New host range for Hematodinium in southern Australia and novel tools for sensitive detection of parasitic dinoflagellates

Abstract

Hematodinium is a parasitic dinoflagellate and emerging pathogen of crustaceans. It preferably manifests in haemolymph of marine decapod crustaceans, killing a large variety of genera with significant impacts on fisheries worldwide. There is, however, evidence that some crustacean stocks harbor high prevalence, low intensity infections that may not result in widespread host mortality and are therefore hard to detect. The most widely used methods for detection of Hematodinium are conventional blood smears and polymerase chain reaction (PCR) against ribosomal RNAs. Blood smears demand a trained investigator, are labor intensive and not readily scalable for high-throughput sampling. PCRs only detect parasite DNA and can also suffer from false negatives and positives. In order to develop alternative detection tools for Hematodinium cells in decapod crustaceans we employed an immunological approach against a newly identified, abundant dinoflagellate-specific nuclear protein--Dinoflagellate/Viral NucleoProtein (DVNP). Both immunofluorescence assay (IFA) and Western blot methods against DVNP showed high sensitivity of detection. The Western blot detects Hematodinium parasites to levels of 25 parasites per milliliter of crustacean haemolymph, with the potential for sample pooling and screening of large samples. Using both PCR and these new tools, we have identified Hematodinium cells present in three new host crab taxa, at high prevalence but with no sign of pathogenesis. This extends the known range of Hematodinium to southern Australia.

Figure 1. Diff-quick^®-stained haemolymph smears of O. australiensis from Port Philip Bay.

(A) Aggregation of Hematodinium sp. cells displaying a Hematodinium-typical nuclear appearance (black arrow) (B) O. australiensis haemocyte. Note the different appearance of the chromatin in comparison to the parasite cells. Scale-bar: 15 µm.

Figure 2. DVNP IFAs of haemolymph parasite mixes.

The DNA is shown in red and anti-DVNP is shown in green. (A) Haemolymph smear of artificially infected sand crab O. australiensis showing two DVNP-positive cells (white arrowsheads) within a large number of haemocytes. (B) Haemolymph smear of artificially infected spider crabs L. gaimardii showing one DVNP-positive Hematodinium cell amongst haemocytes.

Figure 3. DVNP IFAs of ethanol-fixed haemolymph samples obtained from heavily infected blue swimmer crabs Calinectes sapidus from Chesapeake Bay (USA).

DNA is shown in red and anti-DVNP is shown in green. (A1) IFA (left) and brightfield overlay (right) of a DVNP-negative C. sapidus hyalinocyte blood cell. (A2) IFA (left) and brightfield overlay (right) of a DVNP-negative C. sapidus granulocyte blood cell. (A3) IFA (left) and brightfield overlay (right) of a DVNP-positive Hematodinium cell. (B1) Hematodinium cells are strongly labeled around the edge of their nuclei while the DNA stain concentrates in the center of the nuclei. (B2) DVNP-labeling is absent in C. sapidus cells (red nuclei) and prominent in Hematodinium cells (green-yellow nuclei).

Figure 4. DVNP IFAs of fixed haemolymph from PCR-positive L. gaimardii from Port Phillip Bay, Australia.

(A) Brightfield image (left) and IFA (right) of a DVNP-negative L. gaimardii haemocytes. (B) Brightfield image (left) and IFA (right) of a DVNP-positive Hematodinium cell. (C) IFA image of one DVNP-positive Hematodinium cell (white arrowhead) amongst L. gaimardii haemocytes.

Figure 5. Western blot detection of DVNP in haemolymph-parasite mixes after target enrichment using acid extraction.

Samples containing 800, 500, 200, 100, 50, 25, 12.5, 5, 3 and 0 parasites ml^-1 of haemolymph.