Absence of Wolbachia endobacteria in the non-filariid nematodes Angiostrongylus cantonensis and A. costaricensis

Abstract

The majority of filarial nematodes harbour Wolbachia endobacteria, including the major pathogenic species in humans, Onchocerca volvulus, Brugia malayi and Wuchereria bancrofti. These obligate endosymbionts have never been demonstrated unequivocally in any non-filariid nematode. However, a recent report described the detection by PCR of Wolbachia in the metastrongylid nematode, Angiostrongylus cantonensis (rat lungworm), a leading cause of eosinophilic meningitis in humans. To address the intriguing possibility of Wolbachia infection in nematode species distinct from the Family Onchocercidae, we used both PCR and immunohistochemistry to screen samples of A. cantonensis and A. costaricensis for the presence of this endosymbiont. We were unable to detect Wolbachia in either species using these methodologies. In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes. This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples. FindingsWolbachia endobacteria infect most species of insect and are present in other arthropod groups as well as in most filarial nematode species. Phylogenetic analyses currently indicate as many as eight distinct Wolbachia lineages, designated supergroups A to H, along with some other lineages whose taxonomic position remains unresolved [1,2]. Arthropod Wolbachia are found in all supergroups except C and D, with the majority of insect Wolbachia strains in supergroups A and B. Wolbachia from filarial nematodes are exclusively in supergroups C and D with the exception of endosymbionts from Mansonella spp., which are in supergroup F along with the Wolbachia from certain termites.The association between Wolbachia and filarial nematodes appears to be one of mutualism, probably of an obligatory nature [3]. Elimination of the endosymbionts by antibiotic treatment disrupts embryogenesis and hence microfilarial production, disrupts growth and development, and leads to macrofilaricidal effects. Wolbachia are also implicated in the immunopathology of infected per-

Endosymbiotic bacteria associated with nematodes, ticks and amoebae

“…Surveys of nonfilarial nematodes have failed to detect Wolbachia outside of this group (Bordenstein et al ., ), although some evidence for divergent Wolbachia ‐like sequences and structurally distinct bacteria has been reported in the plant parasitic Tylenchid nematode, Radopholus similis (Haegeman et al ., ). Reports of PCR amplification of Wolbachia sequence from the metastrongylid nematode Angiostrongylus cantonensis (Tsai et al ., ) have not been reproduced and appear to be because of laboratory contamination (Foster et al ., ). A more in‐depth survey of subfamilies of the Onchocercidae supports the view that Wolbachia arose late in the divergence of filarial nematodes.…”

“…Reductive evolution among allopatric symbionts (of ticks & nematodes) and large genomes for sympatric symbionts present in amoebae Moliner & Raoult (2010) *Adapted from Greub & Raoult (2004). (Foster et al, 2008). A more in-depth survey of subfamilies of the Onchocercidae supports the view that Wolbachia arose late in the divergence of filarial nematodes.…”

The evolution of parasitism in Nematoda

“…The other Wolbachia found in nematodes are much less well-studied. Some onchocercid nematodes carry a Wolbachia that is placed in supergroup F, alongside Wolbachia from termites, fleas and bedbugs (Bordenstein et al 2003 ; Duron and Gavotte, 2007 ; Foster et al 2008 ; Jacob et al 2008 ; Haegeman et al 2009 ; Comandatore et al 2013 ). Initial analyses suggested that the R. similis Wolbachia was distantly related to any other supergroup (Jacob et al 2008 ; Haegeman et al 2009 ), but this result is questionable (Koutsovoulos et al 2014 ).…”

Absence of Wolbachia endobacteria in Sri Lankan isolates of the nematode parasite of animals Setaria digitata

“…The suitability of the extracted S. digitata gDNA for PCR was examined via the attempted amplification of a portion of the S. digitata single copy gene (SDNP) using the following primers: UNF2 (5 -ATT TTC TCG CTG ACC AC-3 ) and UNR (5 -TCA GTA ATT AAT CAA ATT CGG AAG TCT-3 ) (Rodrigo et al, 2014). S. digitata genomic DNA was initially amplified by PCR using primers that were designed for Wolbachia surface protein (WSP) and the 16S rRNA genes of Wolbachia (Foster et al, 2008) and subsequently using primers that were employed for the multilocus sequence typing (MLST) for Wolbachia (Fig. 1).…”