A Proteomic Study of Hemocyte Proteins from Mud Crab (Scylla paramamosain) Infected with White Spot Syndrome Virus or Vibrio alginolyticus

Abstract

In this study, we investigated the hemocytes’ immune response to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection at the protein level. The differential proteomes from crab hemocytes infected with WSSV or V. alginolyticus were analyzed using the isobaric tags for relative and absolute quantitation approach immediately after infection. Using this approach, we identified 1,799 proteins by their by LC–MS/MS spectra and sequencing data. These included 157 upregulated proteins and 164 downregulated proteins after WSSV infection. Similarly, 243 proteins were determined to be differentially expressed during V. alginolyticus infection, of these, 121 were upregulated and 122 were downregulated after infection. Interestingly, among these differentially expressed proteins, 106 were up- or downregulated significantly in both WSSV and V. alginolyticus infection. Six genes, β-actin, myosin-9, anti-lipopolysaccharide factor isoform 4, anti-lipopolysaccharide factor 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were chosen for further study. The expression of these genes all showed a trend of upregulation at 24 h post-WSSV or V. alginolyticus infection except for myosin-9 in response to WSSV. To confirm the protective effects of the six genes, crabs were injected with specific dsRNAs before WSSV or V. alginolyticus challenge. The results showed that the knockdown of these genes led to an increase in the morbidity and mortality (P < 0.01) rate, and a decrease in infection time in WSSV-infected crabs. During the first 84 h, knockdown of these genes also led to an increase in the morbidity rates in V. alginolyticus -infected crabs, and results of four genes showed a higher mortality rate than that of the control after they were knocked down. This is the first report of the proteome response in crab hemocytes during WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crabs.

Evaluation of Antimicrobial Activities of Seaweed Resources from Zhejiang Coast, China

“…Crabs weighing approximately 300 g were reared in a 50-L tank equipped with an aerated pump at room temperature, and were randomized into 20 groups (n = 16 per group). The crabs were infected with the white spot syndrome virus (WSSV), as described previously [40][41][42]. Both healthy and WSSV-challenged crabs were injected with 1 mg/kg, 5 mg/kg, and 10 mg/kg seaweed extracts dissolved in phosphate buffer solution (PBS) (test groups).…”

“…As previously described [40,41], 100 µL of hemolymph was withdrawn into a 1 mL of sterile syringe containing 0.9 mL of anticoagulant solution (trisodium citrate 30 mM, sodium chloride 0.34 M, ethylenediaminetetraacetic acid 10 mM, pH 7.55), placed on a hemocytometer and THC was performed.…”

“…Prophenoloxidase activity was measured spectrometrically, as previously described [40,41]. The hemolymph was briefly centrifuged at 800× g at 4 • C for 20 min to separate hemocytes and serum.…”

“…SOD activity was measured using nitro blue tetrazolium (NBT) chloride in the presence of riboflavin, as described previously [40,41]. Briefly, 100 mL of hemolymph was homogenized with 0.5 mL of phosphate buffer (50 mM, pH 7.8), and the homogenate was centrifuged at 5724× g for 5 min at 4 • C. The supernatant was heated at 65 • C for 5 min, centrifuged again, and the supernatants were stored at −20 • C for later use.…”

UBXN1 interacts with the S1 protein of transmissible gastroenteritis coronavirus and plays a role in viral replication

“…Calcium-binding protein 2-like regulates its own biological activities via a reversible interaction of its specific protein-binding sites with Ca 2+ [26]. Ca 2+ is a common intracellular signal carrier that regulates many cellular processes from cell birth, differentiation, and reproduction to apoptosis [27].…”