Differential expression of immune-related genes and transposable elements in black tiger shrimp (Penaeus monodon) exposed to a range of environmental stressors.

The health of aquatic species is dependent on interactions between the environment, pathogens and the host. Under intensive shrimp aquaculture, environmental conditions can degrade, causing significant stress to the cultured organisms. To investigate the effect of environmental stress on shrimp hemocyte gene expression profiles, we applied suppression subtractive hybridization (SSH) in juvenile Penaeus monodon exposed to hyperthermal, hypoxic or hyposmotic conditions. Random sequencing of 258 clones from the SSH revealed 176 distinct sequences of which 58 shared high similarity to sequences in the public databases. The three most common groups of identifiable unique sequences in the SSH libraries were the POL region of non-LTR retrotransposons (31%), genes with immune or potential immune functions (30%), and genes involved in protein synthesis and processing (18%). Stress-regulated differential expression was further verified by quantitative qRT-PCR, with seven out of eight randomly selected genes showing qRT-PCR profiles that conformed to the patterns predicted by SSH. Hence this work provides a list of genes which appear to be up- or down-regulated in response to stress, providing a basis for studying the genetic response of shrimp to environmental stress.

Stress-induced gene expression profiling in the black tiger shrimp Penaeus monodon.

Cultured shrimp are continuously exposed to variable environmental conditions that have been associated with stress and subsequent outbreaks of disease. To investigate the effect of environmental stress on Penaeus monodon gene expression, a 3,853 random cDNA microarray chip was generated with clones originating from six stress-enriched hemocyte libraries generated by suppression subtractive hybridization and a normal hemocyte cDNA library. Changes in temporal gene expression were analyzed from shrimp exposed to hypoxic, hyperthermic, and hypoosmotic conditions; 3.1% of the cDNAs were differentially expressed in response to at least one of the environmental stressors, and 72% of the differentially expressed clones had no significant sequence similarity to previously known genes. Among those genes with high identity to known sequences, the most common functional groups were immune-related genes and non-long terminal repeat retrotransposons. 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. Hypoxic and hyperthermic stressors induced the most severe short-term response in terms of gene regulation, and the osmotic stress had the least variation in expression profiles relative to the control. These expression data agree with observed differences in shrimp physical appearance and behavior following exposure to stress conditions.

Esterified astaxanthin levels in lobster epithelia correlate with shell colour intensity: potential role in crustacean shell colour formation.

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton.

The gusBC genes of Escherichia coli encode a glucuronide transport system.

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon.

Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 x 10(9) and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon.