Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei
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Litopenaeus
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Hemolymph
genomic DNA
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Exposure to thermal stress was shown to have a significant effect on the osmotic pressure of the hemolymph, glucose levels, total count of hemocyte (TCH), and proPO activity in adult white shrimp Litopenaeus vannamei. Exposure of the shrimp to CTMax significantly increased the osmotic pressure of the hemolymph relative to the control group. In organisms reaching CTMax, temperature elicited a secondary stress response that included an increase in hemolymph glucose of 31 mg mL−1. Metabolites in hemolymph such as cholesterol, acylglycerides, and total protein were not significantly affected by exposure to CTMax. CTMax exposure affected several immunological parameters causing decreases in TCH and proPO activity. We suggested that biomarkers such as osmolality, glucose levels, TCH, and proPO activity could be used as sensitive predictors of exposure to CTMax in white shrimp.
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In order to investigate the effects of injecting phosphatidyl serine(PS)on hemocyanin synthesis and hemocyanin-derived phenol oxidase(Hd-PO)and probe into the pathway and activating mechanism of PS as a possible native elicitor for Hd-PO in white shrimp(Litopenaeus vannamei),we injected adult white shrimps different PS with 5,10,20 μg/mL,and assayed hemocyanin concentration in plasma,expression of hemocyanin subunits(p75 and p77)and mRNA in hepatopancreas,along with Hd-PO activity in plasma from white shrimp.They were investigated by spectrophotometry,native-PAGE,SDS-PAGE and RT-PCR in vivo with different sampling time at 0,6,12,24,36,48 and 60 h respectively,and we set the control group and parallel group.The results showed that hemocyanin concentration declined slightly during 6 h after injecting PS and afterwards changed as a peak by 36th h after injection with a maximum at 24th h(P0.05);expression of hemocyanin subunits and mRNA all had peak changes during 36 h after injecting PS,and likewise,all reaching their ceiling values at 12th h(P0.05);Hd-PO activity had a peak change within 36 h after injecting PS,with its climax at 12th h(P0.05).The research revealed that PS could activate hemocyanin to show phenol oxidase activity with significant time-dose effects,and meanwhile brought down hemocyanin concentration in a short time at first and subsequently started the hemocyanin synthesis,indicating that PS is the elicitor of Hd-PO in white shrimp,which lays the theoretical foundation for the study on immunological function of hemocyanin in shrimp.
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Abstract Crustacean phenoloxidase (PO) and hemocyanin (Hc) are classified as type 3 copper proteins. PO catalyzes the oxidation of mono- and di-phenol compounds, which is the rate-limiting step of melanization, while Hc generally functions as a dioxygen-transporting protein in the hemolymph of arthropods. To date, many studies have shown PO activity in Hc, which is inspired by their structural similarity. Here, the source of PO activity in crustaceans was re-examined by purifying Hc and PO exclusively from the hemolymph of kuruma prawn. The conventional procedure for the preparation of arthropod Hc, which includes precipitation of Hc by ultracentrifugation and subsequent purification by size exclusion chromatography, was not able to completely remove hemolymph-type PO from Hc. In contrast, fractionation with 50% saturation of ammonium sulfate and subsequent hydrophobic chromatography yielded sufficiently pure Hc, which contained no detectable PO protein and virtually no PO enzymatic activity. These results indicate that the main source of PO activity in the hemolymph of kuruma prawn is hemolymph-type PO and that the improved purification method of Hc is preferable for evaluating the PO activity of Hc.
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Abstract The SDS‐PAGE of Squilla mantis hemolymph reveals 16 major protein bands. Three of them, showing a molecular weight (MW) around 125 kdal, are female specific proteins (FSP) to be considered as vitellogenins because their amount increases with ovarian maturity. The MW of the hemocyanin subunit is confirmed around 70 kdal. Low MW proteins appear or intensify at molt. Hemolymph incubation with 35S‐Methionine and subsequent autoradiography of SDS‐PAGE slabs show that hemocyanin and vitellogenins are not synthesized by the circulating hemocytes, while some proteins connected with molt are heavily labeled by the tracer.
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Hemocyanin
Hepatopancreas
Prophenoloxidase
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Carcinus maenas
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