Abstract
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.
Highlights
Crustaceans, like other ecdysozoans, experience repeated molting processes during their life cycle, as they complete their growth, sexual maturation, and reproduction [1,2]
Three types of hemocytes are found in the hemolymph of C. sapidus (n = 6 crabs): granulocytes, semigranulocytes and hyaline cells (S4A and S4B Fig)
This study provides evidence that PPO present in hemocytes is an essential component in the shell-hardness of C sapidus
Summary
Crustaceans, like other ecdysozoans, experience repeated molting processes during their life cycle, as they complete their growth, sexual maturation, and reproduction [1,2]. Successful molting is achieved through the coordinated actions of multiple hormones and neurohormones [3,4,5,6,7]. The cuticle undergoes dramatic structural and morpohological changes during the molt cycle, from being soft to becoming hard. The hardening process of the crustacean new cuticle differs from that of insects and involves two sequential processes: sclerotization and mineralization [8,9,10,11,12]. The sclerotization occurs in the new soft and pliable cuticle immediately after ecdysis [13,14,15]. Mineralization, as the PLOS ONE | DOI:10.1371/journal.pone.0136916. Mineralization, as the PLOS ONE | DOI:10.1371/journal.pone.0136916 September 22, 2015
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