Heteroplasmy is the presence of multiple genotypes in an individual and is commonly associated with mitochondria where thousands of copies of the genome can be present in a single organelle. Even in low frequencies, it can cause severe illness or death and strong selective pressures usually result in a low occurrence. Despite this, cloning and sequencing of a portion of the open reading frames of the nad2, nad4, and coI mitochondrial loci in the blue crab Callinectes sapidus (Rathbun, 1896) revealed abnormally high levels of heteroplasmy at all loci with as many as 24 haplotypes in a single individual and the dominant haplotype accounting for as little as 43.9% of the total sequences. A minimum spanning network comparing these individuals at each locus demonstrated an overlap of haplotypes between the female and themegalopa with almost no overlap of themale sequences. Illumina sequencing ofmuscle and testes froma single individual usingmultiple differentDNA-based amplificationmethods as well as cDNA amplification confirmed heteroplasmic circular mitochondrial DNA templates with portions of the reads containing unmapped highly repetitive regions at specific sequence positions in both tissue types. Thus, the blue crab appears to possess a highly degenerate and possibly fragmented mitochondrial genome that is present in germline tissue and linearly inherited. A general lack of fragmentation present in the cDNA sequences as well as site-specific sequence changes argues for mitochondrial editing as a way of maintaining sequence integrity. This is the first evidence for mitochondrial editing in a crustacean and a potential explanation for observations of extremely high population diversity when using blue crab mitochondrial markers.

Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.

Invasive aquatic species can have damaging effects on fisheries and aquaculture through significant, and irreversible, effects on biodiversity. Human health may also be affected. To combat this threat the International Maritime Organization (IMO) Convention for Ballast Water and Sediments (BWMC) came into force in September 2017. U.S. Federal and IMO ballast water standards for discharged organisms stipulate discharge limits for different size classes of organisms. Several studies including recent trials aboard Great Lakes freighters have shown that many phytoplankton found in ballast water do not fall into the regulated 10–50 µM size class. Such issues illustrate the need for new methods of assessing microorganism populations that will supersede laborious microscopy requiring rare technical expertise. Recent progress has been made in the use of DNA (deoxyribose nucleic acid)-based methods as a means of identifying the appearance of invasive species in aquatic environments. A significant advance has been the development of high throughput sequencing (HTS), which has expanded DNA barcoding, relating to an individual organism, into second generation sequencing (metabarcoding), capable of mapping whole populations of organisms in an environmental sample. Several recent studies of HTS in ships’ ballast water, have shown that the technique has the capacity for detecting potentially harmful taxonomic groups and is capable of differentiating among water from different sources. The current study was undertaken to investigate the suitability (or otherwise) of HTS as a tool for ballast water management. Possible applications include improved risk assessment relating to invasive species. Feasibility for indicative testing for ballast water treatment efficacy was also addressed. However, pending analysis of treated samples, the current study was confined to a comparison of HTS and microscope counts in untreated samples. A correlation of visual and molecular taxonomic assignments of microorganisms found in the ballast water from different ports and during different seasons indicated that such a comparison was best conducted at Family level, although Principal Components Analysis showed that the two methods differed qualitatively among major taxonomic groups.

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen “core” dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known.