Culex quinquefasciatus carrying Wolbachia is less susceptible to entomopathogenic bacteria

Abstract

In an attempt to evaluate the susceptibility of the mosquito Culex quinquefasciatus to bacterial agents, a population naturally infected with a Wolbachia pipientis wPipSJ native strain was tested against the action of three bacterial mosquitocides, Bacillus thuringiensis subsp. israelensis, Bacillus wiedmannii biovar thuringiensis and Lysinibacillus sphaericus. Tests were carried out on mosquito larvae with and without Wolbachia (controls). Cx. quinquefasciatus naturally infected with the native wPipSJ strain proved to be more resistant to the pathogenic action of the three mosquitocidal bacterial strains. Additionally, wPipSJ was fully characterised using metagenome-assembled genomics, PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and MLST (MultiLocus Sequence Typing) analyses. This Wolbachia strain wPipSJ belongs to haplotype I, group wPip-III and supergroup B, clustering with other mosquito wPip strains, such as wPip PEL, wPip JHB, wPip Mol, and wAlbB; showing the southernmost distribution in America. The cytoplasmic incompatibility phenotype of this strain was revealed via crosses between wildtype (Wolbachia⁺) and antibiotic treated mosquito populations. The results of the tests with the bacterial agents suggest that Cx. quinquefasciatus naturally infected with wPipSJ is less susceptible to the pathogenic action of mosquitocidal bacterial strains when compared with the antibiotic-treated mosquito isoline, and is more susceptible to B. thuringiensis subsp. israelensis than to the other two mosquitocidal agents.

Figure 1

Wolbachia effect on fitness of Culex quinquefasciatus. (a) Mean of percentage of hatched eggs. N_eggs: total eggs analyzed per line. (b) Kaplan–Meier survival curves of Wolbachia, (w⁺)- or (w⁻)-lines.

Figure 2

Effect of Bacillus thuringiensis sp. israelensis (Bti H14), Bacillus wiedmannii biovar thuringiensis (Bwt FCC 41) and Lysinibacillus sphaericus (Ls 2362) on (w⁺) or (w⁻) larvae. (a, c, e) Average of dead larvae (w⁺ and w^-) by entomophatogenic bacteria strain. Means with different letters are significantly different. (b, d, f) Linear regressions of mortality per concentration between w⁺ and w⁻ lines with different entomopathogenic bacteria strain [Inset b: magnification of the curves at initial concentrations to visualize differences].

Figure 3

Maximum-likelihood phylogenetic tree reconstruction performed with GET_PHYLOMARKERS and IQ-TREE (500 ultrafast boostrap replicates) according to the Wolbachia core genome from 30 available genomes. Numbers in the branches indicate ultrafast bootstrap value, and letters on the right indicate the Wolbachia supergroup. The symbol // indicates a trimmed branch.

Figure 4

Complete unidirectional cytoplasmic incompatibility in Culex quinquefasciatus lines. Egg hatching percentage in crosses between the wild type (w⁺PipSJ) and tetracycline-treated (w⁻PipSJ) strains of Cx. quinquefasciatus. Sixteen couples were used in each type of crossing. Each dot represents a mosquito egg raft from a unique female, carrying approximately 150 eggs per raft. The median number of hatched eggs/egg-raft are shown in horizontal bars.