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Seasonal changes in water temperature directly affect the aquatic ecosystem. The blue crab, Callinectes sapidus, inhabiting the Chesapeake Bay has been adapted to seasonal changes of the environmental conditions. In this, the animals halt their physiological process of the growth and reproduction during colder months while they resume these processes as water temperatures increase. We aimed to understand the effect of the elevated temperatures on a disease progression of reo-like virus (CsRLV) and innate immunity of adult female C. sapidus. Following a rise in water temperature from 10 to 23 °C, CsRLV levels in infected crabs rose significantly in hemocytes and multiple organs. However, in hemocytes, the elevated temperature had no effect on the levels of three innate immune genes: Cas-ecCuZnSOD-2, CasPPO and CasLpR three carbohydrate metabolic genes: CasTPS, CasGlyP; and CasTreh and the total hemocyte counts (THC). Interestingly, the hemocytes of CsRLV infected animals exposed to 23 °C for 10 days had significantly elevated levels of Cas-ecCuZnSOD-2 and CasTPS, compared to those of the uninfected ones also exposed to the same condition and compared to hatchery-raised females kept at 23 °C. Despite the lack of changes in THC, the types of hemocytes from the animals with high CsRLV levels differed from those of uninfected ones and from hatchery animals kept at 23 °C: CsRLV-infected crabs had hemocytes of smaller size with less cytosolic complexity than uninfected crabs. It therefore appears that the change in temperature influences rapid replication of CsRLV in all internal tissues examined. This implies that CsRLV may have broad tissue tropism. Interestingly, the digestive tract (mid- and hindgut) contains significantly higher levels of CsRLV than hemocytes while hepatopancreas and ovary have lower levels than hemocytes. Innate immune responses differ by tissue: midgut and hepatopancreas with upregulated Cas-ecCuZnSOD-2 similar to that found in hemocytes. By contrast, hepatopancreas showed a down-regulated CasTPS, suggesting carbohydrate stress during infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process., (Copyright © 2013 Elsevier Ltd. All rights reserved.)