Differential immune responses of the green neon shrimp (Neocaridina denticulate) to dipropyl phthalate.

This study set out to understand the sublethal effect of xenobiotic phthalate esters (PAEs) on the relationship between the susceptibility of shrimp to bacterial infection and the immune response of the shrimp. Neocaridina denticulate were exposed to different concentrations of the PAE dipropyl phthalate (DPrP), and mortality and six immune parameters were measured on days 1, 3, 5, and 10 after exposure. On days 1 and 3 after exposure, shrimp exposed to 0, 1, 5, 10, and 50 mg/L of DPrP and challenged with Aeromonas veronii experienced 14% and 16%, 16% and 16%, 18% and 18%, 34% and 24%, and 38% and 26% mortality, respectively. On day 1, five immune parameters (acid phosphatase, AcP; ß-glucuronidase, ß-Glu; phenoloxidase, PO; superoxide dismutase, SOD; and haemocyanin mRNA) were significantly altered in the all of the groups treated with DPrP compared to the untreated shrimp and were elevated in the 10 mg/L- and 50 mg/L-treated groups. Beta-Glu activity and haemocyanin mRNA levels were significantly increased in a dose-dependent manner. These results suggest that the increased susceptibility of N. denticulate exposed to DPrP is short-term and may be related to the increased expression of DPrP-induced immune mediators.

A long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis.

Hemocytes play important roles in crustacean immune responses. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines have been demonstrated to regulate hematopoiesis and immune responses. In invertebrates, however, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine molecule from hemocytic cDNA of tiger shrimp (Penaeus monodon) which was 1509 bp in length with a 5'-UTR of 143 bp, a coding region of 375 bp and a 3'-UTR of 991 bp. The present clone (GenBank accession no. EU980444) showed to be a longer form of astakine transcript with an extra insert of 671 bp in the 3'-UTR than the NCBI-recorded shrimp astakine cDNA sequence (GenBank accession no. AY787657). The deduced protein had 124 amino acid residues, including a signal peptide and one prokineticin domain. The calculated molecular weight (MW) of the mature peptide was 11,295 Da and pI was 5.2. Phylogenetically, this molecule is most similar to astakine-related molecules of arthropod including tiger shrimp astakine, crayfish astakines 1, 2a and 2b, aphid astakine-like molecule and parasitic wasp astakine-like molecule. Nested RT-PCR showed that astakine mRNA is expressed in many tissues and organs of the shrimp such as eyestalk, subcuticular epithelium, gills, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR further revealed that astakine mRNA is expressed mainly in the hemocytes. The astakine transcript is not inducible in the hemocytes until 24 h post LPS injection of shrimp. The recombinant protein of shrimp astakine (rPmAst) was synthesized using insect cell-baculovirus expression system. The authenticity of rPmAst protein was examined by MALDI-MS/MS spectrometry. Using ESI-MS it was determined that the MW of C-terminally histidine-tagged recombinant protein is 12,107 Da. It is 10 Da less than the computer-predicted MW (12,117 Da), allowing the formation of five pairs of disulfide bonds. Using BrdU incorporation assay it was demonstrated that the injection of rPmAst to the shrimp promoted cell proliferation in hematopoietic tissues. Therefore, we conclude that shrimp astakine functions as a cytokine that influences cell proliferation in the hematopoietic tissues.