Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut

Abstract

Protozoa, of the taxon Gregarinasina, are a heterogeneous group of Apicomplexa that includes ~1600 species. They are parasites of a large variety of both marine and terrestrial invertebrates, mainly annelids, arthropods and mollusks. Unlike coccidians and heamosporidians, gregarines have not proven to have a negative effect on human welfare; thus, they have been poorly investigated. This study focuses on the molecular identification and phylogeny of the gregarine species found in the midgut of two insect species that are considered as an alternative source of animal proteins for the human diet: the mealworm Tenebrio molitor, and the super-worm Zophobas atratus (Coleoptera: Tenebrionidae). Gregarine specimens were isolated from the gut of both larval and adult stages of T. molitor specimens, as well as from Z. atratus larvae. The morphological analyses were restricted to the identification of the different parasite morpho-types, likely corresponding either to different life-cycle stages or to alternative gregarine species. The samples were also used for the DNA extraction necessary for their genetic characterization. Finally, the efficiency of different molecular markers (i.e., 18S rDNA gene alone or combined with the Internal Transcribed Spacer 1) was assessed when applied either to gregarine species identification and to phylogenetic inference.

Integrative taxonomy confirms that Gregarina garnhami and G. acridiorum (Apicomplexa, Gregarinidae), parasites of Schistocerca gregaria and Locusta migratoria (Insecta, Orthoptera), are distinct species

“…The 1.5% genetic distance between the two sequences is in agreement with previously described inter-specific levels of genetic divergence that, for example, distinguish Gregarina niphandrodes from Gregarina polymorpha (1.44%) [31]. However, it should be noted that, according to the same authors, such "low" genetic divergence could also correspond to "intra-specific" variability [31]. Certainly, additional studies will be needed to clarify this issue, but we recently demonstrated that two marine gregarines with an almost identical SSU rDNA sequence (1 bp difference for 1702 positions, i.e.~0.05% divergence) displayed~10% overall nucleic acid divergence at the genomic level, preventing genetic crossing, i.e.…”

“…Overall, 22 different bases were identified in this 1638 bp region that could be used to delimit the species. The 1.5% genetic distance between the two sequences is in agreement with previously described inter-specific levels of genetic divergence that, for example, distinguish Gregarina niphandrodes from Gregarina polymorpha (1.44%) [31]. However, it should be noted that, according to the same authors, such "low" genetic divergence could also correspond to "intra-specific" variability [31].…”

“…Molecular characterization, based on the partial SSU rDNA marker (V1-V8 region [18]) of all gregarines studied, unambiguously demonstrated that all S. gregaria hostsregardless of their subspecies and raising facilitiesare infected by the same gregarine species (presumably G. garnhami), whereas all L. migratoria hosts are infected by a distinct species (presumably G. acridiorum). Both gregarine sequences clustered in the previously reported Gregarinoidea clade [11,29,31]. [11].…”

First record of gregarine protists (Apicomplexa: Sporozoa) in Asian fungus-growing termite Macrotermes barneyi (Blattaria: Termitidae)

“…These protozoan parasites may also good materials for studies on parasite-host coevolution and biological control of insects 3 , 4 . However, the gregarines have only been poorly explored compared to those much better studied vertebrate pathogens 5 , 6 .…”