Trypanosomatid biodiversity in Costa Rica: genotyping of parasites from Heteroptera using the spliced leader RNA gene

Abstract

The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS-EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.

Selective recovery of the cultivation-prone components from mixed trypanosomatid infections: a case of several novel species isolated from Neotropical Heteroptera

“…Insect hosts were collected and dissected as described previously (Westenberger et al, 2004;Maslov et al, 2007). Primary cultures of the parasites were established in the field by inoculating the infected gut material into 1.5 ml Eppendorf tubes filled with 1 ml of SDM79 medium supplemented with antibiotics, as described previously (Westenberger et al, 2004) and by Brun & Schönenberger (1979). The cultures were then brought to the University of California -Riverside (UCR) laboratory where they were checked regularly by using light microscopy for the presence and propagation of trypanosomatids.…”

“…Insect gut DNA samples ('environmental' DNA samples) were obtained in the field by placing an aliquot of the gut material in 0.5 ml 1 % SDS, 100 mM EDTA. The material was kept at ambient temperature for 1-3 weeks before transfer to the UCR laboratory, wherein DNA was isolated by using a standard phenol/ chloroform procedure as described previously (Westenberger et al, 2004;Maslov et al, 2007). In addition, the DNA samples were purified with InstaGene matrix (Bio-Rad), according to the manufacturer's protocol.…”

“…For cluster analysis of the SL RNA gene repeats, the alignment included only the most conserved region encompassing the intron, exon and upstream sequence at positions from 2100 to 21 (Westenberger et al, 2004). Neighbour-joining clustering was performed with PAUP* 4.0 beta version (Swofford, 1998) using the Kimura two-parameter distances.…”

“…nov.) and the respective insect gut samples (env.). The dendrogram also contains sequences representing trypanosomatid typing units TU1 to TU48, shown by numbers in boxes, as well as a set of reference trypanosomatid species used previously (Maslov et al, 2007;Westenberger et al, 2004) and supplemented with the sequences determined in this work (Table 1) Etymology. The species name reflects the findings of this trypanosomatid in two species of Neopamera from Costa Rica and Ecuador.…”

“…We have developed a methodology to analyse spliced leader RNA (SL RNA) gene sequences directly from parasites in insect gut samples (Westenberger et al, 2004;Maslov et al, 2007). Such 'environmental' trypanosomatid sequences were obtained from 60 of 170 species of Neotropical Heteroptera investigated, a number representing a relatively small fraction of all potential hosts.…”