Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme.     

Male-killing Wolbachia in a flour beetle

The bacteria in the genus Wolbachia are cytoplasmically inherited symbionts of arthropods. Infection often causes profound changes in host reproduction, enhancing bacterial transmission and spread in a population. The reproductive alterations known to result from Wolbachia infection include cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males, fecundity enhancement, male killing and, perhaps, lethality Here, we report male killing in a third insect, the black flour beetle Tribolium madens, based on highly female-biased sex ratios of progeny from females infected with Wolbachia. The bias is cytoplasmic in nature as shown by repeated backcrossing of infected females with males of a naturally uninfected strain. Infection also lowers the egg hatch rates significantly to approximately half of those observed for uninfected females. Treatment of the host with antibiotics eliminated infection, reverted the sex ratio to unbiased levels and increased the percentage hatch. Typically Wolbachia infection is transmitted from mother to progeny, regardless of the sex of the progeny; however, infected T. madens males are never found. Virgin females are sterile, suggesting that the sex-ratio distortion in T. madens results from embryonic male killing rather than parthenogenesis. Based on DNA sequence data, the male-killing strain of Wolbachia in T. madens was indistinguishable from the CI-inducing Wolbachia in Tribolium confusum, a closely related beetle. Our findings suggest that host symbiont interaction effects may play an important role in the induction of Wolbachia reproductive phenotypes.     

橘小实蝇遗传性别品系的建立及雄性不育技术

为了更好地应用昆虫不育技术防治橘小实蝇,建立了以蛹色区分雌雄性别的橘小实蝇遗传性别品系。经过连续测交表明遗传品系能稳定遗传。质量监测表明遗传性别品系平均孵化率、蛹重、羽化率、飞出率、存活率等指标与普通种无显著差异。取遗传性别品系羽化前1、2、3天（分别用-1 d、-2 d、-3 d表示）的雄蛹,经过100 Gy的⁶⁰Co辐射后,分别与野生雌虫杂交,F1代卵的孵化率均大大低于对照组。对经过辐射处理-1 d、-2 d、-3 d的雄蛹分别进行质量检测,飞出率、存活率与对照组均无显著差异;-1 d、-2 d的羽化率之间及与对照组之间无显著差异,-3 d的羽化率与-1 d、-2 d及对照组差异均显著。取遗传性别品系的-2d的雄蛹辐射100 Gy的⁶⁰Co,获得不育雄虫,和野生雄虫一起竞争与野生雌虫交配的机会,相对不育系数为0.4923,表明不育雄虫与野生雄虫交配能力相当。田间扩散能力研究表明,不育雄虫在田间存在丰富的寄主植物的情况下不会大量扩散很远的距离。     

Pheromone trapping models for pest control: Effects of mating patterns and immigration

Several models are presented which examine pest population behaviour with the release of female sex pheromones for the attraction and annihilation of males. These models include male polygamy and female monogamy, various mating frequencies, delayed mating of females, immigration of one or all individual types, and differential survivorship of males and females. In all the models there are two steady states, a stable s.s. at the origin and an unstable s.s. in the positive domain for a given value of pheromone release rate. In all the models, control relies on the reduced ability of males to fertilize virgin females following trapping and male annihilation. As such, control is very sensitive to mating frequency, being very difficult when males mate frequently. Control is also very difficult with the immigration of even a moderate number of fertilized females. Control is much easier when mating is delayed, especially if survivorship is low, or with density dependent population regulation.     

Genetic sexing through the use of Y-linked transgenes

Sterile insect technique (SIT)-based pest control programs rely on the mass release of sterile insects to reduce the wild target population. In many cases, it is desirable to release only males. Sterile females may cause damage, e.g., disease transmission by mosquitoes or crop damage via oviposition by the Mediterranean fruit fly (Medfly). Also, sterile females may decrease the effectiveness of released males by distracting them from seeking out wild females. To eliminate females from the release population, a suitable sexual dimorphism is required. For several pest species, genetic sexing strains have been constructed in which such a dimorphism has been induced by genetics. Classical strains were based on the translocation to the Y chromosome of a selectable marker, which is therefore expressed only in males. Recently, several prototype strains have been constructed using sex-specific expression of markers or conditional lethal genes from autosomal insertions of transgenes. Here, we describe a novel genetic sexing strategy based on the use of Y-linked transgenes expressing fluorescent proteins. We demonstrate the feasibility of this strategy in a major pest species, Ceratitis capitata (Wiedemann), and discuss the advantages and disadvantages relative to other genetic sexing methods and potential applicability to other species.     

Pest control by the release of insects carrying a female-killing allele on multiple loci

With recent advances in genetics, many new strategies for pest control have become feasible. This is the second article in which we model new techniques for pest control based on the mass release of genetically modified insects. In this article we model the release of insects carrying a dominant and redundant female killing or sterilizing (FK) allele on multiple genetic loci. If such insects are released into a target population, the FK allele can become widely spread in the population through the males while reducing the population each generation by killing females. We allow the number of loci used to vary from 1 to 20. We also allow the FK allele to carry a fitness cost in males due to the gene insertions. Using a model, we explore the effectiveness and optimal strategies for such releases. In the most ideal circumstances (no density-dependence and released insects equal in fitness to wild ones), FK releases are several orders of magnitude more effective than equal sized sterile male releases. For example, a single release of 19 FK-bearing males for every two wild males, with the released males carrying the FK allele on 10 loci, reduces the target population to 0.002% of no-release size. An equal sized sterile release reduces the target population to 5% of no-release size. We also show how the effectiveness of the technique decreases as the fitness cost of the FK alleles in males increases. For example, the above mentioned release reduces the target population to 0.7% of no-release size if each FK allele carries a fitness cost in males of 5%. Adding a simple model for density-dependence and assuming that each of the released males carries the FK allele on six loci, we show that the release size necessary to reduce the target population to 1/100 of no-release size in 10 generations of releases varies from 0.44:1 to 4:1 (depending on parameter values). We also calculate the optimal number of loci on which to put the FK allele under various circumstances.     

Aromatherapy on a large scale: exposing entire adult holding rooms to ginger root oil increases the mating competitiveness of sterile males of the Mediterranean fruit fly in field cage trials

The sterile insect technique (SIT) is widely used in integrated programs against fruit fly pests, particularly the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in male mating competitiveness. One potential solution involves the pre-release exposure of males to specific attractants. In particular, male exposure to ginger root oil [Zingiber officinale Roscoe (Zingiberaceae); hereafter GRO] has been shown to increase mating success dramatically in field cage trials. Initial studies exposed small groups of males (25 individuals), but more recent work has demonstrated that GRO exposure involving standard storage boxes (containing ≈ 36 000 males) also results in enhanced mating performance. The objective of the present study was to determine whether aromatization of entire trailers, holding ≈ 14 million sterile males from a genetic sexing [temperature sensitive lethal (tsl)] strain, increases male mating success. Independent of the total dose, spatial distribution, or type of dispenser used, sterile males exposed to GRO for a 24-h period displayed greater mating success than non-exposed males in mating cage trials (in which tsl males competed against males from a standard, bisexual strain for females from this same standard strain). Averaged over all experiments, tsl males exposed to GRO obtained 54% of all matings compared to 38% for non-exposed tsl males, an increase of 42%. The implications of these findings for SIT programs against C. capitata are discussed.     

Sexual competitiveness of a transgenic sexing strain of the Mediterranean fruit fly, Ceratitis capitata

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.     

Sex separation strategies: past experience and new approaches

The success of the sterile insect technique (SIT) and other genetic strategies designed to eliminate large populations of insects relies on the efficient inundative releases of competitive, sterile males into the natural habitat of the target species. As released sterile females do not contribute to the sterility in the field population, systems for the efficient mass production and separation of males from females are needed. For vector species like mosquitoes, in which only females bite and transmit diseases, the thorough removal of females before release while leaving males competent to mate is a stringent prerequisite. Biological, genetic and transgenic approaches have been developed that permit efficient male-female separation for some species considered for SIT. However, most sex separation methods have drawbacks and many of these methods are not directly transferable to mosquitoes. Unlike genetic and transgenic systems, biological methods that rely on sexually dimorphic characters, such as size or development rate, are subject to natural variation, requiring regular adjustment and re-calibration of the sorting systems used. The yield can be improved with the optimization of rearing, but the scale of mass production places practical limits on what is achievable, resulting in a poor rearing to output ratio. High throughput separation is best achieved with scalable genetic or transgenic approaches.     

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.     

High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions

ABSTRACT: BACKGROUND: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. METHODS: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. RESULTS: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. CONCLUSIONS: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.     

Insect quality control: synchronized sex,mating system,and biological rhythm

The sterile insect technique (SIT) is a method of eradicating insects by releasing mass-reared sterilized males into fields to reduce the hatchability of eggs laid by wild females that have mated with the sterile males. SIT requires mass-production of the target insect, and maintenance of the quality of the mass-reared insects. The most important factor is successful mating between wild females and sterile males because SIT depends on their synchronized copulation. Therefore, understanding the mating systems and fertilization processes of target insects is prerequisite. Insect behavior often has circadian rhythms that are controlled by a biological clock. However, very few studies of relationships between sterile insect quality and circadian rhythm have been performed compared with the amount of research on the mating ability of target insects. The timing of male copulation attempts with receptivity of females is key to successful mating between released males and wild females. Therefore, we should focus on the mechanisms controlling the timing of mating in target insects. On the other hand, in biological control projects, precise timing of the release of natural enemies to attack pest species is required because behavior of pests and control agents are affected by their circadian rhythms. Involving both chronobiologists and applied entomologists might produce novel ideas for sterile insect quality control by synchronized sex between mass-reared and wild flies, and for biological control agent quality by matching timing in activity between predator activity and prey behavior. Control of the biological clocks in sterile insects or biological control agents is required for advanced quality control of rearing insects.     

Male mating competitiveness of a Wolbachia-introgressed Aedes polynesiensis strain under semi-field conditions

Background

Lymphatic filariasis (LF), a global public health problem affecting approximately 120 million people worldwide, is a leading cause of disability in the developing world including the South Pacific. Despite decades of ongoing mass drug administration (MDA) in the region, some island nations have not yet achieved the threshold levels of microfilaremia established by the World Health Organization for eliminating transmission. Previously, the generation of a novel Aedes polynesiensis strain (CP) infected with an exogenous type of Wolbachia has been described. The CP mosquito is cytoplasmically incompatible (i.e., effectively sterile) when mated with wildtype mosquitoes, and a strategy was proposed for the control of A. polynesiensis populations by repeated, inundative releases of CP males to disrupt fertility of wild females. Such a strategy could lead to suppression of the vector population and subsequently lead to a reduction in the transmission of filarial worms.

Methodology/Principal Findings

CP males and F1 male offspring from wild-caught A. polynesiensis females exhibit near equal mating competitiveness with F1 females under semi-field conditions.

Conclusions/Significance

While laboratory experiments are important, prior projects have demonstrated the need for additional testing under semi-field conditions in order to recognize problems before field implementation. The results reported here from semi-field experiments encourage forward progression toward small-scale field releases.     

Male mating performance and cytoplasmic incompatibility in a wPip Wolbachia trans‐infected line of Aedes albopictus (Stegomyia albopicta)

Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae) is a maternally inherited endosymbiont of a large number of insects and other arthropods that induces various effects on host reproductive biology. Among these, cytoplasmic incompatibility (CI) is a form of sterility induced in eggs produced by mating between infected males and females uninfected or infected by an incompatible Wolbachia strain. This phenomenon has been proposed as a potential way to produce functionally sterile males to be used in genetic control programmes. In this paper, we report on experiments carried out to evaluate the mating performances of males of an Aedes albopictus (Stegomyia albopicta) (Diptera: Culicidae) line (ARwP), harbouring a new Wolbachia infection [the wPip strain from Culex pipiens Linnaeus (Diptera: Culicidae)], in comparison with naturally infected males (SR line). ARwP males did not differ from SR males with regard to insemination capacity. Mating competitiveness did not differ significantly between lines in either laboratory or greenhouse conditions. Moreover, crosses with SR females were characterized by a 100% CI regardless of ARwP male age. All of these findings suggest that ARwP males may represent a very efficient tool for control programmes against Ae. albopictus based on the release of functionally sterile males.     

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.     

Sex, bugs and Haldane's rule: the nematode genus Pristionchus in the United States

The haplodiploid sex determining mechanism in Hymenoptera (males are haploid, females are diploid) has played an important role in the evolution of this insect order. In Hymenoptera sex is usually determined by a single locus, heterozygotes are female and hemizygotes are male. Under inbreeding, homozygous diploid and sterile males occur which form a genetic burden for a population. We review life history and genetical traits that may overcome the disadvantages of single locus complementary sex determination (sl-CSD). Behavioural adaptations to avoid matings between relatives include active dispersal from natal patches and mating preferences for non-relatives. In non-social species, temporal and spatial segregation of male and female offspring reduces the burden of sl-CSD. In social species, diploid males are produced at the expense of workers and female reproductives. In some social species, diploid males and diploid male producing queens are killed by workers. Diploid male production may have played a role in the evolution or maintenance of polygyny (multiple queens) and polyandry (multiple mating). Some forms of thelytoky (parthenogenetic female production) increase homozygosity and are therefore incompatible with sl-CSD. We discuss a number of hypothetical adaptations to sl-CSD which should be considered in future studies of this insect order.     

Raspberry ketone-supplemented diet has no effect on fitness parameters or lure responsiveness in male melon flies,Zeugodacus cucurbitae (Diptera: Tephritidae)

The male annihilation technique (MAT) and sterile insect technique (SIT) are often used to control pestiferous tephritid fruit flies (Diptera: Tephritidae). MAT involves the deployment of traps containing a male attractant and insecticide with the goal of drastically reducing male abundance and ultimately eliminating the entire population. SIT, which involves the mass production, sterilization, and release of the target species, may also be implemented to achieve final extirpation. Generally, simultaneous implementation of MAT and SIT is counterproductive, because the presence of large numbers of male-specific traps in the environment (MAT) would greatly reduce the number of sterile males available for copulating with wild females (SIT). However, studies on the Queensland fruit fly, Bactrocera tryoni (Froggatt), indicate that concurrent use of MAT and SIT may be feasible. Sexually mature males of B. tryoni are attracted to the raspberry ketone and its synthetic analogue cue-lure. Males of B. tryoni fed raspberry-ketone-supplemented diet when newly emerged showed lower attraction to cue-lure baited traps than control males. In addition, newly emerged males provided this diet displayed accelerated sexual maturation, which would allow the early release of sterile males and reduce pre-release holding costs. Here, we examined whether the addition of raspberry ketone to the adult diet of male melon flies, Zeugodacus cucurbitae (Coquillett), produced effects similar to those observed for B. tryoni. Despite using similar methods, no significant effect of raspberry ketone-supplemented diet on time to sexual maturity, survival, mating competitiveness, or attraction to cue-lure baited traps in mass-reared Z. cucurbitae males.     

Laboratory adaptation reduces female mating resistance in the sweet potato weevil

Selection for genetic adaptation might occur whenever an animal colony is maintained in the laboratory. The laboratory adaptation of behavior such as foraging, dispersal ability, and mating competitiveness often causes difficulties in the maintenance of biological control agents and other beneficial organisms used in procedures such as the sterile insect technique (SIT). Sweet potato weevil, Cylas formicarius (Summers) (Coleoptera: Brentidae), is an important pest in sub‐tropical and tropical regions. An eradication program targeting C. formicarius using SIT was initiated in Japan with weevils being mass‐reared for 95 generations to obtain sufficient sterile males. The mass‐reared strain of C. formicarius exhibits weaker female resistance to male mating attempts compared with the wild strain. This could affect the success of SIT programs because mating persistence of mass‐reared males might be expected to decrease in response to weak female resistance. We show that high success of sperm transfer to mass‐reared females was due to weak female resistance to male mating attempts. However, the mating behavior of mass‐reared males did not change. In C. formicarius, the trait of male persistence to mate was not correlated with the female resistance traits. Our results suggest that mass‐rearing conditions do not have negative effects on the mating ability of the sterile males of this species, and thus that the current mass‐rearing procedures are suitable for production of sterile males for the weevil eradication program.     

Genetic control of insect pests: growth industry or lead balloon?

Genetic control is a form of biological control of pest species which exploits the insect's mate-seeking expertise to introduce genetic abnormalities (typically, but not necessarily, dominant lethal mutations) into the eggs of the wild population. The effectiveness of radiation-sterilized males depends on the mating competitiveness of released males being adequate in relation to the recovery potential of and rate of immigration into the target population. This technique is now being applied on a very large scale against agricultural pests especially in Mexico, Egypt and Japan. Variants on this technique, which may have advantages, include novel means of generating genetic loads in populations of Lepidioptera and sheep blowflies and the introduction into mosquito populations of genes making them unable to transmit malaria.     

Historical applications of induced sterilisation in field populations of mosquitoes

Research on sterile mosquito technology from 1955 to the 1980s provided a substantial body of knowledge on propagation and release of sterile mosquitoes. Radiation sterilisation and chemosterilisation have been used effectively to induce dominant lethality and thereby sterilise important mosquito vectors in the laboratory. Experimental releases of chemosterilised males provided complete control of Anopheles albimanus in a small breeding population (14-15 sq km) in El Salvador. Releases of radiation sterilised males failed to control either Aedes aegypti or Anopheles quadrimaculatus in the USA. Releases of radiation-sterilised and chemosterilised male Culex quinquefasciatus in the USA and India were successful in some instances. Development of genetic sexing systems for Anopheles and improved physical separation methods for Culex have made it possible to rear and release males almost exclusively (> 99%) minimizing the release of potential vectors, the females. Factors that affected efficacy in some field programmes included reduction of competitiveness by radiation, immigration of fertilized females from outside the release zones, and inability of laboratory-bred males to perform in the wild. Despite significant progress, institutional commitments to carry the process further were generally lacking in the late 1970s and until recently. Now, with renewed interest and support for further assessment of this technology, this paper summarizes the current knowledge base, prioritizes some areas of investigation, and challenges scientists and administrators to maintain an awareness of progress, remain realistic about the interpretation of new findings, and make decisions about the sterile insect technique on the basis of informed scientific documentation. Areas recommended for priority research status include the establishment of genetic sexing mechanisms that can be transferred to other mosquito species, re-examination of radiation sterilisation, aerial release technology and mass rearing.