The Wolbachia mobilome in Culex pipiens includes a putative plasmid

Abstract

Wolbachia is a genus of obligate intracellular bacteria found in nematodes and arthropods worldwide, including insect vectors that transmit dengue, West Nile, and Zika viruses. Wolbachia's unique ability to alter host reproductive behavior through its temperate bacteriophage WO has enabled the development of new vector control strategies. However, our understanding of Wolbachia's mobilome beyond its bacteriophages is incomplete. Here, we reconstruct near-complete Wolbachia genomes from individual ovary metagenomes of four wild Culex pipiens mosquitoes captured in France. In addition to viral genes missing from the Wolbachia reference genome, we identify a putative plasmid (pWCP), consisting of a 9.23-kbp circular element with 14 genes. We validate its presence in additional Culex pipiens mosquitoes using PCR, long-read sequencing, and screening of existing metagenomes. The discovery of this previously unrecognized extrachromosomal element opens additional possibilities for genetic manipulation of Wolbachia.

Fig. 1

The artificially circularized genome of putative plasmid pWCP. a illustrates Contig O11_A and IS110 transposable element (TE) identified in our assembly. The red rectangles are regions of 100% nucleotide identity between the two contigs. Outward PCR primers were designed to amplify and confirm circularity of the sequence. b–e Gels for PCR tests to confirm a Wolbachia-associated circular genome. To verify the presence of arthropod DNA in our four Culex pipiens samples and the tetracycline-treated (TC) Culex quinquefasciatus samples, we PCR amplified a 708-bp sequence using LCO1490 and HCO2198 primers (b). A 438-bp fragment of the Wolbachia 16S rRNA gene (c), an approximately 1800 bp sequence amplified with the outward primers designed to support circularity of the genome, as illustrated in top panel (d) and a 431-bp of IS110 TE (e) were obtained in wild C. pipiens samples O03-O07-O11-O12 while no amplification was observed in Wolbachia-free samples. NC corresponds to negative control. f illustrates the complete genome. Each arrow represents an open reading frame (ORF). ORFs with no homology to a known function are shown in grey. ParA-like (green), RelBEtoxin–antitoxin operon (blue), and DnaB_C replicative DNA helicase (orange) that is disrupted by the ISWpi12 TE of the IS110 family (purple) are represented by arrows (with an e value < e⁻¹² from an NCBI Conserved Domain or Pfam Search). Black squares represent the location of (1) the variable number tandem repeat (VNTR) and (2) the extragenic palindrome (EP) region

Fig. 2

Alignment of MinION long reads to pWCP. a The alignment of long reads that cover >50% of the pWCP genome (only 12 of 13 total long reads are shown; we omitted 1 from this display solely due to space considerations). Each rectangular figure shows high scoring pairs (HSPs) and their alignments between pWCP and long reads. The broken HSPs that are parallel to each other are due to low-quality regions in long read sequences, and they are shown in different shades. Concentric circles around pWCP demonstrate the alignment of each long read and their start and stop positions. b The alignment of long reads that cover <50% of the pWCP genome (only 5 of 6 are shown). Every long read shown in the figure has a hit to the IS110 TE

Fig. 3

pWCP contains a variable number tandem repeat (VNTR) region and extragenic palindrome (EP) sequence. a A VNTR region is located between parA and uncharacterized gene in the circular genome. While the number of repeats varies across individuals (b), the 36- and 33-bp spacers are conserved. Each black arrow represents a 16-nt repeat. The predicted DNA structure of the EP sequence is illustrated in c where color indicates probability of each base pairing. Red represents the strongest probability, whereas blue is the lowest

Fig. 4

Wolbachia metapangenome in the context of wPip Pel genome synteny. The figure shows the presence–absence of 1166 gene clusters in the pangenome of four Wolbachia metagenome-assembled genomes (MAGs) and the reference genome wPip Pel. The gene clusters (i.e., groups of homologous genes based on amino acid sequence identity) are organized based on wPip Pel synteny. Each MAG is represented by two layers, where the first layer indicates the presence or absence of a gene cluster in a given MAG, and the other shows the average coverage of each Wolbachia MAG O07 gene cluster in the corresponding C. pipiens metagenome. The second to last layer shows whether genes in a given gene cluster have a match in NCBI’s COGs. The outermost layer associates gene clusters with previously identified prophage regions in the wPip Pel genome. The number of gene clusters assigned to WOPip prophage regions are indicated in parenthesis