The health of aquatic species is dependent on interactions between the environment, pathogens and the host organism. In Australia, most cultured and wild caught Penaeus monodon are chronically infected with gill-associated virus (GAV). This subclinical GAV infection is restricted to the lymphoid organ and causes no major impact on prawn production. Theacute infection state involves other tissue types and has been associated with morbidity and mortalities. At present the physiological mechanisms responsible for this transition from a chronic to acute infection remain poorly understood but appear to be triggered by environmental stress.
This study explores the interaction between host, pathogen and environment by applying a series of molecular techniques to analyse the effect of exposing P. monodon chronically infected with GAV, to environmental and anthropogenic stressors. A real-time quantitative RTPCR (qRT-PCR) assay for GAV was developed to measure viral load in infected prawns. This was applied to track GAV load in chronically infected prawns injected with either a viral inoculum or a placebo (0.9% NaCI).which were then bled at successive time points. A rapid increase of viral load in both treatments revealed that repetitive bleeding and handling stress alone can stimulate GAV proliferation in chronically infected P.monodon.
To further understand the effect of environmental stress on the prawn immune system and GAV replication and onset of disease, I have assessed juvenile prawns that were exposed to a short-term hypoxic, hyperthermic and osmotic stress twice over a 1 -week period by measuring their total hemocyte counts (THC), phenoloxidase activity (PO). Respiratory burst, levels of heat shock protein (HSP) 70 expression and GAV load. No significant differences were detected in survival and THC between stressed and control prawns (P>0.05). Significant differences were observed for PO activity, HSP 70 expression and GAV load (P<0.05). PO activity increased, while respiratory burst was reduced in hypoxic stressed prawns (P<0.05). HSP 70 levels were higher in the hyperthermic-treated prawns compared to other treatments (P<0.05). GAV load increased throughout the 21-day experiment for all groups and treatments except for hyperthermic stress, revealing a reduction of GAV replication rate following a short-term increase in water temperature.
To further assess in detail the impact of these stressors on the health and physiology of prawns, six hemocyte cDNA libraries were constructed using a suppression subtractive hybridisation (SSH) technique. These were enriched for genes differentially expressed in prawns exposed to three different environmental stressors. Random sequencing of 263 clones from the SSH libraries revealed low redundancy and high treatment specificity. Significant database matches occurred in 37% of the genes. The most common differentially expressed gene was the POL region of non-long terminal repeat (non-L TR) retrotransposons, followed by immune related genes and those involved in protein synthesis and processing. Quantitative RT-PCR confirmed most genes in these subtractive libraries (seven out of nine genes tested), were differentially expressed.
Extending these analyses further, I constructed a 4000 clone cDNA microarray (consisting of random cDNAs from the six subtracted libraries, one unsubtracted hemocyte cDNA library and PCR products from various prawn viruses and hybridisation controls) and used it to analyse temporal changes of gene expression profiles in a second group of prawns exposed to the same types of environmental stress. 3.1% of the cDNAs in the microarray were differentially expressed (DE) in response to at least one of the environmental stressors. Hierarchical clustering revealed a set of cDNAs with temporal and stress-specific gene expression profiles as well as a set of cDNAs indicating a common stress response between stressors. 70% of the DE clones had no significant sequence similarity to previously known genes, and a further 1 1 % were similar to genes in the public databases with unknown function. The most common functional groups among the DE clones were immune related genes and non-L TR retrotransposons.
Random sequencing of the SSH libraries identified 5 cDNA clones with significant sequence similarity (E value < 1 0-5) to 4 different open reading frames (ORF) of White Spot Syndrome Virus (WSSV), a pathogen considered exotic in Australia. A negative result with the OlE WSSV nested PCR diagnostic test revealed that these prawns were not infected with any of the known strains of WSSV. Sequencing of the flanking regions from one of these clones (ED72) revealed a 1 kb DNA fragment with 54% amino acid identity match and E value of 1 "63 to ORF167 of WSSV. PCR analysis using several DNA samples from various prawn species suggested that ED72 is universally present in Australian prawns. qPCR of ED72 in muscle samples from various prawns revealed that there is no significant differences in template concentration between individuals, although there is a higher template concentration in ovaries than in muscleas determined by qPCR and in situ hybridisation. This preliminary data suggests that these WSSV-like sequences are ancestral viral sequences integrated into the prawn genome and could originate from a previous infection with a WSSV related virus.