Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique

Abstract

Among Australian endemic tephritid fruit flies, the sibling species Bactrocera tryoni and Bactrocera neohumeralis have been serious horticultural pests since the introduction of horticulture in the nineteenth century. More recently, Bactrocera jarvisi has also been declared a pest in northern Australia. After several decades of genetic research there is now a range of classical and molecular genetic tools that can be used to develop improved Sterile Insect Technique (SIT) strains for control of these pests. Four-way crossing strategies have the potential to overcome the problem of inbreeding in mass-reared strains of B. tryoni. The ability to produce hybrids between B. tryoni and the other two species in the laboratory has proved useful for the development of genetically marked strains. The identification of Y-chromosome markers in B. jarvisi means that male and female embryos can be distinguished in any strain that carries a B. jarvisi Y chromosome. This has enabled the study of homologues of the sex-determination genes during development of B jarvisi and B. tryoni, which is necessary for the generation of genetic-sexing strains. Germ-line transformation has been established and a draft genome sequence for B. tryoni released. Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline. Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment. More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

Figure 1

The distributions of B. tryoni, B. neohumeralis and B. jarvisi in Australia. (a) The distribution of B. neohumeralis is entirely within the broader distribution of B. tryoni. The ovals show the populations of B. tryoni that are differentiated by microsatellite analyses. Grey shading is the FFEZ (the Fruit Fly Exclusion Zone). Top right, B. tryoni, bottom right, B. neohumeralis. (b) The distribution of B. jarvisi and, inset, the distribution of its native host the Cocky apple (Planchonia careya). Top right, B. jarvisi.

Figure 2

The 4-way crossing scheme to produce an outbred Factory strain [9]. Three domesticated (inbred) strains are maintained in the Factory and crossed following the illustrated scheme with the fourth strain coming from the mass-reared flies. Genetic markers can be incorporated via one of the inbred strains (1*). If the marker is mitochondrial DNA, then 1* must be all female and in the second generation cross the females must be the daughters of the marked females in generation 1.

Figure 3

Fluoresence in testes and sperm from B. tryoni transformed with vector 1261. (a) A testis dissected from a 1261 transformed fly. Top, white light; bottom, with dsRED Ultra filter. (b) and (c) Confocal images of sperm from a 1261 transformed male (b) and a non-transformed male (c). Sperm were observed using an inverted Zeiss LSM 5 laser scanning confocal microscope. In each case left is the fluorescence image, the right image is bright field.

Figure 4

Mating scheme for the transfer of B. jarvisi markers into a B. tryoni background. (a) Mating scheme for transfer of B. jarvisi (white) mitochondria into B. tryoni (black) background. The mitochondrial type is indicated by small circle (white - B. jarvisi origin; black - B. tryoni origin). (b) Mating scheme for transfer of B. jarvisi (white) Y chromosome into B. tryoni (black) background. P, parents; F1, first filial generation; B1, backcross generation and number; and BF7, intercross of offspring from backcross generation 6. The proportion of the parental genomic contribution in the hybrids is indicated by the size of the corresponding black and white segments.

Figure 5

RNAi in B. tryoni with transformer and transformer-2. (a) Diagramatic representation of dsRNA used to inject B. tryoni early embryos. Four different dsRNA fragments were designed to cover either overlapping thirds or the entire transcript of both female-specific tra and non-sex specific tra-2. (b) Preliminary results for injections of dsRNA fragments of both tra and tra-2 into either 3 hour or 7 hour embryos of B. tryoni. (c) Results of crosses between transformed males and wildtype females. In two cases (1) all progeny that resulted were female. In other cases (2) normal 1:1 male:female ratios were observed.