Structure and expression of a shrimp prohormone convertase 2.

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P<0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.

Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis.

Scattered reports of viral inserts in shrimp and insect genomes led to the hypothesis that random, autonomous insertion of such sequences occurs in these organisms and leads to specific, heritable immunity. To test the prediction regarding random insertion of viral sequences into the shrimp genome, we examined the giant tiger shrimp for random genomic insertions of Penaeus stylirostris densovirus (also called IHHNV). By PCR analysis using a set of 7 overlapping primer pairs to cover the whole IHHNV genome (4 kb), PCR failure with some pairs indicated sequence gaps that revealed a random pattern of putative viral inserts in the genomes of individual shrimp. Targeting a putative insert from one arbitrarily selected specimen, we used genome walking to reveal a viral insert linked to a host microsattelite-like fragment. This differed from 2 previously reported inserted fragments of IHHNV in P. monodon. In one specimen, 2 slightly different inserts were revealed, probably on paired chromosomes. By design and use of chimeric shrimp/virus primer pairs we proved that similar insertions occurred in several shrimp specimens, including those infected with IHHNV but showing no signs of disease. For the infected specimens, the inserts gave false positive PCR test results using 309F/R primers and a new IQ2000 test protocol currently recommended for detection of infectious IHHNV. This is the first experimental support for the hypothesis-based prediction that a random number and length of sequence fragments from a single virus genome may occur in the shrimp genome. Since some inserts can give false positive results for infectious IHHNV with the recommended methods above, they may have a negative effect on international seafood trade. In addition, discard of domesticated shrimp breeding stocks based on such false positive results might have negative consequences, if such inserts are related to shrimp viral disease tolerance, as also hypothesized.