Genetic diversity of parasitic dinoflagellates in the genus amoebophrya and its relationship to parasite biology and biogeography

We determined 18S rRNA gene sequences of Amoebophrya strains infecting the thecate dinoflagellates Alexandrium affine and Gonyaulax polygramma from Korean coastal waters and compared those data with previously reported sequences of Amoebophrya from cultures, infected cells concentrated from field samples, and environmental 18S rRNA gene sequences obtained from a variety of marine environments. Further, we used these data to examine genetic diversity in Amoebophrya strains relative to geographic origin, host phylogeny, site of infection, and host specificity. In our analyses of known dinoflagellate taxa, the 13 available Amoebophrya sequences clustered together within the dinoflagellates as three groups forming a monophyletic group with high bootstrap support (maximum likelihood, ML: 100%) or a posterior probability (PP) of 1. When the Amoebophrya sequences were analyzed along with environmental sequences associated with Marine Alveolate Group II, nine subgroups formed a monophyletic group with high bootstrap support (ML: 100%) and PP of 1. Sequences known to be from Amoebophrya spp. infecting dinoflagellate hosts were distributed in seven of those subgroups. Despite differences in host species and geographic origin (Korea, United States, and Europe), Amoebophrya strains (Group II) from Gymnodinium instriatum, A. affine, Ceratium tripos (AY208892), Prorocentrum micans, and Ceratium lineatum grouped together by all of our tree construction methods, even after adding the environmental sequences. By contrast, strains within Groups I and III divided into several lineages following inclusion of environmental sequences. While Amoebophrya strains within Group II mostly developed within the host cytoplasm, strains in Groups I and III formed infections inside the host nucleus, a trait that appeared across several of the subgroups. Host specificity varied from moderately to extremely species-specific within groups, including Group II. Taken together, our results imply that genetic diversity in Amoebophrya strains does not always reflect parasite biology or biogeography.     

Multiple strains of the parasitic dinoflagellate Amoebophrya exist in Chesapeake Bay

Small subunit rRNA sequences were amplified from Amoebophrya strains infecting Karlodinium micrum, Gymnodinium instriatum and an unidentified Scrippsiella species in Chesapeake Bay. The alignable parts of the sequences differed from each other and from the previously reported rRNA sequence of the Amoebophrya strain infecting Akashiwo sanguinea in Chesapeake Bay by 4 to 10%. This is a greater degree of difference than sometimes found between sequences from separate genera of free-living dinoflagellates. These sequence differences indicate that the Amoebophrya strains parasitizing dinoflagellates in Chesapeake Bay do not all belong to the same species. In spite of their relative dissimilarity, the sequences do group together into a single clade with high bootstrap support in phylogenetic trees constructed from the sequences.     

Multiple species of the dinophagous dinoflagellate genus Amoebophrya infect the same host species

Populations of the dinoflagellate Dinophysis norvegica in the Baltic Sea and in the adjacent North Sea are infected by the endoparasite Amoebophrya sp. The high diversity recently unveiled within the genus Amoebophrya brings uncertainty about their identities. We applied molecular biology techniques--18S rDNA sequencing and fluorescent in situ hybridization (FISH)--to compare this host-parasite system from both environments. The North Sea Amoebophrya sp. 18S rDNA sequence was 89% identical to the previously described Baltic Sea Amoebophrya sp. sequence, suggesting they are different species. In spite of that, a phylogenetical analysis placed the North Sea parasite sequence in a well-supported cluster with other Amoebophrya sp. sequences. The D. norvegica 18S rDNA sequences from both environments were 100% identical, indicating that the hosts have not evolved independently. A DNA probe designed for the Baltic Sea Amoebophrya sp. 18S rRNA was used in FISH assays on infected D. norvegica populations from both environments. The probe stained all infected cells from the Baltic sample, whereas none from the North Sea were stained. The results indicate that D. norvegica is released from one parasite when entering the Baltic Sea, and become less infected by an alternative parasite species.     

The Phylogenetic Position of Amoebophrya sp. Infecting Gymnodinium sanguineum

The small-subunit rRNA sequence of a species of Amoebophrya infecting Gymnodinium sanguineum in Chesapeake Bay was obtained and compared to the small subunit rRNA sequences of other protists. Phylogenetic trees constructed with the new sequence place Amoebophrya between the remaining dinoflagellates and other protists.     

Sterols of the syndinian dinoflagellate Amoebophrya sp., a parasite of the dinoflagellate Alexandrium tamarense (Dinophyceae)

Several harmful photosynthetic dinoflagellates have been examined over past decades for unique chemical biomarker sterols. Little emphasis has been placed on important heterotrophic genera, such as Amoebophrya, an obligate, intracellular parasite of other, often harmful, dinoflagellates with the ability to control host populations naturally. Therefore, the sterol composition of Amoebophrya was examined throughout the course of an infective cycle within its host dinoflagellate, Alexandrium tamarense, with the primary intent of identifying potential sterol biomarkers. Amoebophrya possessed two primary C(27) sterols, cholesterol and cholesta-5,22Z-dien-3beta-ol (cis-22-dehydrocholesterol), which are not unique to this genus, but were found in high relative percentages that are uncommon to other genera of dinoflagellates. Because the host also possesses cholesterol as one of its major sterols, carbon-stable isotope ratio characterization of cholesterol was performed in order to determine whether it was produced by Amoebophrya or derived intact from the host. Results indicated that cholesterol was not derived intact from the host. A comparison of the sterol profile of Amoebophrya to published sterol profiles of phylogenetic relatives revealed that its sterol profile most closely resembles that of the (proto)dinoflagellate Oxyrrhis marina rather than other extant genera.     

Interplay between the parasite Amoebophrya sp. (Alveolata) and the cyst formation of the red tide dinoflagellate Scrippsiella trochoidea

Syndiniales (Alveolata) are marine parasites of a wide range of hosts, from unicellular organisms to Metazoa. Many Syndiniales obligatorily kill their hosts to accomplish their life cycle. This is the case for Amoebophrya spp. infecting dinoflagellates. However, several dinoflagellate species known to be infected by these parasites produce diploid resting cysts as part of their life history. These resting cysts may survive several seasons in the sediment before germinating. How these parasites survive during the dormancy of their host remained an open question. We successfully established infections by Amoebophrya sp. in the red tide dinoflagellate Scrippsiella trochoidea. This host strain was homothallic and able to continuously produce typical calcified cysts covered by calcareous spines. Presence of the parasite significantly speeded up the host cyst production, and cysts produced were the only cells to resist infections. However, some of them were clearly infected, probably earlier in their formation. After 10 months, cysts produced in presence of the parasite were able to germinate and new infective cycles of the parasite were rapidly observed. Thus, a very novel relationship for protists is demonstrated, one in which parasite and host simultaneously enter dormancy, emerging months later to propagate both species.     

Alveolate phylogeny inferred using concatenated ribosomal proteins

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.     

102 Characterization of an Arsenate Reductase Encoded by ORF slr0946 from the Cyanobacterium Synechocystis SP. PCC6803

Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom-forming dinoflagellates, including the toxic species Karlodinium micrum . Unlike non-toxic hosts, K. micrum is partially resistant to infection, a trait that may be related to toxin production. Here we tested the hypothesis that parasitism of K. micrum is inversely related to toxin concentration in the culture medium. Time-course studies were conducted to determine the influence of extracted toxin and toxin carrier (methanol) on host growth, parasite prevalence, and parasite load. Results indicate that methanol concentrations below 0.1% have no effect on these variables. When methanol concentration was maintained below 0.1%, extracted toxin equivalent to 100 to 10,000 K. micrum per ml had no effect on host abundance. We are currently analyzing sample to assess the fate of Amoebophrya dinospores when exposed to K. micrum toxin. We will also consider the effect of intracellular host toxin on parasite success, by examining the fate of Amoebophrya dinospores when inoculated to K. micrum cultures that express different levels of toxin production. Understanding the effect of toxins on parasite success will contribute to our knowledge of host-parasite biology and provide insight into the role of dinoflagellate toxins as a defense against parasitism.     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

Infection of Gymnodinium sanguineum by the dinoflagellate Amoebophrya sp.: effect of nutrient environment on parasite generation time, reproduction, and infectivity

Preliminary attempts to culture Amoebophrya sp., a parasite of Gymnodinium sanguineum from Chesapeake Bay, indicated that success may be influenced by water quality. To explore that possibility, we determined development time, reproductive output, and infectivity of progeny (i.e. dinospores) for Amoebophyra sp. maintained on G. sanguineum grown in four different culture media. The duration of the parasite's intracellular growth phase showed no significant difference among treatments; however, the time required for completion of multiple parasite generations did, with elapsed time to the middle of the third generation being shorter in nutrient-replete media. Parasites of hosts grown in nutrient-replete medium also produced three to four times more dinospores than those infecting hosts under low-nutrient conditions, with mean values of 380 and 130 dinospores/host, respectively. Dinospore production relative to host biovolume also differed, with peak values of 7.4 per 1,000 microm3 host for nutrient-replete medium and 4.8 per 1,000 microm3 host for nutrient-limited medium. Furthermore, dinospores produced by "high-nutrient" parasites had a higher success rate than those formed by "low-nutrient" parasites. Results suggest that Amoebophrya sp. is well adapted to exploit G. sanguineum populations in nutrient-enriched environments.     

Effect of the endoparasite Amoebophrya sp. on toxin content and composition in the paralytic shellfish poisoning dinoflagellate Alexandrium fundyense (Dinophyceae)

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing “edible food” for consumers of the marine microbial food-web during toxic algal bloom event.     

101 Effect of Karlodinium Micrum Toxin on Success of the Parasitic Dinoflagellate Amoebophyra SP.

Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom‐forming dinoflagellates, including the toxic species Karlodinium micrum. Unlike non‐toxic hosts, K. micrum is partially resistant to infection, a trait that may be related to toxin production. Here we tested the hypothesis that parasitism of K. micrum is inversely related to toxin concentration in the culture medium. Time‐course studies were conducted to determine the influence of extracted toxin and toxin carrier (methanol) on host growth, parasite prevalence, and parasite load. Results indicate that methanol concentrations below 0.1% have no effect on these variables. When methanol concentration was maintained below 0.1%, extracted toxin equivalent to 100 to 10,000 K. micrum per ml had no effect on host abundance. We are currently analyzing sample to assess the fate of Amoebophrya dinospores when exposed to K. micrum toxin. We will also consider the effect of intracellular host toxin on parasite success, by examining the fate of Amoebophrya dinospores when inoculated to K. micrum cultures that express different levels of toxin production. Understanding the effect of toxins on parasite success will contribute to our knowledge of host‐parasite biology and provide insight into the role of dinoflagellate toxins as a defense against parasitism.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

Social parasitism in fire ants (Solenopsis spp.): a potential mechanism for interspecies transfer of Wolbachia

One possible mechanism for interspecific transfer of Wolbachia is through the intimate contact between parasites and their hosts. We surveyed 10 species of fly parasitoids (Pseudacteon spp.) and one inquiline social parasite, Solenopsis daguerrei, for the presence and sequence identity (wsp gene) of Wolbachia. Two Wolbachia variants infecting S. daguerrei were identical to known variants infecting the two common ant host species, Solenopsis invicta and Solenopsis richteri, suggesting possible transfers of Wolbachia between this parasite and their hosts have occurred. Our data also revealed an unexpectedly high diversity of Wolbachia variants within S. daguerrei: up to eight variants were found within each individual, which, to our knowledge, is the highest reported number of Wolbachia variants infecting a single individual of any host species.     

Syndinean dinoflagellates of the genus Euduboscquella are paraphyletic

Syndinean dinoflagellates of the genus Euduboscquella infect marine ciliates and dinoflagellates. Euduboscquella species infecting dinoflagellates are understudied relative to congeners infecting ciliates and their molecular phylogeny remains uncertain. Morphology, development, and rRNA gene sequences of intracellular parasites infecting heterotrophic dinoflagellates from coastal waters of Busan, Republic of Korea in summer to fall of 2019–2021 indicate that Cucumeridinium coeruleum, Gyrodinium cf. ochraceum, and two unidentified species of Gyrodinium were each infected by a different Euduboscquella species. Morphological features including shield structure, shape and color of the mature trophont, and sporogenic process distinguished each of the four parasites from the 10 previously described species of Euduboscquella. Our molecular and phylogenetic analyses showed considerably greater genetic distance of SSU and ITS-LSU rRNA gene regions among Euduboscquella species infecting dinoflagellates than among those infecting ciliates. Rather than clustering as a group with Euduboscquella species infecting ciliates, SSU rRNA sequences of the four novel parasites spread out across the syndinean Group I phylogeny, occurring in two different clades and a new lineage. Placement of our novel parasites in multiple clades that encompass Ichythyodinium chabelardi strongly indicates that the genus Euduboscquella is paraphyletic.     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

Phylogenetic position of the copepod-infesting parasite Syndinium turbo (Dinoflagellata, Syndinea)

Sequences were determined for the nuclear-encoded small subunit (SSU) rRNA and 5.8S rRNA genes as well as the internal transcribed spacers ITS1 and ITS2 of the parasitic dinoflagellate genus Syndinium from two different marine copepod hosts. Syndinium developed a multicellular plasmodium inside its host and at maturity free-swimming zoospores were released. Syndinium plasmodia in the copepod Paracalanus parvus produced zoospores of three different morphological types. However, full SSU rDNA sequences for the three morphotypes were 100% identical and also their ITS1-ITS2 sequences were identical except for four base pairs. It was concluded that the three morphotypes belong to a single species that was identified as Syndinium turbo, the type species of the dinoflagellate subdivision Syndinea. The SSU rDNA sequence of another Syndinium species infecting Corycaeus sp. was similar to Syndinium turbo except for three base pairs and the ITS1-ITS2 sequences of the two species differed at 34-35 positions. Phylogenetic analyses placed Syndinium as a sister taxon to the blue crab parasite Hematodinium sp. and both parasites were affiliated with the so-called marine alveolate Group II. This corroborates the hypothesis that marine alveolate Group II is Syndinea.