Identification of a Divergent Environmental DNA Sequence Clade Using the Phylogeny of Gregarine Parasites (Apicomplexa) from Crustacean Hosts

Abstract

BackgroundEnvironmental SSU rDNA surveys have significantly improved our understanding of microeukaryotic diversity. Many of the sequences acquired using this approach are closely related to lineages previously characterized at both morphological and molecular levels, making interpretation of these data relatively straightforward. Some sequences, by contrast, appear to be phylogenetic orphans and are sometimes inferred to represent “novel lineages” of unknown cellular identity. Consequently, interpretation of environmental DNA surveys of cellular diversity rely on an adequately comprehensive database of DNA sequences derived from identified species. Several major taxa of microeukaryotes, however, are still very poorly represented in these databases, and this is especially true for diverse groups of single-celled parasites, such as gregarine apicomplexans.Methodology/Principal FindingsThis study attempts to address this paucity of DNA sequence data by characterizing four different gregarine species, isolated from the intestines of crustaceans, at both morphological and molecular levels: Thiriotia pugettiae sp. n. from the graceful kelp crab (Pugettia gracilis), Cephaloidophora cf. communis from two different species of barnacles (Balanus glandula and B. balanus), Heliospora cf. longissima from two different species of freshwater amphipods (Eulimnogammarus verrucosus and E. vittatus), and Heliospora caprellae comb. n. from a skeleton shrimp (Caprella alaskana). SSU rDNA sequences were acquired from isolates of these gregarine species and added to a global apicomplexan alignment containing all major groups of gregarines characterized so far. Molecular phylogenetic analyses of these data demonstrated that all of the gregarines collected from crustacean hosts formed a very strongly supported clade with 48 previously unidentified environmental DNA sequences.Conclusions/SignificanceThis expanded molecular phylogenetic context enabled us to establish a major clade of intestinal gregarine parasites and infer the cellular identities of several previously unidentified environmental SSU rDNA sequences, including several sequences that have formerly been discussed broadly in the literature as a suspected “novel” lineage of eukaryotes.

A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarineAncora sagittata(Leuckart, 1860) Labbé, 1899 (Apicomplexa: Eugregarinida)

“…Gregarines have been also detected in environmental sequence surveys from various marine and freshwater samples, possibly because oocysts are stable in the environment (Rueckert et al, 2011; Janouškovec et al, 2015). A large majority of these environmental sequences cannot be taxonomically assigned to a specific gregarine family.…”

Life without a Host Cell: What is Cryptosporidium ?

“…Those most closely related now include the rhytidocystid (e.g., Rhytidocystis), trichotokarid (e.g., Trichotokaria), and polyplicariid (e.g., Polyplicarium) orthogregarines [21]. The few descriptions of these protozoa have revealed some fascinating similarities to Cryptosporidium [54][55][56][57][58][59][60] but more studies are now clearly warranted. Such comparative data are needed to better understand these parasitic organisms, to identify conserved and/or diverged traits and behaviors, and to clarify their relationships to each other.…”

Krill feeding on sediment in the Gulf of Maine (North Atlantic)

“…Operational taxonomic unit (OTU) A, the most common prey item found in krill guts, was sequenced for full length 18S rDNA using universal primers (Medlin et al 1988, Rueckert et al 2011) and OTU Aspecific primers (Table 2) from individual krill gut contents and from sediment samples. OTU A-specific primers were designed based on an alignment of 18 sequences of varying divergence from OTU A (MegAlign, IDT Oligo Analyzer).…”

“…We used only the foregut, the first portion of the krill's digestive system, to detect recently consumed prey. This organ contains the gastric mill and consists of teeth, spines, and armored areas and is used by the krill to further macerate prey (Mauchline & Fisher 1969, Suh & Nemoto 1988, Schmidt 2010; it is therefore not an environment conducive to soft-bodied symbionts, such as gregarines (Rueckert et al 2011). OTU A was not in krill guts as a parasite of copepods or other metazoan prey since it and related prey sequences OTUs F and G were found in krill guts at Stn 7 where no metazoan prey were found in krill guts.…”

“…Most gregarines still remain un known (Leander 2008), and thus this group may include free-living organisms in addition to gut symbionts of larger animals. With their soft bodies and characteristic gliding motility (Rueckert et al 2011), the marine sediment surface might be a likely environment for any such as yet Table 3. Sequences showing similarity with operational taxonomic unit (OTU) A. SI = sequence identity with OTU A unknown free-living gregarines.…”