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. 2012 Jun 19:10:51.
doi: 10.1186/1741-7007-10-51.

Control of the olive fruit fly using genetics-enhanced sterile insect technique

Thomas Ant  1 , Martha Koukidou, Polychronis Rempoulakis, Hong-Fei Gong, Aris Economopoulos, John Vontas, Luke Alphey
Affiliations

Control of the olive fruit fly using genetics-enhanced sterile insect technique

Thomas Ant et al. BMC Biol. .

Abstract

Background: The olive fruit fly, Bactrocera oleae, is the major arthropod pest of commercial olive production, causing extensive damage to olive crops worldwide. Current control techniques rely on spraying of chemical insecticides. The sterile insect technique (SIT) presents an alternative, environmentally friendly and species-specific method of population control. Although SIT has been very successful against other tephritid pests, previous SIT trials on olive fly have produced disappointing results. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, resulting in asynchronous mating activity between the wild and released sterile populations, and low competitiveness of the radiation-sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is considered an essential prerequisite for successful olive fly SIT.

Results: We developed a set of conditional female-lethal strains of olive fly (named Release of Insects carrying a Dominant Lethal; RIDL®), providing highly penetrant female-specific lethality, dominant fluorescent marking, and genetic sterility. We found that males of the lead strain, OX3097D-Bol, 1) are strongly sexually competitive with wild olive flies, 2) display synchronous mating activity with wild females, and 3) induce appropriate refractoriness to wild female re-mating. Furthermore, we showed, through a large proof-of-principle experiment, that weekly releases of OX3097D-Bol males into stable populations of caged wild-type olive fly could cause rapid population collapse and eventual eradication.

Conclusions: The observed mating characteristics strongly suggest that an approach based on the release of OX3097D-Bol males will overcome the key difficulties encountered in previous olive fly SIT attempts. Although field confirmation is required, the proof-of-principle suppression and elimination of caged wild-type olive fly populations through OX3097D-Bol male releases provides evidence for the female-specific RIDL approach as a viable method of olive fly control. We conclude that the promising characteristics of OX3097D-Bol may finally enable effective SIT-based control of the olive fly.

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Figures

Figure 1
Figure 1
The OX3097 transposon and induced phenotypes in olive fly. (A) Diagrammatic representation of the OX3097 transposon. OX3097 comprises a fluorescent marker (hr5-IE1-DsRed2), and the female-specific tTAV expression system (tetO-Dmhsp70 minimal promoter - Cctra:tTAV) [15]. Sex-specific alternative splicing of the Cctra intron leads to production of tTAV and the initiation of a lethal tTAV positive-feedback loop in females only [14,15,34]. (b) Products of alternative splicing of Cctra:tTAV in (lane 1) male and (lane 2) female OX3097D-Bol olive flies. Three splice variants were detected, corresponding to Cctra transcripts M1, M2 and F1 [18] (identity confirmed by sequencing). Only females produce the F1 splice variant, corresponding to the reconstitution of the tTAV open reading frame and leading to production of functional tTAV. Lane M shows DNA size standards: 200-1,000 bp in 200-bp increments (Eurogentec Smartladder). (C) Penetrance and (D) tetracycline repressibility of female lethality in five OX3097 olive fly lines. Strains OX3097A-D-Bol &F-Bol are five insertion lines of OX3097 in olive fly. Penetrance and repressibility of female-specific lethality was assessed by crossing heterozygous males of each strain to virgin wild-type (WT) females, and collecting eggs on filter paper saturated with water containing either 0 μg/ml tetracycline or 100 μg/ml tetracycline. The sex ratio of adult progeny expressing the DsRed2 fluorescent marker is shown for each strain compared with wild-type (WT) progeny. Lines A, C and D showed fully penetrant female-specific lethality when reared in the absence of tetracycline (off-tet); that is, they produced no female progeny off-tet in this assay. In lines C and D, female-specific lethality was also efficiently repressed on-tet. (E) Fluorescence microscopy allows discrimination of OX3097D-Bol from wild type at larval, pupal, and adult stages. Photomicrographs of OX3097D-Bol and wild-type olive flies under (upper panels) fluorescence and (lower panels) bright-field illumination. Each panel shows OX3097D-Bol to the left and wild-type to the right: OX3097D-Bol and wild-type (1,2) larvae, (3,4) pupae, and (5,6) adults are shown. Expression of DsRed2 is clearly visible all over the OX3097D-Bol larvae and pupae, and in areas of less opaque cuticle (for example, the labellum, upper thorax, leg joints, and anus) of OX3097D-Bol adults.
Figure 2
Figure 2
Mating initiation times and re-mating propensity of OX3097D-Bol with wild olive flies. (A) Copulation initiation times were similar for OX3097D-Bol males and wild males. Copulation initiation times were recorded for all mating pairs; each pair contained a wild female and either an OX3097D-Bol male (left circle, n = 216) or a wild male (right circle, n = 161). Scotophase is the dark phase of a light/dark cycle. Each 'wedge' on the circular graphic represents a time-interval of 45 minutes; the radial length of the wedge indicates the proportion of total matings of that type that occurred in each time segment. Mean copulation initiation time for wild females and either OX3097D-Bol or wild males was 63 and 66 minutes before scotophase respectively. Peak mating activity times were not significantly different between the two types of male (P = 0.45, degrees of freedom (d.f.) = 1, circular statistics F-test). (B) Genotype of first mate (OX3097D-Bol or wild) did not affect female re-mating frequency or genotype of second mate. Of 188 females initially mated to OX3097D-Bol males 32 (17%) re-mated, of which 17 (9%) re-mated to wild males (open portion of left bar), and 13 (8%) to OX3097D-Bol males (solid portion of left bar). Of 296 females initially mated to wild males 55 (19%) re-mated, of which 23 (8%) re-mated to wild males (open portion of right bar) and 32 (11%) to OX3097D-Bol males (solid portion of left bar). Re-mating propensity of wild females initially mated with either an OX3097D-Bol male or a wild male were not significantly different (P = 0.7, d.f. = 1, χ2 test). Furthermore, the re-mating preference of the wild females was not found to differ significantly depending on first-mate choice (P = 0.38, d.f. = 1, χ2 test).
Figure 3
Figure 3
Population elimination by periodic release of OX3097D-Bol males. (A) The average daily egg production for each cage. Weeks 1 to 12 was the population stabilization period with 250 pupae added in the first week, and 200 pupae added to each cage per week thereafter. From week 13, 1,600 OX3097D-Bol pupae were added weekly into cages A and B. After week 13, weekly pupal return to the treatment cages was made proportional to the weekly egg production in the cage relative to the control cages. From 5 weeks after initiation of RIDL introductions, egg production in each treatment cage was consistently lower than in either control cage; the difference increased until eventual extinction of the wild-type population in both treatment cages by week 24 (12 weeks after the first RIDL release). Extinction was defined as 2 weeks of zero egg production. Egg numbers in control cages remained relatively stable. (B) Dead flies were removed from the cages weekly, and the numbers of dead females are shown. From 7 weeks after the initiation of RIDL release, increasingly fewer such females were recovered from the treatment cages than from the control cages. (C) Frequency of DsRed2 in treatment cages. Larvae selected for return were screened for presence of DsRed2 marker by fluorescence microscopy before being returned to the treatment cage (see Methods). The proportion of returning pupae carrying the OX3097D-Bol transgene reached 100% in both treatment cages by week 23 (10 weeks post-RIDL release). Olive fly females typically mate only once [21] (Figure 2B). Females start to lay eggs approximately 2 days after mating, and lay most of their eggs within the next 10 days. Egg to pupa development time was approximately 12 days. These pupae therefore indicate female mating choice of approximately 3 weeks before each measurement.
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