A Novel Genetic Sexing Strain of Anastrepha Ludens for Cost-Effective Sterile Insect Technique Applications: Improved Genetic Stability and Rearing Efficiency

Anastrepha ludens (Loew) is one of the most destructive insect pests damaging several fruits of economic importance. The sterile insect technique (SIT) is used under an area-wide integrated pest management approach, to suppress these pest populations. Mass rearing facilities were initially established to produce sterile males of bi-sexual strains in support of SIT. The first genetic sexing strain (GSS) for A. ludens, Tapachula-7, based on pupal color dimorphism, was a key development since the release of males-only significantly increases the SIT efficiency. In this study, we document the development of a novel pupal color-based GSS. Twelve radiation-induced translocation lines were assessed as potential GSS in terms of recombination rates and rearing efficiency at a small scale. The best one, GUA10, was cytogenetically characterized: it was shown to carry a single translocation between the Y chromosome and chromosome 2, which is known to carry the black pupae marker. This GSS was further evaluated at medium and large scales regarding its genetic stability, productivity and quality versus Tapachula-7. GUA10 presented better genetic stability, fecundity, fertility, production efficiency, flying ability, and male mating, clear indicators that GUA10 GSS can significantly improve the efficacy and cost-effectiveness of SIT applications against this pest species.

Pupation Substrate Type and Volume Affect Pupation, Quality Parameters and Production Costs of a Reproductive Colony of Ceratitis capitata (Diptera: Tephritidae) VIENNA 8 Genetic Sexing Strain

Adequate pupation substrates and substrate volume are critical factors in the mass-rearing of insects for Sterile Insect Technique (SIT) applications. To identify an ideal pupation substrate for a reproductive colony of Ceratitis capitata (Wiedemann) VIENNA 8 genetic sexing strain, we first examined pupation in cellulose from recycled paper (cellulose I), sawdust, fine wheat bran, vermiculite and coconut fiber using a volume of 2.5–12.5 mL of substrate for each 5 mL volume of fly larvae. We found a positive relationship between substrate volume and pupation, with cellulose I generating the highest proportions of pupation and coconut fiber the lowest. Higher proportions of female flies (white pupae) pupated in sawdust. The proportion of female fliers increased as substrate volume rose in sawdust and coconut fiber, whereas it decreased in vermiculite and cellulose. In a second experiment, we tested three types of cellulose differing in physicochemical characteristics (celluloses I, II and III), sawdust, and fine wheat bran using a substrate:larvae ratio of 1:1. The three types of cellulose produced the highest pupation levels. The highest proportions of female fliers were observed in sawdust, and cellulose types III and II. Cellulose III and sawdust at relatively low volumes were more cost-effective to produce one million pupae than other substrates, including fine wheat bran used in a mass-rearing facility in Mexico.

Determining the Sterilization Doses under Hypoxia for the Novel Black Pupae Genetic Sexing Strain of Anastrepha fraterculus (Diptera, Tephritidae)

A common strategy used to maintain sterile fly quality without sacrificing sterility is to irradiate the insects under an oxygen-reduced atmosphere. So far, sterilizing doses for the South American fruit fly Anastrepha fraterculus have only been determined under normoxia. Our study reports for the first time the dose-sterility response under hypoxia for two different A. fraterculus strains. The pupae were derived from a bisexual strain (a Brazilian-1 population) and a recently developed genetic sexing strain (GSS-89). Two hours prior to irradiation, pupae were transferred to sealed glass bottles and irradiated when oxygen concentration was below 3%. Four types of crosses with nonirradiated flies of the bisexual strain were set to assess sterility for each radiation dose. For males from both strains, Weibull dose–response curves between radiation doses and the proportion of egg hatch, egg-to-pupa recovery, and recovery of adults were determined. The GSS males revealed high sterility/mortality levels compared to males from the bisexual strain at doses < 40 Gy, but a dose of 74 Gy reduced egg hatch by 99% regardless of the male strain and was considered the sterilizing dose. The fertility of irradiated females was severely affected even at low doses under hypoxia.

Development and characterization of a pupal-colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae)

Background Area-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of different fruit fly species during the last six decades. In addition, the development of genetic sexing strains (GSS) for different fruit fly species has allowed for sterile male-only releases and has significantly improved the efficacy and cost effectiveness of the SIT applications. The South American Fruit Fly Anastrepha fraterculus (Diptera: Tephritidae) is a major agricultural pest attacking several fruit commodities. This impedes international trade and has a significant negative impact on the local economies. Given the importance of sterile male-only releases, the development of a GSS for A. fraterculus would facilitate the implementation of an efficient and cost-effective SIT operational program against this insect pest species. Results For potential use in a GSS, three new morphological markers (mutants) were isolated in a laboratory strain of A. fraterculus sp. 1, including the black pupae (bp) gene located on chromosome VI. The black pupa phenotype was used as a selectable marker to develop genetic sexing strains by linking the wild type allele (bp+) to the Y-chromosome -via irradiation to induce a reciprocal Y-autosome translocation. Four GSS were established and one of them, namely GSS-89, showed the best genetic stability and the highest fertility. This strain was selected for further characterization and cytogenetic analysis. Conclusions We herein report the development of the first genetic sexing strain of a major agricultural pest, A. fraterculus sp. 1, using as a selectable marker the black pupae genetic locus.

Quantification and Impact of Cold Storage and Heat Exposure on Mass Rearing Program of Bactrocera dorsalis (Diptera:Tephritidae) Genetic Sexing Strain

Cold storage and heat exposure are crucial components of tephritid fruit fly mass-rearing programs, as they influence the development and fitness traits of produced flies. This work investigated the effects of cold storage on the pupal developmental parameters and quality of Bactrocera dorsalis (Hendel) genetic sexing strain (GSS) adults. Furthermore, the impact of short-term thermal exposure on the fecundity of B. dorsalis (GSS) that also underwent pupal cold storage was examined. Our results show that pupal development time, emergence rate, partial emergence rate, flight ability and fecundity were significantly affected by low temperature and pupal age and their interaction. Pupal cold storage did not pose negative impacts on the mating competition and response to methyl eugenol (ME) in the males. In addition, compared with the adults that were subjected to the same pupal storage protocol (five-day-old pupae stored at 13 °C), adult exposure to 41 °C for 1 h showed significant reparative effects on fecundity. In summary, the cold storage procedure of B. dorsalis (GSS) pupae has the potential to improve the flexibility and efficiency of mass-rearing schedules. Furthermore, short-term thermal exposure showed reparative effects on the fecundity costs induced by pupal cold storage in B. dorsalis (GSS).

Improving the Phenotypic Properties of the Ceratitis capitata (Diptera: Tephritidae) Temperature-Sensitive Lethal Genetic Sexing Strain in Support of Sterile Insect Technique Applications

The genetic sexing strain (GSS) of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) Vienna 8D53− is based on a male-linked translocation system and uses two selectable markers for male-only production, the white pupae (wp) and the temperature sensitivity lethal (tsl) genes. In this GSS, males emerge from brown pupae and are resistant to high temperatures while females emerge from white pupae, are sensitive to high temperatures. However, double homozygous females (wp tsl/wp tsl) exhibit a slower development rate compared to heterozygous males (wp+tsl+/wp tsl) during the larval stage, which was attributed to the pleiotropic effects of the tsl gene. We present the first evidence that this slower development is due to a different gene, here namely slow development (sd), which is closely linked to the tsl gene. Taking advantage of recombination phenomena between the two loci, we report the isolation of a novel temperature sensitivity lethal strain using the wp mutation as a morphological marker, which showed faster development (wp tsl FD) during the larval stage and increased in its temperature sensitivity compared with the normal tsl strain. Moreover, the introgression of this novel wp tsl FD combined trait into the Vienna 8D53− GSS, resulted in a novel Vienna 8D53− FD GSS, where females showed differences in the thermal sensibility, larval development speed, and productivity profiles. The modification of these traits and their impact on the mass rearing of the GSS for sterile insect technique applications are discussed.

Pre- and Post-Copulatory Competitiveness of the Genetic Sexing Strain Tapachula-7 of Anastrepha ludens (Diptera: Tephritidae)

Anastrepha ludens (Loew) (Diptera: Tephritidae) is an endemic pest of Mexico, attacking several fruits of economic importance. The Sterile Insect Technique (SIT), consisting of mass-rearing, irradiation and release of adults, is used to control this pest in affected areas. Currently, a genetic sexing strain (Tapachula 7, Tap-7) consisting only of A. ludens males is being released, yet we lack information on its sexual performance at the early ages when they are released and on its post copulatory behavior in terms of sperm transfer and mating inhibition. Here, sexual competitiveness at young ages and ability to inhibit female re-mating was compared between sterile Tap-7, Standard Bisexual (SB), or wild males both in laboratory and field cage conditions. Sperm stored by females mating with wild, Tap-7, or SB strain males was also compared. Six-day-old sterile Tap-7 males had low mating rates, however, by 7 d of age 80% of males had mated. Tap-7 males were just as likely as wild males to inhibit wild female re-mating. In field cages, sterile Tap-7 males mated faster than wild and sterile males from the SB strain and had comparable mating success to wild males. Females mated to sterile Tap-7 males stored more sperm than those mated to sterile SB males. Females mated to wild males stored more sperm than females mated to any other type of male. Mass-rearing had a greater effect on decreasing sperm stored by females than irradiation. We recommend continuing the release of the GSS strain of A. ludens.

Host Suitability and Fitness-Related Parameters in Coptera haywardi (Hymenoptera: Diapriidae) Reared on Irradiated Ceratitis capitata (Diptera: Tephritidae) Pupae Stemming From the tsl Vienna-8 Genetic Sexing Strain

Coptera haywardi (Ogloblin) is a pupal endoparasitoid of tephritid flies with great potential as a biological control agent worldwide as it does not attack other Diptera. To reach its full potential, its mass rearing needs to be enhanced lowering costs. Here, we focused on the use of irradiated pupae of Ceratitis capitata (Wiedemann) stemming from the temperature-sensitive lethal (tsl) Vienna-8 genetic sexing strain (= CcVienna-8), which is mass-produced in the San Juan Medfly and Parasitoid Mass Rearing Facility in Argentina. Exposure of 1- to 2-d-old CcVienna-8 pupae irradiated at 90 Gy to 6- to 8-d-old C. haywardi females at a 10:1 host/parasitoid ratio for 24 h turned out to be highly successful for the rearing of this parasitoid. High radiation doses (90–100 Gy) did not adversely influence fitness parameters of C. haywardi offspring F1, namely lifetime reproductive rates, adult life expectancy, and survival time. Demographic parameters in C. haywardi F1 from irradiated CcVienna-8 young pupae were improved compared to those values recorded from parasitoid originated from nonirradiated CcVienna-8 pupae. These findings will help to enhance parasitoid mass rearing for augmentative releases against medfly in Argentinean fruit-producing regions.

Evaluation of a Genetic Sexing Strain of the Oriental Fruit Fly as a Candidate for Simultaneous Application of Male Annihilation and Sterile Insect Techniques (Diptera: Tephritidae)

The oriental fruit fly, Bactrocera dorsalis (Hendel), is an important pest of fruits and vegetables, and many countries operate surveillance programs to detect infestations and, if needed, implement costly control efforts. The Male Annihilation Technique (MAT), which involves deployment of the male lure methyl eugenol (ME) to reduce or eliminate the male population, has been used as a stand-alone strategy or as a precursor to the Sterile Insect Technique (SIT), which involves the release of sterile males to generate sterile male by wild female crosses and the production of inviable progeny. Modeling suggests that simultaneous implementation of MAT and SIT, rather than sequential deployment, increases the probability of successful eradication. Previous research has shown that B. dorsalis males that have fed on ME show reduced tendency to re-visit ME sources. Also, males fed ME gain a mating advantage over nonfed males. The goal of the present study was to evaluate the suitability of a genetic sexing strain of B. dorsalis as a candidate for concurrent implementation of MAT and SIT. Evaluation focused on the timing of prerelease exposure to methyl eugenol to identify the male age at which feeding upon the lure both i) reduces postrelease attraction to ME-baited traps (thus allowing the operation of MAT) and ii) enhances postrelease mating competitiveness (thus increasing the effectiveness of SIT). Results indicate that prerelease ME feeding by 6-d-old males, with release the following day, would allow effective, concurrent implementation of MAT and SIT.

Radiation on Medfly Larvae of tsl Vienna-8 Genetic Sexing Strain Displays Reduced Parasitoid Encapsulation in Mass-Reared Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

Improvements in the mass rearing of Diachasmimorpha longicaudata (Ashmead) on larvae of the Vienna-8 temperature-sensitive lethal genetic sexing strain of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (= GSS Vienna-8) at the San Juan biofactory, Argentina, are currently under way. Lowering cost production is a key factor regarding parasitoid rearing. Thus, the variation in mass-reared parasitoid encapsulation levels and the incidence of superparasitism were determined; also, the gamma radiation dose-effect relation on host larvae and the influence of Mediterranean fruit fly strain were considered. Naked Mediterranean fruit fly larvae of both GSS Vienna-8 and a wild bisexual strain (= WBS) aged 6-d-old were irradiated at 0, 20, 40, 60, 80, 100, and 120 Gy, and exposed to parasitoid females. Melanization level was tested for encapsulated parasitoid larval first-instars (= L1). Non-irradiated and irradiated WBS larvae at 20–40 Gy displayed a significantly higher incidence of encapsulation when compared with GSS Vienna-8 larvae. The low melanized level in encapsulated parasitoid L1 was the most common melanization process at 72 h puparium dissection. A high melanized level was only found in non-irradiated WBS larvae. Irradiated GSS Vienna-8 larvae can neutralize the host immunological reactions over irradiated WBS larvae much more quickly. Superparasitism intensity in both Mediterranean fruit fly strains was not affected by radiation doses. High levels of superparasitism seemingly helped to overcome the host’s immune reaction by the surviving parasitoid larva. Parasitoid emergence increased from 60 Gy onwards in both Mediterranean fruit fly strains. Radiation in GSS Vienna-8 larvae may favor host’s antagonistic reactions decrease in relation with D. longicaudata development.