Genetic sexing strains in medfly,Ceratitis capitata,sterile insect technique programmes

The introduction of genetic sexing strains (GSS) into medfly, Ceratitis capitata(Wiedemann), sterile insect technique (SIT) programmes started in 1994 and it was accompanied by extensive evaluation of the strains both in field cages and in open field situations. Two male-linked translocation systems, one based on pupal colour, wp, and the other based on temperature sensitivity, tsl, have been used in medfly SIT programmes and they have quite different impacts on mass rearing strategy. In strains based on tsl, female zygotes are killed using high temperature and for wpstrains, female and male pupae are separated based on their colour. In all these systems the colony females are homozygous for the mutation requiring that the mutation is not too deleterious and the males are also semi-sterile due to the presence of a male-linked translocation. Managing strain stability during large-scale mass rearing has presented some problems that have been essentially solved by selecting particular translocations for GSS and by the introduction of a filter rearing system (FRS). The FRS operates by removing from the colony any recombinant individuals that threaten the integrity of the strain. The use of GSS opens up the possibility of using the SIT for suppression as opposed to eradication and different radiation strategies can be considered. Some of the many field trials of the strains that were carried out before the strains were introduced into operational programmes are reviewed and an overview is given of their current use.     

Remating in Ceratitis capitata sterile males: Implications in sterile insect technique programmes

Sterile insect technique (SIT) is used, among other biological control tools, as a sustainable measure for the management of Ceratitis capitata Wiedemann (Diptera: Tephritidae) in many agricultural regions where this pest can trigger severe economic impacts. The tendency of wild females to remate multiple times has been deeply studied; it has been a common point of controversy when evaluating SIT programmes. Nevertheless, the remating potential of the released sterile males remains unknown. Here, under laboratory conditions, the remating capability of mass-reared sterile males was determined. Wild-type virgin females were offered to sterile males (Vienna-8 strain), which had the opportunity to mate up to four consecutive times. The remating assays were carried out at 24 hr, 48 hr, 4 days and 7 days after the first mating. At the end of each tested time period, males were divided according to their mating response, mated or unmated, and subsequently reused for the next round of mating assays. The frequency of successful remating in each tested time period was obtained. Insemination was confirmed by determining the sperm transfer in mated female spermathecae by quantitative real-time PCR. Our results demonstrate that 73% of the mass-reared sterile males were able to remate 24 hr after the first mating, 55% of which remated again the day after. Close to 25% of the V8 sterile males tended to copulate in all of the four mating opportunities. The qPCR analysis of the spermathecae contents verified an effective transfer of V8 sperm to wild females with every mating; 99% of copulations resulted in sperm transfer. These findings shed light on the remating potential of V8 sterile males, an aspect until now underestimated in many SIT programmes.     

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.     

A novel molecular approach to assess mating success of sterile Ceratitis capitata (Diptera: Tephritidae) males in sterile insect technique programs

Areawide sterile insect technique (SIT) programs against Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), are increasingly implemented worldwide. A key issue in SIT is to assess mating success of released sterile males, which could be currently estimated by egg hatchability and by stored sperm head measurements. We report here on a novel molecular approach that would allow detecting the presence of Mediterranean fruit fly sterile male sperm in the female spermathecae under field conditions, as a precise marker to assess mating performance. The simplicity (only two polymerase chain reactions) and reliability of this method, jointly with the capability to detect Vienna sperm in wild Mediterranean fruit fly maintained in monitoring traps for 7 d under field conditions, suggest that it could be an efficient tool when coupled with areawide SIT programs.     

The isolation of microsatellite loci in the Mediterranean fruitfly Ceratitis capitata (Diptera: Tephritidae) using a biotin/streptavidin enrichment technique

The Medfly (Ceratitis capitata) is a polyphagous dipteran pest which has spread from North Africa to the countries of the Mediterranean Basin and has also invaded tropical and subtropical regions throughout the world. Colonizing populations typically possess low levels of genetic variability. Microsatellites provide an effective means of investigating the population structure of such genetically depauperate populations, however, microsatellite markers traditionally require a long phase of development in new taxa. We used a biotin/streptavidin capture technique to isolate microsatellites directly from C. capitata genomic DNA and we describe here the identification of seven polymorphic microsatellite markers in C. capitata.     

Improving male mating competitiveness and survival in the field for medfly,Ceratitis capitata (Diptera: Tephritidae) SIT programs

The success of the sterile insect technique (SIT) depends critically upon mating between released sterilized males and wild females. In Hawaii, improvements in the efficiency of sterile males were attempted on two separate fronts – mating enhancement and survival improvement. In the former, two methods have been investigated – selective breeding and aromatherapy. In the latter, flies which survived in field cages for several days were selected and bred to produce progeny with enhanced survival ability compared to control flies. Regarding mating selection, standard laboratory-reared males that successfully mated with wild females in field cages were allowed to breed. F1 offspring were inbred, then the selection procedure was repeated for four additional cycles. In the aromatherapy procedure, laboratory-reared males were exposed to ginger root oil for several hours 1 day prior to testing in field cages. Compared to controls, the selected flies improved the mating competitiveness of male flies ca. 3-fold, irradiation reduced this increase to ca. 2.5-fold. Exposing the selected, hybrid strain raised the fitness of the lab males to ca. 9-fold that of wild males. In the ongoing survival selection study, we have obtained lines in which the selected males survived ca. 2-fold better than laboratory control males over several days in an outdoor field cage, with food and water provided. The goal is to combine the traits of higher survival and mating ability into a single strain for SIT release.     

Male position and calling effort together influence male attractiveness in leks of the medfly,Ceratitis capitata (Diptera: Tephritidae)

Despite the close association between lek mating systems and the study of female mate choice, male mating success in leks is often associated with other aspects of sexual selection as well as female choice of male display traits. Males of the medfly Ceratitis capitata form leks on the undersides of leaves of their host plants. By experimentally creating artificial leks, we show that male success at attracting females depends not only on male calling effort (pheromone production dispersed by wing movement), but also on the position of the male within a lek. Males in the highest position in the artificial lek (closest to the light) attracted more females, and received more visits from those females. In our experiment, we deliberately minimized the visual cues that females approaching a male could use and, under these conditions, found no associations between male attractiveness and male size, weight or fluctuating asymmetry, either of the wings or sex setae (a pair of bilateral supra‐fronto‐orbital bristles). The latter result contrasts with earlier studies showing a significant negative association between sex setae fluctuating asymmetry and mating success. Accordingly, we conclude that symmetry of the male sex setae has no role in nonvisual communication (e.g. through directing pheromone plumes). Mating patterns associated with this insect are therefore every bit as complex as those in vertebrate leks. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 479–487.     

Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae)

Sperm storage organs allow females to temporally separate insemination from fertilization, manipulate ejaculates and control fertilization. In the reproductive tract of female fruit flies (Diptera: Tephritidae), sperm are found in two different organs--a pair or triplet of spermathecae, and a "fertilization chamber". In order to understand the specific function of each of these organs, we tested the following hypotheses: (1) Sperm are distributed equally amongst the various sperm storage organs; (2) Both organ types maintain sperm viability; and (3) Sperm used in fertilization come from the fertilization chamber. We counted sperm in spermathecae and fertilization chamber of Mediterranean fruit flies (Ceratitis capitata) every 3 days for 18 days following insemination, and used a live/dead staining technique to determine the viability of sperm in these organs. Finally, by extirpating spermathecae from inseminated females and allowing them to oviposit, we were able to identify the fertilization chamber as the source of fertilizing sperm. Numbers of sperm in the spermathecae declined from an average of 3575 on the day of copulation to 649, 18 days later. Conversely, the fertilization chamber maintained a fairly constant level of sperms, ranging between an average of 207 cells on day 3 to 115 sperms on day 18. Throughout the period we monitored, we found high levels of sperm viability in both organs (> 80%). Sperm viability was similarly high in the fertilization chambers of females without spermathecae. However, fertility of eggs laid by these females declined rapidly, as did the number of sperm in the fertilization chamber. We conclude that both the spermathecae and the fertilization chamber are active sperm storage organs, with separate functions: the spermathecae for long-term storage and the fertilization chamber, periodically filled by the spermathecae, a staging point for fertilizing sperm. We suggest that the use of both organs by females results in sperm economy, which adaptively prolongs the intervals between copulations.     

The feasibility of using the sterile insect technique against Liriomyza trifolii (Diptera: Agromyzidae) infesting greenhouse chrysanthemum

Liriomyza trifolii is a serious pest of chrysanthemum greenhouses and other crops around the world. The larvae feed within the leaves of the host plants and create serpentine mines. The Sterile Insect Technique (SIT) has been successfully used against some Dipteran species in the field, and it is especially efficient against isolated populations like those on islands. Therefore, SIT against pest populations in confined environments such as greenhouses should have high potential for success. The objectives of this study were to determine the optimum gamma‐irradiation dosage required for the sterilisation of L. trifolii adults, and to determine the quality (emergence percentage, flight ability, longevity, copulatory success, and sperm transfer) of the irradiated males compared with that of non‐irradiated (normal) males. We found that sterility (< 0.7 mine per female) was achieved with the dose of 170 ± 5% Gy. The copulatory success and sperm transfer during copulation of the sterile males were not significantly different from those of normal males. Moreover, the longevity, emergence percentage and flight ability of irradiated males were also not significantly different from normal males. The SIT experiments suggest that the release of sterile L. trifolii can significantly reduce the reproductive capacity of a wild leafminer population. Our studies indicate that sterilisation of L. trifolii flies is feasible and that sterile males are of high quality and competitive with normal males. Based on these data, research on the use of SIT against L. trifolii populations in greenhouses is ongoing.     

Improvement of the sterile insect technique for codling moth Cydia pomonella (Linnaeus) (Lepidoptera Tortricidae) to facilitate expansion of field application

The codling moth Cydia pomonella (L.) (Lepidoptera Tortricidae) is a key pest of pome fruit (apple, pear and quince) and walnut orchards in most temperate regions of the world. Efforts to control the codling moth in the past mostly relied on the use of broad spectrum insecticide sprays, which has resulted in the development of insecticide resistance, and the disruption of the control of secondary pests. In addition, the frequent reliance and use of these insecticides are a constant threat to the environment and human health. Consequently, there have been increased demands from the growers for the development of codling moth control tactics that are not only effective but also friendly to the environment. In that respect, the sterile insect technique (SIT) and its derivative, inherited sterility (IS), are, together with mating disruption and granulosis virus, among the options that offer great potential as cost‐effective additions to available control tactics for integration in area‐wide integrated pest‐management approaches. In support of the further development of the SIT/IS for codling moth control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a 5‐year Coordinated Research Project (CRP) entitled ‘Improvement of codling moth SIT to facilitate expansion of field application’. Research focussed on sterile codling moth quality and management (e.g. mobility and life‐history traits in relation to rearing strategy, dispersal, flight ability, radiosensitivity and mating compatibility) and a better understanding of the basic genetics of codling moth to assist the development of genetic sexing strains (e.g. cytogenetics, the development of dominant conditional lethal mutations, molecular characterization of the sex chromosomes, sex identification in embryos and cytogenetic markers). The results of the CRP are presented in this special issue.     

地中海实蝇及其近缘种基因芯片检测研究

本研究选择线粒体DNA (mtDNA) 细胞色素氧化酶Ⅰ基因（COⅠ）为分子标记基因,以双翅目实蝇科昆虫DNA序列为目标,建立了我国进境植物检疫害虫地中海实蝇Ceratitis capitata、芒果小条实蝇C. cosyra和纳塔尔小条实蝇C. rosa等生物芯片检测方法。地中海实蝇及其近缘种检测芯片由检测探针（实蝇科通用探针1条,小条实蝇属通用探针1条,地中海实蝇、芒果小条实蝇和纳塔尔小条实蝇近缘种探针2条和种特异探针4条）、质控探针（定位点探针、阳性质控、阴性质控和空白对照探针各1条）组成。芯片检测结果表明,检测探针特异性强,能实现上述3种实蝇的种类快速区分和准确鉴定; 检测方法稳定性好,地中海实蝇不同虫态（卵、幼虫、蛹和成虫）和不同地理种群检测结果完全一致。地中海实蝇生物芯片检测技术将为我国进口果蔬中检疫性实蝇快速筛查和种类鉴定提供检测方法,同时,还可应用到其他属的实蝇以及相关害虫的检疫中,为有害生物的快速鉴定提供了新方法。     

Impact of lek mating on the sterile insect technique: A modeling study

Modelling studies are presented which describe the effect of lek mating on the control of a wild population by sterile male release. The mixed leks are assumed to follow a Poisson-binomial distribution and the system includes three parts: territory defense, matings inside a lek and matings outside a lek. The effects of parameters on the hatchability are discussed. Among the parameters, sterile type effect (W_s), female choice (f_s) and mating competitiveness (C_m) are the most important. The application to determining the effects of sterile male release and on the proportion of sterile males required for eradication are also discussed.     

Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae)

The Sterile Insect Technique (SIT) involving area-wide release of mass-reared and sterilized pest insects has proven successful to reduce, control and eradicate economically important pest species, such as the Mediterranean fruit fly (medfly). For the efficient application, effective monitoring to assess the number and mating success of the released medflies is essential. Here, we report sperm-specific marking systems based on the spermatogenesis-specific Ceratitis capitata beta2-tubulin (Ccbeta2t) promoter. Fluorescent sperm can be isolated from testes or spermathecae. The marking does not cause general disadvantages in preliminary laboratory competitiveness assays. Therefore, transgenic sperm marking could serve as a major improvement for monitoring medfly SIT programs. The use of such harmless transgenic markers will serve as an ideal initial condition to transfer insect transgenesis technology from the laboratory to field applications. Moreover, effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive biology which will help to further improve SIT programs.     

Potential for controlling codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) in Argentina using the sterile insect technique and egg parasitoids

Codling moth is the main pest affecting apples and pears worldwide. Most pest control strategies used against this insect have relied on the use of broad‐spectrum insecticides which have led to non‐desirable effects like pesticide resistance, residues in the environment, human health concerns and the reduction of access to international markets. Therefore, alternative pest control strategies that would result in sustainable fruit production systems while taking care of the environment are strongly promoted. The use of the sterile insect technique has proven to be a valuable pest control tactic within area‐wide integrated pest management strategies, and its synergistic effect for Lepidoptera pests when combined with other biological control tactics such as parasitoids has been documented. The purposes of this research were to evaluate the response of an Argentinean codling moth strain to a sub‐sterilizing radiation dose of 100 Gy and to assess the acceptability and suitability of sterile codling moth eggs by the egg parasitoids, Trichogramma cacoeciae (Marchal) and Trichogramma nerudai (Pintureau and Gerding). Irradiated female moths survived better than irradiated male moths and non‐irradiated male and female moths. Also, the fecundity of irradiated female moths was reduced by more than 30% as compared to non‐irradiated ones whereas their fertility was close to zero. The F₁ generation was male biased with a lower fertility (inherited sterility) than the parental generation. Trichogramma cacoeciae and T. nerudai parasitized both fertile and sterile eggs. However, there was a significant reduction in acceptability for sterile eggs. Trichogramma nerudai parasitized more eggs than T. cacoeciae, but egg acceptability for this species was proportionally lower than for T. cacoeciae especially on eggs oviposited by irradiated females. Development to adult of both parasitoids species was not substantially affected by the origin of the eggs and the wasps had acceptable levels of adult emergence, survival and fecundity. These results provided useful information on the potential for controlling the codling moth using egg parasitoids and the sterile insect technique in Argentina.     

Sexual competitiveness of sterile Ceratitis capitata males exposed to essential oils from non-host plant species native to Argentina

The sterile insect technique (SIT) is used to control fruit fly pests, such as the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Enhancing sexual competitiveness of mass-reared sterile males can contribute to making this technique more effective. It has been shown that exposure to volatiles from essential oils (EOs), such as ginger root oil (GRO) and those from host fruits, increases male mating success. We evaluated the effect of EOs from non-host species native to Argentina, Schinus polygama (Cav.) Cabrera (Anacardiaceae) and Baccharis spartioides (Hook. & Arn.) Remy (Asteraceae), on the sexual competitiveness of sterile C. capitata males. In field cage experiments, sterile males exposed to S. polygama EO increased their mating success. In addition, sterile males exposed to this EO achieved more matings on trees than non-exposed males, thus suggesting the former are more prone to locate and defend the pheromone-calling territory. Deprivation of water and/or food significantly reduced males' life span, but exposure to S. polygama EO and GRO did not affect their survival. Schinus polygama EO is composed of mono- and sesquiterpenes with behavioral and electroantennographic responses in medflies, indicating that semiochemicals that boost medfly sexual competitiveness combine in this EO.     

Inability of Ceratitis capitata (Diptera: Tephritidae) to overwinter in the Judean hills

The overwintering potential of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in cold winter areas within its northern distribution is a key element in understanding its ecology. Recent studies have suggested that although originating in tropical Africa, the fly has become adapted to the cold weather that prevails within its northernmost areas of distribution. We address the question of whether the Mediterranean fruit fly has expanded its overwintering range to include the mountains of central Israel. Doing so would imply that the fly has developed either a behavioral or a physiological mechanism to cope with low temperature and/or damp conditions in combination with cold. We monitored adult populations year round, sampling fruit, calculating expected emergence days for overwintering flies, and studying adults captured within dense and sparse apple orchards. We also performed several manipulative experiments to study preimago ability to survive the winter under natural or seminatural conditions. The study was conducted in the central mountains of Israel at 700-m altitude from 1994 to 2003. Comparison experiments also were conducted at 400 m and at sea level. Our results show 1) no adults captured during the winter and spring, 2) an absence of new infestations during the winter and spring, and 3) inability of preimago stages to overwinter in the central mountains of Israel. Thus, we conclude that the fly does not overwinter in the central mountains of Israel. We discuss the ecological and applied significance of our findings.     

Multispectral imaging for quality control of laboratory‐reared Anastrepha fraterculus (Diptera: Tephritidae) pupae

The sterile insect technique (SIT) has been widely used to suppress several fruit fly species. In southern Brazil, millions of sterile flies of the South American fruit fly, Anastrepha fraterculus Wiedemann (Dipetra: Tephritidae), will be produced in a mass‐rearing facility called MOSCASUL to suppress wild populations from commercial apple orchards. In spite of standard rearing conditions, the quality of pupal batches can be inconsistent due to various factors. The quantification of poor quality material (e.g. empty pupae, dead pupae or larvae) is necessary to track down rearing issues, and pupal samples must be taken randomly and evaluated individually. To speed up the inspection of pupal samples by replacing the manual testing with the mechanized one, this study assessed a multispectral imaging (MSI) system to distinguish the variations in quality of A. fraterculus pupae and to quantify the variations based on reflectance patterns. Image acquisition and analyses were performed by the VideometerLab4 system on 7‐d‐old pupae by using 19 wavelengths ranging from 375 to 970 nm. The image representing the near infrared wavelength of 880 nm clearly distinguished among high‐quality pupae and the other four classes (i.e. low‐quality pupae, empty pupae, dead pupae and larvae). The blind validation test indicated that the MSI system can classify the fruit fly pupae with high accuracy. Therefore, MSI‐based classification of A. fraterculus pupae can be used for future pupal quality assessments of fruit flies in mass‐rearing facilities.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.