Formalin Bath Treatment on Blood Glucose in the Giant Tiger PrawnPenaeus monodon

Abstract—JuvenilePenaeus monodonprawns were bathed in seawater‐formaldehyde concentrations of 312, 625, and 1,250 ppm for up to 6 h. The tests were conducted at concentrations in excess of those previously published, in order to examine maximum tolerance levels to formaldehyde. Physiological stress was monitored by measurement of blood glucose levels. Under controlled laboratory conditions, 12 h food deprived juvenile prawns exposed to formaldehyde concentrations of 312 ppm did not exhibit signs of stress. However, bathing in formaldehyde concentrations of 625 and 1,250 ppm, caused significant increases (P< 0.05) in blood glucose levels after 2 and 4 h, respectively. There were no mortalities in any treatment group. In contrast, juvenile prawns from grow‐out ponds bathed in 625 ppm of formaldehyde immediately after 2.5 h of transport did not exhibit significant increases in blood glucose. This may be due to their nutritional status and these results are discussed. No significant change in blood glucose levels was observed in bilaterally eyestalk ablated prawns after exposure to 1,250 ppm formaldehyde. The increase of blood glucose due to formaldehyde treatment is presumably mediated by a stress response, which includes crustacean hyperglycemic hormone (CHH), and not a result of non‐specific cell lysis and death.

Estimates for the heritability of size in juvenile Penaeus monodon prawns from half-sib matings

Eighteen half-sib groups of the giant tiger prawn, Penaeus monodon, were obtained using artificial insemination of two females by each male. The total length (mm) and wet weight (mg) of the young were measured at 6 and 10 weeks after hatching. Sire component estimates were approximately 0.10 for both total length and wet weight at each age. Dam components were greater suggesting large non-additive genetic effects and/or common environment effects that could not be differentiated on the available data. A reduction in the dam component for both total length and wet weight from approximately 0.5–0.6 at 6 weeks to 0.3–0.4 at 10 weeks suggested significant maternal effects were present. The results emphasised the strong environmental controls on prawn growth, particularly potential maternal effects that are likely to play a role earlier in development. These influences resulted in highly variable data, emphasising the need for higher degrees of replication of sire and dam groups in order to detect significant sire effects in P. monodon.