Sterile males of Ceratitis capitata (Diptera: Tephritidae) as disseminators of Beauveria bassiana conidia for IPM strategies

Sterile Mediterranean fruit fly, Ceratitis capitata (Wied.), males were evaluated as vectors to spread Beauveria bassiana (Bals) conidia to wild C. capitata populations under field conditions. The inoculated sterile males were released by air, using the chilled adult technique over 7000 ha of coffee growing in Chimaltenango, Guatemala, Central America. The impact of releases was determined using dry traps baited with a food attractant. The effects of these releases on Apis mellifera, Linnaeus (honey bee), Hypothenemus hampei, Ferrari (coffee berry borer) and the parasitic mite Varroa destructor (Oudeman) were also evaluated. Inoculated sterile males were able to transmit fungal spores to 44% of the wild C. capitata flies captured in traps, which likely were infected through intra- and intersexual interactions during leks, mating or mating attempts. There was no transmission of the fungal spores to non-target insect species such as coffee berry borer, honey bees or varroa. We conclude that sterile males of Mediterranean fruit fly inoculated with B. bassiana can act as effective vectors of conidia to wild populations, constituting a safe, environmentally friendly and selective alternative for suppressing the medfly under a Sterile Insect Technique-based IPM approach.     

Do sterile females affect the sexual performance of sterile males of Anastrepha ludens (Diptera: Tephritidae)?

The release of sterile males to suppress the Mexican fruit fly Anastrepha ludens (Loew) population was simulated in field cages to compare the sexual performance of sterile males with wild females in the presence and absence of sterile females. Using different release ratios of sterile and wild individuals, this study showed that the presence of sterile females negatively affected the sexual performance of sterile males in mating with wild females. We found that the sterility induced was proportional to the magnitude of the sterile/fertile ratio. These results indicate that the release of sterile males without sterile females can significantly improve the efficiency of the sterile insect technique for the control of A. ludens.     

Seasonal variation of the capture and field distribution of sterile Anastrepha ludens in Tamaulipas,Mexico

Anastrepha ludens (Loew) (Diptera: Tephritidae) is one of the most important citrus pest in Mexico. The sterile insect technique (SIT) is used against pest populations of fruit flies for suppression, eradication, containment and prevention to reduce damages in fruit‐growing areas. In this study, we analyzed the seasonal variation of captures and field distribution of sterile A. ludens released in different seasons of the year in north‐eastern Mexico. Chilled releases were conducted by air at constant densities per ha on a citrus area for a period of 32 weeks that included the coldest and warmest seasons that is winter, spring and summer. Multilure traps baited with torula yeast pellets were used to capture sterile flies. Fly capture data were compared over the three seasons and correlated with climate. The lowest number of captures of the sterile insect occurred in the summer and the highest in winter and spring. High and low temperatures were negatively correlated with fly captures. Field distribution was also negatively correlated with high temperatures in summer, but no relationships were observed in winter and spring. No relationships were observed between rainfall with capture and field distribution of sterile flies. These results indicate that summer is a season involving agro‐ecological and environmental constraints for the capture and field distribution of sterile flies. This study may be useful for enhancing release strategies and optimizing economic resources in north‐eastern Mexico. Further research on the behaviour of sterile flies under stressful environments is suggested.     

Mapping the spatial variability of the field distribution of sterile Anastrepha ludens over a release area

The sterile insect technique (SIT) is an environmental-friendly method used against Anastrepha ludens Loew (Diptera: Tephritidae) populations. This study aimed to perform an analysis of the spatial variability of the field distribution of sterile A. ludens using a geostatistical approach along with Geographic Information Systems (GIS). Field data on captures of sterile A. ludens during a Valencia orange season over a release area were analysed using spherical, exponential and Gaussian variograms. Such variograms were evaluated by criteria such as the mean absolute error, average standard error, root mean square error and the coefficient of determination. Results revealed a spatially structured distribution of sterile A. ludens across the release area. Interpolated models by Ordinary Kriging technique exhibited continuous surfaces evidencing spatial heterogeneity of the distribution of flies. Such a result evidenced that the spatial dynamics of flies significantly varied despite the planned uniform coverage of the release. The GIS led to integrating spatial information of the spatial dynamics through one single model. The release activity should be improved westward of the studied area, as the final model suggested that the ratio sterile: wild is lower than that in the east. This study provides insights into the spatial analysis of the distribution of sterile flies further than one single geographical point. Moreover, it highlights geostatistical techniques and GIS as useful tools for the assessment of the impact and quality of the release activity over fruit-growing areas subjected to an area-wide integrated pest management approach.     

Mating behaviour of Dacus ciliatus (Loew) [Diptera:Tephritidae]: comparisons between a laboratory and a wild population

As part of the development of a sterile insect technique (SIT) application for the Ethiopian fruit fly, Dacus ciliatus, we studied the mating behaviour of a laboratory‐adapted strain (a 4‐year‐old colony kept for more than 40 generations) and a wild population. Effects of laboratory rearing and irradiation were assessed by carrying out mating compatibility and male mating competitiveness tests using a 1:1 ratio between irradiated (120 Gy) laboratory males and non‐irradiated wild males. Mating behaviour was studied on host and non‐host plants under field cage conditions. To assess the effect of mass rearing upon male performance, we repeated the mating competitiveness test using non‐irradiated laboratory insects. The findings indicated a high degree of compatibility among the two populations and satisfactory competitiveness of the irradiated laboratory males (ca. 35%). The competitiveness of non‐irradiated laboratory males was also ca. 35%, suggesting that no adverse effects resulted from their irradiation. Mating occurred only at twilight and mainly on the underside of leaves of non‐host plants (lemon trees). Findings are discussed in view of their implications for a future application of SIT against this fruit fly pest.     

CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni

The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a polyphagous horticultural pest in Australia that is capable of causing significant damage to more than 100 different host fruits and vegetables. Chemical applications and ecological control strategies, such as the sterile insect technique (SIT), are commonly used to suppress established populations and eradicate invasive outbreaks following migration. The recently published B. tryoni draft genome provides new opportunities to identify candidate genes for targeted genome modification in order to generate advanced genetic strains for management using sterile insect strategies. Here, we demonstrate CRISPR/Cas‐mediated mutagenesis in B. tryoni through generating a series of frame‐shift mutations in the ATP‐dependent binding cassette transporter, white, causing a classic white‐eye phenotype. This work establishes methods for CRISPR/Cas genome editing in tephritids and demonstrates its potential for developing genetic sexing strains which could be used for SIT‐based pest control.     

Different host plant species modifies the susceptibility of Bactericera cockerelli to the entomopathogenic fungus Beauveria bassiana

Bactericera cockerelli (Sulc.) is a serious pest of solanaceous crops and a vector of the plant pathogen Candidatus Liberibacter psyllaurous. Entomopathogenic fungi are the most important biological control alternatives for this pest. Host plant species, however, can modify the outcomes of insect–pathogen interactions. We conducted laboratory experiments to quantify the virulence of two isolates of the entomopathogenic fungus Beauveria bassiana (Bals. [Vuill.]), BB40 and BB42, against third instar B. cockerelli nymphs maintained on chilli pepper plants. Owing to the lack of difference in virulence against B. cockerelli nymphs on chilli pepper between the two B. bassiana isolates, only BB42 was used to: compare virulence against nymphs maintained on either chilli pepper, potato or tomato; and in vivo conidia production from nymphs maintained on different host plants. Virulence of the two B. bassiana isolates against B. cockerelli nymphs was similar. Bactericera cockerelli nymphs maintained on tomato were more susceptible to B. bassiana than nymphs maintained on potato or chilli peppers. Infected nymphs maintained on chilli peppers produced the greatest number of conidia followed by infected nymphs maintained on tomato and potato. Host plant affected the susceptibility of B. cockerelli to B. bassiana isolate BB42 and subsequent conidia production. The implications of our results for microbial control of B. cockerelli by B. bassiana are discussed.     

Precocious Mexican fruit fly methoprene‐fed males inhibit female receptivity and perform sexually as mature males

The sterile insect technique (SIT) has been used successfully for the control of fruit flies. The efficiency of this technique can be significantly reduced when sterile released insects are exposed to adverse conditions and predators, as a great number of sterile insects die before reaching sexual maturity and thus fail to mate with wild females. Treatments with juvenile hormone (JH) analogues such as methoprene (M) significantly reduce the time to reach sexual maturity by sterile Anastrepha ludens (Loew) (Diptera: Tephritidae) males. In this study, we compared the sexual performance of non‐treated sexually mature males with young males that had been sexually accelerated with M. Furthermore, we compared the ability of M‐fed males in inhibiting female remating compared with sexually mature males. Results showed that at 5 days M‐fed males had lower mating success than mature males; however, 6‐day‐old (0.1%) M‐fed males had the same amount of matings as mature 13‐day‐old males. Young 5‐ to 10‐day‐old M‐fed males also had similar number of matings as mature non‐treated 12‐ to 17‐day‐old males. There were no differences in copula duration between treatments. Moreover, there were no differences between the fertility, fecundity or refractory period of females mated with either young male fed M or normal sexually mature males. These results indicated that young males that were sexually accelerated with M have the same sexual performance as non‐treated sexually mature males. Implications of using M as a pre‐release treatment for A. ludens controlled through SIT are discussed.     

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of Bactrocera oleae populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass‐rearing techniques impedes the successful application of area‐wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera – such as Erwinia, Providencia, Enterobacter, and Klebsiella – were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community.     

Transgenic sexing system for Ceratitis capitata (Diptera: Tephritidae) based on female-specific embryonic lethality

Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hid^Ala5 to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early-acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early-acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.     

Pupal X-ray irradiation influences protein expression in adults of the oriental fruit fly,Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is a pest of fruit in the Asia–Pacific region and also, due to quarantine restrictions, a threat to California fruit production. Area-wide suppression of B. dorsalis integrated several approaches including the sterile insect technique (SIT). SIT involves exposing juveniles to gamma radiation and releasing sterile males in substantial numbers, where they successfully compete for wild females. The resulting infertile eggs lead to reduction of the pest populations. Although these protocols are well documented, arising issues about the international transport and distribution of radioactive products is creating difficulties in use of radioactive sources for sterilizing radiation. This led to a shift toward use of X-ray irradiation, which also sterilizes male and female insects. However, use of X-ray technologies is in its infancy and there is virtually no information on the effects of irradiation, other than sterilization, at the physiological and molecular levels of fruit fly biology. We posed the hypothesis that sterilizing male oriental fruit flies via radiation treatment also influences protein expression in the flies. We found that exposing pupae to X-ray irradiation impacted expression of 26 proteins in adult females and 31 proteins in adult males. Seven proteins (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, larval cuticle protein 2, sarcoplasmic calcium-binding protein alpha-B and A chains, general odorant-binding protein 99b, polyubiquitin, and protein disulfide-isomerase) were impacted in both sexes. Some of the proteins act in central energy-generating and in pheromone-signal processing pathways; we infer that males sterilized by X-ray irradiation may be enfeebled in their ability to compete with wild males for females in nature.     

Understanding Bactrocera dorsalis trapping to calibrate area-wide management

Knowing the dispersal of released insects and estimating the size of field populations are keys to the successful implementation of area-wide management (AWM) programmes based on the sterile insect technique (SIT), as they determine the release strategy of sterile males. Mark–release–recapture (MRR) is a common method used to estimate field populations and spatiotemporal dynamics. However, the extent to which the pest is attracted to lures is often difficult to identify, thereby biasing extrapolation to movement patterns and population size. We performed MRR experiments on the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in a fruit-growing area in Senegal. Methyl eugenol and protein baits were used to trap males and females, respectively. After studying the suitability of marking B. dorsalis with fluorescent pigments at the laboratory, two releases of marked sterile flies were organized in the centre of an organic mango orchard, first in the late mango fruiting stage and second in the fruit setting stage. Traps were placed symmetrically along a 250 and 500 m radius from the release point. A very small fraction of the released individuals was recaptured: 0.45% in the first release and 3.08% in the second. Trapping of both sterile and wild flies was completely anisotropic. Sterile flies were mostly trapped at a relatively short distance (250 m) from the release point, in the first two days. Male trapping using methyl eugenol was highly effective, whereas the response of females to food bait traps was low. The direction of the wind was the main driver of recapture, with flies heading upwind. The results underline the importance of taking the odour plume around the traps into account when estimating populations, and the heterogeneous spread of the wild population in the landscape for the set-up of the release strategy of sterile insects for SIT-based AWM.     

Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by Botrytis cinerea

The entomopathogenic fungus Beauveria bassiana is widely used for insect pest control and can produce three distinct infective unit types under different nutritional and environmental conditions: aerial conidia, blastospores, and submerged conidia. Here, we identified endophytic colonization ability and existing forms of the three types of B. bassiana infective units after inoculating Arabidopsis plants via soil drenching, and tested their effects on their presence mold disease caused by Botrytis cinerea. We found that all B. bassiana infective unit types colonized Arabidopsis leaves, with germinating and producing hyphae by hydrophilic blastopores and submerged conidia; further, we showed that blastospores were more effective in defending against B. cinerea, compared with aerial conidia. These findings suggest that in addition to aerial conidia, the colonization of other two types of entomopathogenic fungal infective units could also have important impacts on plant resistance. This study contributes to better understanding on the function of B. bassiana as fungal endophytes, which could lead to a new paradigm for how to successfully use these organisms in biological control against plant diseases.     

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

Background  

The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.     

Female presence enhances sexual performance of sterile Anastrepha ludens males of the Tapachula-7 GSS strain

The sterile insect technique (SIT), used for the control of many tephritid fly pests, is based on the rearing and release of large numbers of sexually competitive sterile insects into a wild population. In the interest of reducing expenses and increasing SIT effectiveness, genetic sexing strains (GSS) have been developed. These strains allow the production and release of only males. The objective of our study was to assess the effects of pre-release adult exposure to methoprene and to females on the mating propensity and mating competitiveness of GSS sterile males of Anastrepha ludens (Loew) (Diptera: Tephritidae). GSS sterile males were kept on a protein-sugar (protein-fed) or a protein-sugar-methoprene diet and were exposed to different proportions of females for the normal pre-release period of 5 days. Using laboratory and field-cage bioassays, we examined the influence of methoprene and female presence on the mating success of sterile males of 3–9 days old, in competition for wild females with untreated males and with wild males. Methoprene and female exposure had no significant effects on male mating success in the laboratory, whereas age had a positive relationship with the number of copulations observed. However, in field-cage bioassays, males exposed to females obtained a higher number of copulations than unexposed control males. Possible implications of these findings for programs that use GSS and especially for the campaign against Mexican fruit flies are discussed.     

Bacterially enriched diet improves sexual performance of sterile male Mediterranean fruit flies

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), (medfly) is a polyphagous and cosmopolitan agricultural pest, targeted in many areas for control by the Sterile Insect Technique (SIT). Our objective in the present study was to test the hypothesis that a bacterially enriched diet provided to sterile males will improve their sexual performance in competitive settings that emulate natural conditions. Specifically we determined how feeding on diets enriched with Klebsiella oxytoca affected the ability of sterile males to compete for wild females against wild males, their ability to inhibit female receptivity, and their survival. We found that enriching the sterile male diet with K.oxytoca significantly improved mating competitiveness in the laboratory and in field cages. In addition, bacterially enriched sterile males inhibited female receptivity more efficiently than sugar fed males and survived longer duration of starvation. We conclude that inoculating mass reared sterile flies with bacteria prior to their release is a valid approach to improve the efficacy of SIT.     

Disparity in sexual behaviour between wild and mass‐reared Mexican fruit flies

The sterile insect technique (SIT) is currently used to control Mexican fruit fly Anastrepha ludens Loew (Diptera: Tephritidae). However, mass‐rearing can alter the quality of released males. If males that are mass‐reared have behaviours different from those of their wild counterparts, then this may diminish the effectiveness of SIT. Questions remain as to whether wild females may be able to detect the male condition before, during and/or after copulation with a mass‐reared male. In the present study, copula duration, female remating, female fecundity and fertility of both mass‐reared and wild A. ludens are evaluated. Marked differences are found between mass‐reared and wild females. Specifically, mating latency is longer and copula duration is shorter for wild females compared with mass‐reared females. Importantly, there are no significant differences in mating latency, copula duration or remating probability between wild females paired with either mass‐reared or wild males. All mass‐reared females remate, whereas only approximately half of the wild females remate after first mating with either a wild or mass‐reared male. Fecundity of wild females mated to either wild or mass‐reared males is approximately one‐third lower than that of mass‐reared females, confirming that mass‐reared females may have been selected for high fecundity and are adapted to laboratory conditions. Fertility of females that mate with a wild male for only 10 min is not significantly different from that achieved via a full‐length copulation. By contrast, females mating with mass‐reared males need copulation durations of at least 40 min to achieve fertility comparable with that achieved via a full‐length copulation. The findings of the present study have important implications for A. ludens controlled through SIT and broaden our understanding on the copulatory and post‐copulatory behaviours between wild females and mass‐reared males.     

Light stimulates conidiation of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a commercially important entomopathogenic fungus. Like other insect fungal pathogens, B. bassiana usually produces asexual reproductive bodies, conidia, for dispersal, transmission and infection of insects. Adequate mass-production of high quality conidia is crucial to development of an efficient B. bassiana insecticide. However, little is known about details of conidiation in this fungus in response to environmental signals, which limits understanding of the mechanism of conidiation and improvement in conidia production. Here, morphologenetic changes of B. bassiana under different light conditions are reported. When cultured in total darkness, B. bassiana hyphae can grow continuously with few reproductive structures differentiated, while illumination with white light resulted in prolific formation of conidiophores bearing abundant conidia, indicating that light could stimulate conidiation of B. bassiana. Among the single colour lights tested, blue light was the most effective to stimulating sporulation. Colonies became adapted for blue light stimulus only after hyphae had grown in total darkness for at least 96 h, whereas the photoadaptation obviously declined after 144 h. For the exposure time, 3 min of blue light pulse was enough to stimulate conidiation in the photoadapted mycelia, while prolonged light exposure over 3 min resulted in a decrease in conidia yield. Our results provided useful clues for understanding the mechanism of conidiation mediated by light in B. bassiana.     

Mate choice by wild and mass‐reared females of the Mediterranean fruit fly

The sterile insect technique (SIT) is used to control Mediterranean fruit fly, Ceratitis capitata (Wiedemann), but its effectiveness is limited by low sexual competitiveness of mass‐reared males. This study investigated whether wild and mass‐reared [from a temperature sensitive lethal (tsl) genetic sexing strain] females display similar mate preferences and thus exert similar selective forces on the evolution of male courtship behaviour. Wild females preferred wild males over tsl males, whereas tsl females mated indiscriminately. The probability that mounting resulted in copulation was related to the duration of pre‐mount courtship for wild females, and wild males performed longer courtships than tsl males. Copulation occurred independently of courtship duration in tsl females. Counter to the aim of the SIT, female choice by tsl females appears to promote the evolution of male behaviour disfavoured by wild females.     

Capture of mass‐reared vs. wild‐like males of Ceratitis capitata (Dipt., Tephritidae) in trimedlure‐baited traps

The sterile insect technique (SIT) is widely used to suppress or eradicate infestations of the Mediterranean fruit fly, Ceratitis capitata (Wied.), an insect whose broad polyphagy poses a serious threat to fruit and vegetable crops. The SIT involves the production, sterilization and release of sterile insects to obtain sterile male by wild female matings, thus yielding infertile eggs. Mass‐rearing over many generations is known to produce dramatic changes in the behaviour and life history of C. capitata. This study investigated the possibility that mass‐rearing also alters male response to trimedlure, a sex‐specific attractant widely used in detection and monitoring programmes. We compared captures of released males from a mass‐reared strain and a recently established colony of wild flies in trimedlure‐baited Jackson traps at three spatial scales – open field, large field enclosures (75 m²) and small field cages (7 m²) – in two separate years. In the first year, males were used independently of flight ability, while in the second year only males with demonstrated flight capability were used. Trap capture was scored 2 days after release for the open field and the large field enclosures but either 1 h or 1 day after release in the small field cages. The findings were consistent across these different experiments: wild‐like males were captured in significantly greater numbers than mass‐reared males in both years of study, except in the trials lasting 1 day in the small field cages where significantly more wild than mass‐reared males were captured in 1 year but not the other. These results are compared with other studies, and their implications for SIT are discussed.