Table 1.
Properties of the different hemocyte types of C. sapidus and other decapod crustaceans.
Table 2.
List of primers used for dsRNA template amplification and qPCR assay.
Fig 1.
Effects of multiple injections of CasPPO-hemo-dsRNA on shell-hardness.
Intermolt crabs (n = 7/ group) were injected with 10 μg of CasPPO-hemo-dsRNA, control dsRNA, or saline every other day until ecdysis. (A) Levels of CasPPO-hemo transcripts, 24 hrs after ecdysis, (bars) in hemocytes (n = 7) and levels of CasAK transcirpts (line) in the same cDNA samples that are assayed as a reference gene. Different letters denotes the significant differences at P < 0.05. (B) CasHLS-PO activity at postmolt (24 hrs after ecdysis, n = 7). (C) The western blot analysis (c’) identifies the presence of CasPPO-hemo in CasHLS in the samples. n.d. = no statistical difference.
Fig 2.
Shell-hardness of the new cuticle during 48 hrs after ecdysis.
Intermolt crabs (n = 7) were injected with 10 μg of CasPPO-hemo-dsRNA every other day until ecdysis and the hardness of the postmolt cuticle was measured by a hand-held durometer (durometer scale = 0–100 DU). Dashed line = CasPPO-hemo-dsRNA injected group (R2 = 0.93), solid line = control saline (R2 = 0.84), and dashed-dot line = control dsRNA (R2 = 0.94). ANCOVA analysis of general linear models (Y = a + bX) shows a significant difference (P < 0.05, noted with different letters) in the hardening of the cuticle of PPO-dsRNA injected and control crabs (n = 7). The data are shown as mean ± SE DU.
Fig 3.
Levels of CasPPO-hemo in hemocytes after multiple injections of CasPPO-hemo-dsRNA.
(A) Fluorescent intensity analysis, (B) ICC (left to right = control saline, control dsRNA, CasPPO-hemo dsRNA), and (C) flow cytometry analysis of hemocytes from CasPPO-hemo-dsRNA and control groups. The positive α-CasPPO-hemo immnunopositve staining of the cells obtained at 24 hrs after ecydsis was determined for fluorescent intensity, which was calculated using ImageJ. The insertion in the ICC pictures shows a positive hyaline cell (a) and granulocyte (a’ and a”). The flow cytometry indicates no changes in the cytosolic complexity and granularity of hemocytes in the CasPPO-hemo-dsRNA injected group. The values of cytosolic complexity (SSC-H) are shown as mean ± SE (n = 4–5). Different letters indicate significant differences at P < 0.05: B and C) Two way ANOVA. n.d. = no significant differences. Bar sacle = 10 μm. G = granulocytes; SG = semigranulocytes; H = hyaline cells.
Fig 4.
Differential hemocyte counting (DHC) during the molt cycle of C. sapidus using the flow cytometry analysis.
The values of THC (solid line) do not significantly differ among the different molt stages (n = 18). Each bar represent a hemocyte type. n.d. = no difference; the ‘*’ and ‘**’ denote significant difference at P < 0.05 and P < 0.01 (two way ANOVA), respectively. G = granulocytes; SG = semigranulocytes; H = hyaline cells.
Table 3.
Changes in different cell types in hemocyte populations of the hemolymph of C. sapidus during the molt cycle.
Fig 5.
Identification of CasPPO-hemo in hemocytes during the molt cycle of C. sapidus.
Immunostaining of hemocytes at different molt stages using a specific α-CasPPO-hemo. Granulocytes contain PPO during all the molt stages, while semigranulocytes and hyaline cells show a PPO protein expression which is molt-stage dependent. Signals: + PPO and–PPO. (A) ICC (n = 5–6) and (B) fluorescent intensity of the positive signal of α-CasPPO-hemo staining using ImageJ. (C) Immunostaining of CasPPO-hemo by flow cytometry analysis: At intermolt stage, ~14% of hemocytes are PPO positive, while at postmolt, the PPO positive hemocytes are over 90% (n = 5–8). Bar scale = 10 μm. G = granulocytes; SG = semigranulocytes; H = hyaline cells.
Fig 6.
A proposed model for the involvement of hemocytes and its PPO (CasPPO-hemo) in the shell-hardening process of the blue crab, C. sapidus.
The diagram summarizes the results obtained from the current study. Changes in hemocyte numbers are shown with bar plots. The positive (+) and negative (-) symbols indicate the presence or absence of PPO expression in each type of hemocytes, respectively. Blue arrows indicate the potential transport of PPO from hemocytes into the cuticle, as described [38]. Red arrows indicate the specific hemocytes and PPO role in the immunity system [40,41]. Asterisks denote that the studies were carried out by other researchers.