Integrative omics analysis highlights the immunomodulatory effects of the parasitic dinoflagellate hhematodinium on crustacean hemocytes.

Parasitic dinoflagellates in genus Hematodinium have caused substantial economic losses to multiple commercially valuable marine crustaceans around the world. Recent efforts to better understand the life cycle and biology of the parasite have improved our understanding of the disease ecology. However, studies on the host-parasite interaction, especially how Hematodinium parasites evade the host immune response are lacking. To address this shortfall, we used the comprehensive omics approaches (miRNA transcriptomics, iTRAQ-based proteomics) to get insights into the host-parasite interaction between hemocytes from Portunus trituberculatus and Hematodinium perezi in the present study. The parasitic dinoflagellate H. perezi remodeled the miRNome and proteome of hemocytes from challenged hosts, modulated the host immune response at both post-transcriptional and translational levels and caused post-transcriptional regulation to the host immune response. Multiple important cellular and humoral immune-related pathways (ex. Apoptosis, Endocytosis, ECM-receptor interaction, proPO activation pathway, Toll-like signaling pathway, Jak-STAT signaling pathway) were significantly affected by Hematodinium parasites. Through modulation of the host miRNome, the host immune responses of nodulation, proPO activation and antimicrobial peptides were significantly suppressed. Cellular homeostasis was imbalanced via post-transcriptional dysregulation of the phagosome and peroxisome pathways. Cellular structure and communication was seriously impacted by post-transcriptional downregulation of ECM-receptor interaction and focal adhesion pathways. In conclusion, H. perezi parasites could trigger striking changes in the miRNome and proteome of crustacean hemocytes, and this parasite exhibited multifaceted immunomodulatory effects and potential immune-suppressive mechanisms in crustacean hosts.

The parasitic dinoflagellate Hematodinium infects multiple crustaceans in the polyculture systems of Shandong Province, China.

The parasitic dinoflagellates of the Hematodinium genus have impacted wild and cultured stocks of commercial crustaceans worldwide. In the past decade, outbreaks of Hematodinium epizootics resulted in substantial mortalities in cultured Chinese swimming crabs Portunus trituberculatus in the polyculture ponds located in Shandong Peninsula, whereas the source and transmission of the parasite in the polyculture pond system remains to be determined. During April to December of 2018, 2034 crabs and 108 shrimps were collected from the polyculture pond systems in the highly endemic area of Hematodinium diseases in Qingdao, Shandong Province. Among those, 188 individuals of the 6 crab species were infected by the parasite, including 4 novel host species (Uca arcuate, Hemigrapsus penicillatus, Helice wuana and Macrophthalmus japonicas). No infection was identified in Penaeus monodon. Further phylogenetic analyses indicated that the Hematodinium isolate infecting the six crab hosts, together with other isolates reported from China, composed the genotype II of Hematodinium perezi. The parasite was more infectious to cultured Portunus trituberculatus and the dominant wild crab Helice tientsinensis dwelling in the waterways connecting to the polyculture ponds, even though it was found to be a host generalist pathogen. The prevalence of Hematodinium perezi infection in Helice tientsinensis was higher than that of other wild crabs and showed significant positive correlation with that of the cultured Portunus trituberculatus. The results indicated that the wild crabs, particularly Helice tientsinensis, were the important alternate hosts closely involved in transmission and spreading of the Hematodinium disease in the polyculture pond systems.

The parasitic dinoflagellate Hematodinium perezi infecting mudflat crabs, Helice tientsinensis, in polyculture system in China.

Since 2012, frequent outbreaks of Hematodinium diseases have significantly impacted sustainable culture of marine crabs Portunus trituberculatus in the coastal areas of Shandong Peninsula. The mechanisms of the Hematodinium parasite epizootics in polyculture pond systems remain to be explored and alternate crustacean hosts are speculated to play important roles in transmission and epizootiology of the disease. To investigate their possible role in transmission, the common wild mudflat crabs Helice tientsinensis were sampled from the waterway connecting to polyculture ponds in Huangdao, Qingdao, China and diagnosed for Hematodinium infection. Hematodinium infection was found in H. tientsinensis collected in the waterway from April-November 2018, with a prevalence of 5.8-31.7%. In addition, 23.1% of H. tientsinensis sampled from the adjacent polyculture pond were infected during the peak of the Hematodinium epizootic in July. Amoeboid trophonts or prespores were observed in the hemolymph of the Hematodinium-infected crabs and histopathological changes were observed in major organs (e.g. hepatopancreas, heart, gill and muscle). The ITS1 rRNA of Hematodinium sp. infecting H. tientsinensis shared 99-100% similarity to isolates infecting P. trituberculatus and Penaeus monodon in local polyculture ponds, and are included in a monophylogenetic clade, Hematodinium perezi genotype II, in the phylogenetic tree. The results further showed that this generalist parasite was infecting various types of marine crustaceans in the coastal waters of China, and that mudflat crabs H. tientsinensis may serve as an important alternate host during epizootics of Hematodinium parasites in the polyculture system.

Characterization of the Parasitic Dinoflagellate Amoebophrya sp. Infecting Akashiwo sanguinea in Coastal Waters of China.

The endoparasitic dinoflagellate Amoebophrya infects a number of free-living marine dinoflagellates, including harmful algal bloom species. The parasitoid eventually kills its host and has been proposed to be a significant loss factor for dinoflagellate blooms in restricted coastal waters. For several decades, the difficulties of culturing host-parasitoid systems have been a great obstacle for further research on the biology of Amoebophrya. Here, we established an Akashiwo sanguinea-Amoebophrya sp. coculture from Chinese coastal waters and studied the parasitoid's generation time, dinospore survival and infectivity, as well as its host specificity. The lifespan of Amoebophrya sp. ex. A. sanguinea was approximately 58 h. The infective dinospores can survive up to 78 h in ambient waters but gradually lose their infectivity. The parasitoid was unable to infect other dinoflagellate species, its infection rate reached as high as 91% when the ratio of dinospores to host cells was 20:1. The high infectivity of dinospores suggests that the Amoebophrya strain was capable of removing a considerable fraction of host biomass within a short period, but that it is probably unable to maintain high infection levels under nonbloom conditions of its host, due to limited survival and time constraints in encountering host cells.

Differential expression of microRNAs in Portunus trituberculatus in response to Hematodinium parasites.

Latest studies have indicated that microRNAs (miRNAs) play important roles in defending against bacterial and viral infections in marine crustacean, whereas little is known regarding the immunological roles of crustacean miRNAs in response to parasitic infection. To further reveal the host-parasite interactions between the parasitic dinoflagellate Hematodinium and its crustacean hosts, we applied the high-throughput sequencing technology to identify and characterize miRNAs in the Chinese swimming crab Portunus trituberculatus challenged with the Hematodinium parasite at a timescale of 16 days (d). A total of 168 miRNAs were identified and 51 miRNAs were differentially expressed in the hepatopancreas tissues of affected hosts. Eleven of the differentially expressed miRNAs were selected and verified by the quantitative real-time RT-PCR (qRT-PCR), manifesting the consistency between the high throughout sequencing and qRT-PCR assays. Further analysis of the putative target genes indicated that various immune-related pathways (e.g. endocytosis, Fc gamma R-mediated phagocytosis, lysosome, ECM-receptor interaction, complement and coagulation cascades, antigen processing and presentation, focal adhesion, etc.) and signal transduction pathways (e.g. JAK-STAT signaling pathway, MAPK signaling pathway, p53 signaling pathway, etc.) were mediated by the differentially expressed miRNAs. The results presented fundamental knowledge on the immunological roles of crustacean miRNAs and contributed to the better understanding of hosts' miRNAs-mediated 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.

Synthesis and in vitro antiproliferative evaluation of novel nonsymmetrical disulfides bearing 1,3,4-oxadiazole moiety.

A series of novel nonsymmetrical disulfides bearing 1,3,4-oxadiazole moiety were designed, synthesized and evaluated for their in vitro antiproliferative activities against SMMC-7721, Hela and A549 human cancer cell lines by CCK-8 assay. The preliminary bioassay results demonstrated that all tested compounds 7a-7o exhibited antiproliferation with different degrees, and some compounds showed better effects than positive control 5-fluorouracil against various cancer cell lines. Among these compounds, compound 7j showed significant antiproliferative activity against SMMC-7721 cells with IC50 value of 3.40µM. Compound 7a displayed highly effective biological activity against Hela cells with IC50 value of 4.26µM. Compound 7g exhibited the best inhibitory effect against A549 cells with IC50 value of 6.26µM.

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.

The role of arousal in the estimation of time-to-collision of threatening stimuli.

The accurate estimation of time-to-collision (TTC) is essential for the survival of organisms. Previous studies have revealed that the emotional properties of approaching stimuli can influence the estimation of TTC, indicating that approaching threatening stimuli are perceived to collide with the observers earlier than they actually do, and earlier than non-threatening stimuli. However, not only are threatening stimuli more negative in valence, but they also have higher arousal compared to non-threatening stimuli. Up to now, the effect of arousal on TTC estimation remains unclear. In addition, inconsistent findings may result from the different experimental settings employed in previous studies. To investigate whether the underestimation of TTC is attributed to threat or high arousal, three experiments with the same settings were conducted. In Experiment 1, the underestimation of TTC estimation of threatening stimuli was replicated when arousal was not controlled, in comparison to non-threatening stimuli. In Experiments 2 and 3, the underestimation effect of threatening stimuli disappeared when compared to positive stimuli with similar arousal. These findings suggest that being threatening alone is not sufficient to explain the underestimation effect, and arousal also plays a significant role in the TTC estimation of approaching stimuli. Further studies are required to validate the effect of arousal on TTC estimation, as no difference was observed in Experiment 3 between the estimated TTC of high and low arousal stimuli.

CPEB2 inhibits preeclampsia progression by regulating SSTR3 translation through polyadenylation.

AIMS: Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear. METHODS: The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining. RESULTS: CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression. CONCLUSION: CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.

Synthesis and antitumor-evaluation of 1,3-selenazole-containing 1,3,4-thiadiazole derivatives.

A series of novel 1,3-selenazole-containing 1,3,4-thiadiazole derivatives bearing Schiff base moieties were synthesized and evaluated for their in vitro antiproliferative activities against human breast cancer cell MCF-7 and mouse lymphocyte leukemia cell L1210 by CCK-8 assay. The majority of the compounds showed better activity against MCF-7 cell, compared with lead compound PCS. In particular, compound 6c was the most potent compound with IC50 value of 4.02 µM.

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Improving the physicochemical and pharmacokinetic properties of olaparib through cocrystallization strategy.

Olaparib (OLA) is the first PARP inhibitor worldwide used for the treatment of ovarian cancer. However, the oral absorption of OLA is extremely limited by its poor solubility. Herein, pharmaceutical cocrystallization strategy was employed to optimize the physicochemical and pharmacokinetic properties. Four cocrystals of OLA with oxalic acid (OLA-OA), malonic acid (OLA-MA), fumaric acid (OLA-FA) and maleic acid (OLA-MLA) were successfully discovered and characterized. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the formation of cocrystals rather than salts, and the possible hydrogen bonding patterns were analyzed through molecular surface electrostatic potential calculations. The in vitro and in vivo evaluations indicate that all of the cocrystals demonstrate significantly improved dissolution performance, oral absorption and tabletability compared to pure OLA. Among them, OLA-FA exhibit sufficient stability and the most increased Cmax and AUC0-24h values that were 11.6 and 6.1 times of free OLA, respectively, which has great potential to be developed into the improved solid preparations of OLA.

Synthesis and antitumor-evaluation of 1,3,4-thiadiazole-containing benzisoselenazolone derivatives.

A series of novel 1,3,4-thiadiazole-containing benzisoselenazolone derivatives were prepared by the condensation of 2-chloroselenobenzoyl chloride and 2-amino-5-substituted-1,3,4-thiadiazole. Their in vitro antiproliferative activities were evaluated in SSMC-7721, MCF-7 and A-549 cells. The results suggest that, in different tumor cells, some compounds have good antiproliferative activity, certain selectivity and potential value of further research.

Synthesis and in vitro antitumor activity of 1,3,4-oxadiazole derivatives based on benzisoselenazolone.

A series of novel 1,3,4-oxadiazole derivatives based on benzisoselenazolone has been prepared and tested for antiproliferative activity in vitro against the cells of human cancer cell lines: SSMC-7721 (human liver cancer cell), MCF-7 (human breast cancer cell) and A549 (human lung cancer cell). All the compounds obtained exhibited antiproliferative activity and showed selective cytotoxicity against different cancer cells. Compounds 7d and 7i showed significant antiproliferative activities against MCF-7 cells, with IC50 values of 1.07 and 1.76 µM respectively. Compound 7d were found to be the most potent compound against SSMC-7721 cells, with IC50 values 4.46 µM.

Full-length transcriptome analysis of the bloom-forming dinoflagellate Akashiwo sanguinea by single-molecule real-time sequencing.

The dinoflagellate Akashiwo sanguinea is a harmful algal species and commonly observed in estuarine and coastal waters around the world. Harmful algal blooms (HABs) caused by this species lead to serious environmental impacts in the coastal waters of China since 1998 followed by huge economic losses. However, the full-length transcriptome information of A. sanguinea is still not fully explored, which hampers basic genetic and functional studies. Herein, single-molecule real-time (SMRT) sequencing technology was performed to characterize the full-length transcript in A. sanguinea. Totally, 83.03 Gb SMRT sequencing clean reads were generated, 983,960 circular consensus sequences (CCS) with average lengths of 3,061 bp were obtained, and 81.71% (804,016) of CCS were full-length non-chimeric reads (FLNC). Furthermore, 26,461 contigs were obtained after being corrected with Illumina library sequencing, with 20,037 (75.72%) successfully annotated in the five public databases. A total of 13,441 long non-coding RNA (lncRNA) transcripts, 3,137 alternative splicing (AS) events, 514 putative transcription factors (TFs) members from 23 TF families, and 4,397 simple sequence repeats (SSRs) were predicted, respectively. Our findings provided a sizable insights into gene sequence characteristics of A. sanguinea, which can be used as a reference sequence resource for A. sanguinea draft genome annotation, and will contribute to further molecular biology research on this harmful bloom algae.

A Sensitive and Portable Double-Layer Microfluidic Biochip for Harmful Algae Detection.

Harmful algal blooms (HABs) are common disastrous ecological anomalies in coastal waters. An effective algae monitoring approach is important for natural disaster warning and environmental governance. However, conducting rapid and sensitive detection of multiple algae is still challenging. Here, we designed an ultrasensitive, rapid and portable double-layer microfluidic biochip for the simultaneous quantitative detection of six species of algae. Specific DNA probes based on the 18S ribosomal DNA (18S rDNA) gene fragments of HABs were designed and labeled with the fluorescent molecule cyanine-3 (Cy3). The biochip had multiple graphene oxide (GO) nanosheets-based reaction units, in which GO nanosheets were applied to transfer target DNA to the fluorescence signal through a photoluminescence detection system. The entire detection process of multiple algae was completed within 45 min with the linear range of fluorescence recovery of 0.1 fM-100 nM, and the detection limit reached 108 aM. The proposed approach has a simple detection process and high detection performance and is feasible to conduct accurate detection with matched portable detection equipment. It will have promising applications in marine natural disaster monitoring and environmental care.

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.

Lack of transmission of Hematodinium sp. in the blue crab Callinectes sapidus through cannibalism.

Hematodinium spp. are parasitic dinoflagellates of marine crustaceans. Outbreaks of Hematodinium sp. have impacted commercial landings of the blue crab Callinectes sapidus in the coastal bays of Virginia and Maryland (USA), with seasonal peaks in prevalence reaching 85%. The life cycle and transmission routes of the parasite in blue crabs are poorly understood. Cannibalism and waterborne transmission may be routes of transmission, although little conclusive evidence has been reported for these modes. We examined cannibalism as a route by a series of experiments wherein we repeatedly fed adult and juvenile crabs the tissues of crabs infected with Hematodinium. In each experiment, feeding was done 3 times over the course of 1 wk. Only 2 of 120 crabs were infected within 7 to 9 d after feeding, and these 2 were likely infected prior to the experimental exposures. Crabs inoculated with hemolymph from infected donors served as positive controls. They developed infections over 11 to 21 d, indicating that the Hematodinium sp. used in the cannibalism trials was infectious at the time of inoculation. Because amphipods also harbor Hematodinium-like infections, we fed tissues of infected crabs to the estuarine amphipod Leptocheirus plumulosus. Hematodinium DNA was detected in amphipods shortly after feeding, but not in animals held for longer periods, nor was it observed in histological preparations. Amphipods did not obtain infections by scavenging infected crab tissues. Our results show that Hematodinium sp. is not effectively transmitted through ingestion of diseased tissues, indicating that cannibalism may not be a major route of transmission for Hematodinium sp. in blue crabs.