Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies

Allele Intersection Analysis: A Novel Tool for Multi Locus Sequence Assignment in Multiply Infected Hosts

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects

Cytoplasmic Incompatibility as a Means of Controlling Culex pipiens quinquefasciatus Mosquito in the Islands of the South-Western Indian Ocean

The use of the bacterium Wolbachia is an attractive alternative method to control vector populations. In mosquitoes, as in members of the Culex pipiens complex, Wolbachia induces a form of embryonic lethality called cytoplasmic incompatibility, a sperm-egg incompatibility occurring when infected males mate either with uninfected females or with females infected with incompatible Wolbachia strain(s). Here we explore the feasibility of the Incompatible Insect Technique (IIT), a species-specific control approach in which field females are sterilized by inundative releases of incompatible males. We show that the Wolbachia wPip(Is) strain, naturally infecting Cx. p. pipiens mosquitoes from Turkey, is a good candidate to control Cx. p. quinquefasciatus populations on four islands of the south-western Indian Ocean (La Réunion, Mauritius, Grande Glorieuse and Mayotte). The wPip(Is) strain was introduced into the nuclear background of Cx. p. quinquefasciatus mosquitoes from La Réunion, leading to the LR[wPip(Is)] line. Total embryonic lethality was observed in crosses between LR[wPip(Is)] males and all tested field females from the four islands. Interestingly, most crosses involving LR[wPip(Is)] females and field males were also incompatible, which is expected to reduce the impact of any accidental release of LR[wPip(Is)] females. Cage experiments demonstrate that LR[wPip(Is)] males are equally competitive with La Réunion males resulting in demographic crash when LR[wPip(Is)] males were introduced into La Réunion laboratory cages. These results, together with the geographic isolation of the four south-western Indian Ocean islands and their limited land area, support the feasibility of an IIT program using LR[wPip(Is)] males and stimulate the implementation of field tests for a Cx. p. quinquefasciatus control strategy on these islands

Wolbachia infection and expression of cytoplasmic incompatibility in Armigeres subalbatus (Diptera: Culicidae).

Polymerase chain reaction screening revealed that Armigeres subalbatus (Coquillett), a vector of filariasis, was infected with the intracellular bacteria Wolbachia. Laboratory crosses between infected males and uninfected females resulted in less than half the number of offspring than control crosses between uninfected individuals when young (2- to 3-d-old) males were used in the cross. However, imcompatibility was lost when old (14- to 17-d-old) males were used. Field-collected females did not show detectable cytoplasmic incompatibility, and this may be because of the age at which males mate in the field. We used head pigment fluorescence levels to age field males collected from mating swarms, and found that 25-63% of swarming males were older than 13 d. Male age may be one factor influencing the observed low levels of cytoplasmic incompatibility detected in the field

Wsp gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences