Snail mucus from the mantle and foot of two land snails, Lissachatina fulica and Hemiplecta distincta, exhibits different protein profile and biological activity.

OBJECTIVE: Snails secrete different types of mucus that serve several functions, and are increasingly being exploited for medical and cosmetic applications. In this study, we explored the protein pattern and compared the biological properties of the mucus secreted from the mantle collar and foot of two snail species, Lissachatina fulica and Hemiplecta distincta. RESULT: Protein profile showed a different pattern between the two species and between the two secretory parts. The mantle-specific protein bands were further characterized and among them was an antibacterial protein, achacin. Accordingly, the mucus from the mantle exhibited the higher antibacterial activity than that from the foot in both snail species. The mucus from H. distincta, first reported here, also showed antibacterial properties, but with a lower activity compared to that for L. fulica. Snail mucus also exhibited anti-tyrosinase activity and antioxidant activity but with no significant difference between the foot and mantle mucus. These results indicate some different protein compositions and biological activities of snail slime from the mantle and foot, which might be associated with their specific functions in the animal and are useful for medical applications.

Penaeus vannamei serine proteinase inhibitor 7 (LvSerpin7) acts as an immune brake by regulating the proPO system in AHPND-affected shrimp.

Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in aqua cultured shrimp that is caused by virulent strains of Vibrio parahaemolyticus (VP). Our previous study used transcriptomics to identify key pathogenic factors in the stomach of AHPND-infected shrimp (Litopenaeus vannamei), and here we used a different subset of the same data to construct a gene-to-gene expression correlation network to identify immune-responsive genes. LvSerpin7 was found to have the highest number of correlations after infection, and it also showed a significant increase in mRNA expression. LvSerpin7 is expressed in all tissues but its expression levels are highest in hemocytes. After successfully silencing LvSerpin7 transcript prior to AHPND challenge, mortality was significantly increased relative to the controls and reached 100% within 36 h post infection. Compared to the controls, the phenoloxidase (PO) activity also increased in both hemolymph and stomach. Recombinant LvSerpin7 inhibited shrimp PO activity in vitro, and we also found that rLvSerpin7 inhibited the growth of AHPND-causing bacteria. These results suggest that LvSerpin7 might reduce the toxic effects that result from unregulated activation of the PO defense system by AHPND-causing bacteria.

The gene structure and hypervariability of the complete Penaeus monodon Dscam gene.

Using two advanced sequencing approaches, Illumina and PacBio, we derive the entire Dscam gene from an M2 assembly of the complete Penaeus monodon genome. The P. monodon Dscam (PmDscam) gene is ~266 kbp, with a total of 44 exons, 5 of which are subject to alternative splicing. PmDscam has a conserved architectural structure consisting of an extracellular region with hypervariable Ig domains, a transmembrane domain, and a cytoplasmic tail. We show that, contrary to a previous report, there are in fact 26, 81 and 26 alternative exons in N-terminal Ig2, N-terminal Ig3 and the entirety of Ig7, respectively. We also identified two alternatively spliced exons in the cytoplasmic tail, with transmembrane domains in exon variants 32.1 and 32.2, and stop codons in exon variants 44.1 and 44.2. This means that alternative splicing is involved in the selection of the stop codon. There are also 7 non-constitutive cytoplasmic tail exons that can either be included or skipped. Alternative splicing and the non-constitutive exons together produce more than 21 million isoform combinations from one PmDscam locus in the P. monodon gene. A public-facing database that allows BLAST searches of all 175 exons in the PmDscam gene has been established at http://pmdscam.dbbs.ncku.edu.tw/ .

Selective expression of a "correct cloud" of Dscam in crayfish survivors after second exposure to the same pathogen.

Arthropod hypervariable Dscam (Down syndrome cell adhesion molecule) may be involved in adaptive-like immune characteristics, namely immune priming, enabling the host to "learn" and "remember" pathogens previously encountered in arthropods. However, expression of Dscam in immune-primed arthropods after a second challenge has apparently not been confirmed. Herein, working with Dscam of Australian freshwater crayfish (Cherax quadricarinatus, i.e. CqDscam), we further investigated whether immune priming is mediated by "clouds" of appropriate (or "correct") CqDscam isoforms. In crayfish that survived a first WSSV challenge (immune priming), long-lasting CqDscam expression remained higher after a second WSSV challenge. Selective CqDscam isoforms were also induced after both challenges. Based on pathogen binding assays, these WSSV-induced CqDscam isoforms had a higher WSSV binding ability, perhaps mainly mediated by Ig3-spliced variants. We therefore hypothesized that in these crayfish survivors, an unknown selection process was generating a "correct cloud" of CqDscam against a previously encountered pathogen.

Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection.

Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus.

Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PlHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.

Role of Penaeus monodon hemocyte homeostasis associated protein (PmHHAP) in regulation of caspase-mediated apoptosis.

The viral responsive protein, PmHHAP, plays an important role in the control of hemocyte homeostasis in shrimps during viral infection. In this study, we further investigate the role of PmHHAP in the regulation of hemocyte apoptosis. RNA interference (RNAi) mediated gene silencing was used to suppress the PmHHAP expression and the change in hemocyte apoptosis was determined in the knockdown shrimp. Within circulating hemocytes, PmHHAP knockdown increased the number of annexin V-positive apoptotic cells and the combined caspase-3/-7 activity and induced the characteristic apoptotic DNA ladder. Furthermore, PmHHAP down-regulation was accompanied by significantly altered expression of apoptosis-related proteins including the effector caspases, PmCaspase and PmCasp. Yeast two-hybrid and co-immunoprecipitation assays showed that PmHHAP binds to the p20 domain of PmCasp. Moreover, the recombinant PmHHAP protein was able to reduce the caspase activity in the actinomycin D-treated hemocyte cells and rPmCasp-treated hemocyte cells. Taken together, our data indicate that PmHHAP regulates hemocyte homeostasis by inhibits apoptotic cell death through caspase activation.