Comparative sequences of the Wolbachia genomes of drug-sensitive and resistant isolates of Dirofilaria immitis

Abstract

The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia.Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8-12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40-75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3,000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected. Phylogenetic analysis suggested that the endosymbionts of the drugsusceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.

Whole-genome reference of Dirofilaria immitis from Australia to determine single nucleotide polymorphisms associated with macrocyclic lactone resistance in the USA

“…The filarial nematode D. immitis harbours bacterial ( Wolbachia ) endosymbionts that could become resistant to the antibiotic doxycycline - an established component for treatment of heartworm-infected dogs ( Belanger & Perkins, 2010 ; Shin et al., 2020 ). The whole genome sequencing approach is suitable for evaluating the genomic sequence of both the nematode as well as its endosymbiont.…”

“…The whole genome sequencing approach is suitable for evaluating the genomic sequence of both the nematode as well as its endosymbiont. The Wolbachia -endosymbiont has been shown to include 67 loci specifically found in resistant D. immitis isolates but not in susceptible D. immitis USA isolates ( Shin et al., 2020 ). Around 3,000 SNPs in the wDi sequences have been found between drug-sensitive and resistant isolates of D. immitis ( Shin et al., 2020 ).…”

“…The Wolbachia -endosymbiont has been shown to include 67 loci specifically found in resistant D. immitis isolates but not in susceptible D. immitis USA isolates ( Shin et al., 2020 ). Around 3,000 SNPs in the wDi sequences have been found between drug-sensitive and resistant isolates of D. immitis ( Shin et al., 2020 ). Further studies including additional D. immitis sampling and genome sequencing are needed to understand why there is almost twice the number of SNPs in Australian wDi.…”

Genome structure and population genomics of the canine heartwormDirofilaria immitis

“…A total of 12 publicly available datasets were used from the following ENA BioProject IDs, including (i) PRJNA681066: data from five adult samples from New South Wales (NSW), Australia (Lau et al, 2021); (ii) PRJNA588995: data from five microfilaria samples from the USA, including two from Georgia (GEO), one each from Illinois (ILL), Louisiana (LOU), and Mississippi (MIP) (Shin et al, 2020); and (iii) PRJEB2554: two adult samples, one from Georgia (GEO), USA, and one from Pavia (PAV), Italy (Godel et al, 2012).…”