Longicorn beetle that vectors pinewood nematode carries many Wolbachia genes on an autosome

Abstract

Monochamus alternatus is the longicorn beetle notorious as a vector of the pinewood nematode that causes the pine wilt disease. When two populations of M. alternatus were subjected to diagnostic polymerase chain reaction (PCR) detection of four Wolbachia genes, only the ftsZ gene was detected from one of the populations. The Wolbachia ftsZ gene persisted even after larvae were fed with a tetracycline-containing diet for six weeks. The inheritance of the ftsZ gene was not maternal but biparental, exhibiting a typical Mendelian pattern. The ftsZ gene titres in homozygotic ftsZ(+) insects were nearly twice as high as those in heterozygotic ftsZ(+) insects. Exhaustive PCR surveys revealed that 31 and 30 of 214 Wolbachia genes examined were detected from the two insect populations, respectively. Many of these Wolbachia genes contained stop codon(s) and/or frame shift(s). Fluorescent in situ hybridization confirmed the location of the Wolbachia genes on an autosome. On the basis of these results, we conclude that a large Wolbachia genomic region has been transferred to and located on an autosome of M. alternatus. The discovery of massive gene transfer from Wolbachia to M. alternatus would provide further insights into the evolution and fate of laterally transferred endosymbiont genes in multicellular host organisms.

Figure 1.

Molecular phylogenetic analysis of the ftsZ gene sequence identified from M. alternatus, together with representatives of the Wolbachia supergroups A and B. A supergroup D sequence (Wuchereria bancrofti) was used as an outgroup. A total of 523 aligned nucleotide sites were subjected to the analysis. A maximum likelihood (ML) tree is shown, and a neighbour-joining (NJ) tree exhibited the same topology. Bootstrap probabilities (ML/NJ) no less than 50 per cent are shown at the nodes. Accession numbers are in brackets.

Figure 2.

Inheritance patterns of the Wolbachia ftsZ gene in M. alternatus. (a) Cross between ftsZ⁺ male and ftsZ⁻ female. (b) Cross between ftsZ⁻ male and ftsZ⁺ female. Shade indicates the presence of the ftsZ gene. Letters in each circle of male or female symbol mean homozygous ftsZ (FF), homozygous no ftsZ (OO) or heterozygous ftsZ (FO). Numbers beneath each male or female symbol show the number of offspring obtained.

Figure 3.

Titres of the ftsZ gene in M. alternatus evaluated by quantitative PCR in terms of ftsZ gene copies per nanogram of total DNA in each sample. Solid columns show the adult individuals produced by the cross between a ftsZ⁺ male and a ftsZ⁺ female, shaded columns show those produced by the cross between a ftsZ⁻ male and a ftsZ⁺ female, and open columns show those produced by the cross between a ftsZ⁺ male and a ftsZ⁻ female. The means and standard deviations of 10 measurements are shown for the ovary, testis, female thoracic muscle and male thoracic muscle. Different letters (a and b) indicate statistically significant differences (Mann–Whitney U-test: p < 0.05 after Bonferroni correction).

Figure 4.

Wolbachia genes detected by PCR from M. alternatus, mapped on the whole genome of the Wolbachia strain wMel from D. melanogaster. Long and short lines indicate PCR-positive and PCR-negative genes, respectively. Arrowheads and arrows indicate the transferred genes that were only detected from the Kasumigaura population and the Miyako population, respectively.

Figure 5.

Fluorescent in situ hybridization of Wolbachia genes on the chromosomes of M. alternatus. (a) Male chromosomes in metaphase II arranged in the order of size. (b) Male chromosomes in the meiotic metaphase I. (c) Sperm nuclei. Wolbachia genes are visualized in yellow, and insect chromosomes are in blue.