Caiwen Li

In vitro culture and developmental cycle of the parasitic dinoflagellate Hematodinium sp. from the blue crab Callinectes sapidus.

Hematodinium is a genus of parasitic dinoflagellates whose species have caused significant mortalities in marine crustacean fisheries worldwide. A species of Hematodinium infects the blue crab, Callinectes sapidus on the eastern seaboard of the USA. The mode of transmission of the parasite in blue crabs is unknown. We established several continuous in vitro cultures of Hematodinium sp. isolated from the haemolymph of infected blue crabs. One isolate has been continuously maintained in our laboratory through serial subcultivation for over 12 months, and is capable of infecting new hosts when inoculated into healthy crabs. Cells of the parasite undergo characteristic developmental changes in vitro consistent with the identifiable stages of Hematodinium sp.: filamentous trophonts, amoeboid trophonts, arachnoid trophonts and sporonts, sporoblasts, prespores and dinospores (macrospores and microspores). Additionally, we describe an unusual shunt in the life cycle wherein presumptive schizonts derived from arachnoid sporonts developed into filamentous and arachnoid trophonts that can then initiate arachnoid sporonts in new cultures. This may explain the rapid proliferation of the parasite in blue crab hosts. We also found that temperature and light intensity affected the growth and development of the parasite in vitro.

Pathology and hematology of the Caribbean spiny lobster experimentally infected with Panulirus argus virus 1 (PaV1).

We examined the histopathological and hematological response of the Caribbean spiny lobster to experimentally induced infections with Panulirus argus Virus 1 (PaV1). The fixed phagocytes in the hepatopancreas were the primary sites of PaV1 infection in spiny lobsters. Fixed phagocytes were activated in early infections. However, as the disease progressed, the fixed phagocytes became infected and eventually lysed. Infected cells were subsequently observed in the hepatopancreas, gill, heart, hindgut, glial cells around the ventral nerves, and in the cuticular epidermis and foregut. In advanced infections, spongy connective tissues were heavily infected, as were glial cells around the optic nerves. The structure of the hepatopancreas was significantly altered as the disease progressed. The hemal sinuses among the hepatopancreatic tubules filled with massive amounts of cellular aggregates, including infected circulating hemocytes and spongy connective tissues. Atrophy of the hepatopancreatic tubules occurred in the late stage of viral infection. The virus caused significant decreases in total hemocyte counts and significantly altered several constituents in the hemolymph lysates of diseased lobsters, including: glucose, phosphorus, and triglycerides.

Effect of salinity on the immune responses and outbreak of white spot syndrome in the shrimp Marsupenaeus japonicus

Abstract In order to understand the role of salinity in outbreaks of white spot syndrome (WSS) in the kuruma shrimp, Marsupenaeus japonicus, the effects of salinity variation on THC (total haemocyte count), phenoloxidase (PO) activities and outbreaks of white spot syndrome were studied. The results showed that salinity variation (increasing or decreasing salinity) could cause the changes of THC and PO activities of M. japonicus. Salinity was also an important environmental factor affecting the immune responses in M. japonicus. The further from the original salinity that M. japonicus were mantained, the weaker were the immune responses of shrimps. M. japonicus were more susceptible to WSSV under salinity stress due to changes in their immune responses.

The Effect of Salinity On Experimental Infections of A Hematodinium SP. In Blue Crabs, Callinectes sapidus

Abstract: The parasitic dinoflagellate Hematodinium sp. parasitizes blue crabs along the Atlantic seaboard of the United States. Infections in blue crabs have only been reported from waters where salinity is >11 practical salinity units (psu). Blue crabs maintain a hyperosmotic internal concentration at low salinities (0–5 psu), roughly comparable to 24 psu, and should be capable of maintaining an infection in low-salinity waters even if Hematodinium spp. cells are intolerant of low salinities. We tested this notion by observing the effect of low salinity on the progression of disease in crabs experimentally infected with the parasite. Blue crabs were acclimated to 5 psu or 30 psu salinity treatments. They were inoculated with Hematodinium sp. and necropsied 3, 7, 10, and 15 days post-inoculation. The low-salinity treatment did not have an effect on the proliferation of Hematodinium sp. infections in blue crabs; moreover, a greater proportion of infections in crabs in the low-salinity treatment developed dinospore stages than did those in the high-salinity treatment, indicating that salinity may affect the development of the parasite. However, dinospores from in vitro cultures rapidly became inactive when held in salinities <15 psu. Our experiments indicate that Hematodinium spp. can develop in blue crabs at low salinities, but that the parasite is incapable of transmission in this environment, which explains the lack of natural infections in crabs at low salinities.

Molecular characterization and expression of a novel Toll gene from the swimming crab Portunus trituberculatus

Tolls/Toll-like receptors (TLRs) are important cell-surface receptors serving as pattern recognition receptors (PRRs) in the Tolls/TLRs signaling pathway of innate immune responses. In the present study, we isolated and characterized a novel Toll gene (PtToll) from Portunus trituberculatus, and further investigated its expression in various tissues of crab hosts challenged with the parasitic dinoflagellate Hematodinium. The full-length cDNA of PtToll was 3745 bp, with a 3012 bp open reading frame (ORF) encoding 1003 amino acids. Conserved domains consist of 15 tandem leucine-rich repeats (LRRs), a single-pass transmembrane segment (TM) and a cytoplasmic Toll/interleukin-1R (TIR) domain. The PtToll protein shared high similarity to other crustacean Tolls and was clustered with the crustacean Tolls in the phylogenetic tree. The PtToll gene was constitutively expressed in various tissues of P. trituberculatus, with the highest expression in hemocytes. After being challenged with the parasite, the transcripts of PtToll reacted immediately with significant alterations in all the tested tissues, and decreased consistently in most of the detected tissues (e.g., hemocytes, gill, heart, and muscle) within 24h. Then the transcripts of PtToll were significantly up-regulated in hemocytes and heart at 48 h, and in hepatopancreas at 48 and 96 h post the parasitic challenge. By 192 h post challenge, the transcriptional level of PtToll indicated a significant suppression or a decreasing trend. The fluctuations of PtToll gene expression suggested that PtToll was closely associated with intrusion of the Hematodinium parasites, and may possess a vital and systematic function in the innate immunity of P. trituberculatus against the parasitic infection.

Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium

The parasitic dinoflagellates in the genus of Hematodinium infect broad range of crustaceans around the world, causing fatal diseases in multiple species of wild and cultured crabs and subsequent economic loss. In order to explore this host-parasite interaction in the early development stage of infection, mRNA transcript levels of eight key immune-related genes, including LGBP, proPO, PPAF, serpin, α2m, and three PTcSPs were quantitatively assessed in Portunus trituberculatus artificially inoculated with the Hematodinium parasites. The fluctuation of proPO gene expression indicated that the host proPO system was disturbed overtly due to the intrusion of the parasites. And as manifested by the suppressed expression of LGBP and PPAF, an immunosuppressive mechanism was likely induced by the parasites against being entrapped or killed by the host proPO system. Furthermore, the significant variations of the transcript levels of serpin, α2m, and the three PTcSPs suggested that the parasites affected the proteinase cascade reactions associated with the immune response by destroying the balance between serine proteinases and the proteinase inhibitors. The hemocytes counts and PO activity varied accordingly over the time course of infection, showing that hemocytes were actively involved in the immune response against the parasitic invasion. This study primarily highlighted the anti-parasitic immune response of crab hosts, and presented the first report of the immune response of P. trituberculatus to the parasitic dinoflagellate Hematodinium.

Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2−- generating and antioxidant systems in host immune responses to Hematodinium

Increasing evidences have established that the nitric oxide synthase (NOS) and NADPH oxidase (NOX) play important roles in host defense system by catalyzing the production of nitrogen oxide (NO) and superoxide anions (O2(-)), respectively. While, there are limited studies to explore the roles of NOS/NOX enzymes in crustacean immunity, and no studies as yet were attempted to elucidate their functions in host immune responses to parasites. In the present study, we cloned a full-length cDNA of NOS and two partial cDNA fragments of NOX and GPx from the economic valuable crab Portunus trituberculatus. The full-length cDNA of NOS was 4002 bp in length that encoded 1203 amino acids containing motifs of the NOS protein and conserved domains. The phylogenetic analysis showed that the NOS protein sequence was clustered together with those of crustacean species in the phylogenetic tree. All of the three novel genes showed high mRNA transcripts in the immune-related tissues (e.g. hemocytes, hepatopancreas) of P. trituberculatus. Striking fluctuation in the transcripts of the critical NO/O2(-)- generating/scavenging related genes (NOS, NOX, CuZnSOD, CAT, GPx) as well as in the enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in the hemocytes and hepatopancreas of P. trituberculatus post challenged with the parasitic dinoflagellate Hematodinium, indicating that the NO/O2(-)- generating and the antioxidant systems played vital roles in the crustacean innate immunity against the parasitic intrusion. The results indicated a novel respect of the host-parasite interaction between the crab host and the parasitic dinoflagellate Hematodinium.

Early transcriptional response to the parasitic dinoflagellate Hematodinium in hepatopancreas of Portunus trituberculatus

The swimming crab Portunus trituberculatus supports a large proportion of crab aquaculture in China. In the last decade, the sustainable culture of this crab was threatened by the parasitic dinoflagellate Hematodinuim, resulting in massive mortality (up to 95%) in severely impacted culture ponds. Previous studies of Hematodinium were mainly focused on histology, molecular characterization, epizootiology, etc., with limited studies conducted to explore this specific host-parasite interaction. Thus, to give a primary insight into the anti-parasitic immune response at the critical stage of infection, the expression levels of 8 immune-related genes together with enzyme activities of phenoloxidase (PO), alkaline phosphatase (AKP), and acid phosphatase (ACP), were evaluated in hepatopancreas during 3-192h (h) post inoculation. Hematoxylin-eosin (H&E) staining showed noticeable pathological changes in hepatopancreas. The enzyme activities of PO, AKP, and ACP were significantly induced after inoculation. The changes of the prophenoloxidase (proPO) transcripts and the constantly enhanced PO activity reflected the critical function of the proPO system in resisting against the parasites. The decreased expression levels of LGBP and PPAF implied an immunosuppressive mechanism of the parasites against the host proPO system. And the significant variations in transcriptional levels of two important proteinase inhibitors (serpin, α2m) and three P. trituberculatus clip-domain serine proteinases (PTcSPs) suggested that the parasites could affect proteinase cascade reactions associated with immune response by destroying the balance between serine proteinases and the inhibitors. Moreover, the results indicated that the hepatopancreas of P. trituberculatus was significantly affected by invasion of the parasite, and hepatopancreas played important roles in the crustacean innate immunity against the parasitic infection.

Hematodinium spp. infections in wild and cultured populations of marine crustaceans along the coast of China

The parasitic dinoflagellate Hematodinium spp. infects a broad range of marine crustaceans. Its epidemics have impacted wild populations of various commercial fishery species around the world and the sustainability of mariculture in China. To study the epidemiology of Hematodinium spp. in marine crustaceans along the coast of China, we conducted a broad survey of wild and cultured stocks of major crustacean species in 2013 to 2015. Hematodinium sp. infections were identified in wild stocks of Portunus trituberculatus from Huludao, Laizhou, Qingdao, Yangtze River Estuary and Zhoushan, and Scylla paramamosain from Shantou; and cultured stocks of Portunus trituberculatus and Penaeus monodon from a polyculture pond in Qingdao. In the polyculture pond, Hematodinium sp. infections were observed in Portunus trituberculatus from June until October, with peak prevalence (up to 90%) observed in late July to early August. Furthermore, Hematodinium sp. infection was identified for the first time in the giant tiger prawn Penaeus monodon in the polyculture system during the disease outbreak. Phylogenetic analysis indicated that the Hematodinium isolate infecting Penaeus monodon was identical to the isolate infecting the co-cultured Portunus trituberculatus, and it was grouped into H. perezi genotype II together with the other isolates reported in China. The Hematodinium sp. isolated from Portunus trituberculatus appeared to have similar life stages as the H. perezi genotype III isolated from the American blue crab Callinectes sapidus. Our study indicates that outbreaks of Hematodinium disease can be a significant threat to the widely used polyculture system for decapods in China that may be particularly vulnerable to such generalist pathogens.

The response of spring phytoplankton assemblage to diluted water and upwelling in the eutrophic Changjiang (Yangtze River) Estuary

A comprehensive study on the phytoplankton standing stocks, species composition and dominant species in the eutrophic Changjiang (Yangtze River) Estuary (CE) was conducted to reveal the response of phytoplankton assemblage to Changjiang Diluted Water (CDW) and upwelling in the spring. Phytoplankton presented peak standing stocks (13.03 μg/L of chlorophyll a, 984.5×103 cells/L of phytoplankton abundance) along the surface isohaline of 25. Sixty-six species in 41 genera of Bacillariophyta and 33 species in 19 genera of Pyrrophyta were identified, as well as 5 species in Chlorophyta and Chrysophyta. Karenia mikimotoi was the most dominant species, followed by Prorocentrum dentatum, Paralia sulcata, Pseudo-nitzschia delicatissima and Skeletonema costatum. A bloom of K. mikimotoi was observed in the stratified stations, where the water was characterized by low nitrate, low phosphate, low turbidity, and specific ranges of temperature (18–22 °C) and salinity (27–32). K. mikimotoi and P. dentatum accumulated densely in the upper layers along the isohaline of 25. S. costatum was distributed in the west of the isohaline of 20. Benthonic P. sulcata presented high abundance near the bottom, while spread upward at upwelling stations. CDW resulted in overt gradients of salinity, turbidity and nutritional condition, determining the spatial distribution of phytoplankton species. The restricted upwelling resulted in the upward transport of P. sulcata and exclusion of S. costatum, K. mikimotoi and P. dentatum. The results suggested that CDW and upwelling were of importance in regulating the structure and distribution of phytoplankton assemblage in the CE and the East China Sea.