A genetic sexing system to improve the sterile insect technique against the Mediterranean fruit fly

The autosomal recessive allele v wing (v) in the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), produces flies that when reared at 30 degrees C have stubby wings. The mutant was used to construct a translocation-based genetic sexing system in an attempt to improve the efficiency and effectiveness of the sterile insect release method for field control.     

Dispersion theory and the sterile insect technique: application to two species of fruit fly

Dispersion theory is applied to the distribution of two kinds of sterile insect, Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann), and Queensland fruit fly (Qfly), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Dispersion theories are an essential basis of sampling theory and sampling plans, but this paper looks at them from another direction and uses data from arrays of sterile insect technique (SIT) monitoring traps to compare the utility of different measures such as coefficient of variation (CV), the exponent b of Taylor's power law, and exponent k of the negative binomial distribution and also derives predictions pertaining to the density (and hence release rate) of sterile insects that would be required to achieve effective coverage of the target area. This is far more useful than reliance on just the mean values of trap catches because such reliance takes no account of the fact that sterile flies distribute themselves unevenly with many patches inadequately covered despite the impression given by the mean. Data were used from recapture rates following either ‘roving releases’ of Medfly or releases from fixed points of Qfly. The relation of recapture rate to CV indicated that a doubling of release rate in order to double average recapture rate from 150 per trap per week to a value of 300 would have very little effect in terms of reducing CV and that there appears to be no practical prospect of reducing CV to below unity with the current methods of release without incurring a manifold increase in cost. Similarly, models derived from the negative binomial equation indicated that a law of diminishing returns applies in terms of the increase in the amount of adequate coverage (such as the percentage of traps catching >50 flies per week) that can be obtained by increasing release rates.     

昆虫不育技术防治柑橘大实蝇研究概况

柑橘大实蝇Bactrocera minax(Enderlein)是柑橘果树上的重要害虫,我国在20世纪80、90年代曾经成功利用昆虫不育技术防治柑橘大实蝇的危害。本文从人工饲养、不育昆虫的获取以及野外释放等方面对利用昆虫不育技术防治柑橘大实蝇进行了综述,以期为我国柑橘大实蝇的可持续治理提供参考。     

The sterile insect technique and the Mediterranean fruit fly: assessing the utility of aromatherapy in large field enclosures

The sterile insect technique (SIT) is widely used in integrated programs against tephritid fruit flies, 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 the male mating competitiveness. One potential solution involves the prerelease exposure of males to particular 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. To evaluate more rigorously the effectiveness of GRO exposure, we here describe two projects that compared levels of egg sterility or pupal yield, respectively, following the release of wild flies and either GRO‐exposed (treated) sterile males or GRO‐deprived (control) sterile males in large field enclosures. In both projects, sterile males from a genetic sexing strain were exposed as adults to GRO for 24 h while held in large storage boxes. In Hawaii, we dissected eggs from fruits to determine the percentage of egg hatch at four overflooding ratios, ranging from 5 : 1 to 60 : 1 (sterile : wild males), and found that, at all four ratios, the proportion of unhatched (sterile) eggs was significantly greater in enclosures containing GRO‐exposed males than control males. In Guatemala, we allowed larvae to develop in fruits and counted the number of pupae produced. At the only overflooding ratio tested (25 : 1), pupal yield was approximately 25% lower for enclosures containing GRO‐exposed males than control males, although this difference was not statistically significant. An explanation for the differing outcomes is proposed, and the implications of these findings for the SIT are discussed.     

Re-engineering the sterile insect technique

The mass release of sterile insects (the Sterile Insect Technique, SIT) is a highly effective area-wide method of pest control with a low environmental impact. SIT relies on the sterilization by irradiation of large numbers of insects. This has unavoidable costs in terms of the fitness of the irradiated insects and the financial requirements of constructing and operating the radiation facility. In many cases it is considered important to release only males, but large-scale sex-separation is also problematic. I have proposed that both of these difficulties can be overcome by using engineered strains of insects carrying a dominant, repressible, lethal gene or genetic system. As a proof of principle, my group and others have constructed strains of Drosophila melanogaster with the required genetic properties.     

Eradication of the screwworm from Libya using the sterile insect technique

The introduction in 1988 of the New World screwworm into Libya presented a serious threat to the livestock and wildlife sectors o f the African continent and the Mediterranean region. In this article, Moisés Vargas-Terán, Brian S. Hursey and Edward P. Cunningham describe the action taken to determine the extent of the problem, to prevent the spread of the infestation and to eradicate the fly from the region using the sterile insect technique.     

Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications

Background

The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males.

Results

AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet.

Conclusions

Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

     

Chemosterilant bait stations coupled with sterile insect technique: an integrated strategy to control the Mediterranean fruit fly (Diptera: Tephritidae)

During 2008 and 2009, the efficacy of the combination of two Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), control techniques, sterile insect technique (SIT) and a chemosterilant bait station system (Adress), was tested in three crops: citrus (Citrus spp.), stone fruit (Prunus spp.), and persimmon (Diospyros spp.). Two thousand sterile males were released per ha each week in the whole trial area (50,000 ha, SIT area). For 3,600 ha, within the whole trial area, 24 Adress traps per ha were hung (SIT + Adress area). Ten SIT + Adress plots and 10 SIT plots in each of three different fruit crops were arranged to assess Mediterranean fruit fly population densities and fruit damage throughout the trial period. To evaluate the efficacy of each treatment, the male and female populations were each monitored from August 2008 to November 2009, and injured fruit was assessed before harvest. Results showed a significant reduction in the C. capitata population in plots treated with both techniques versus plots treated only with the SIT. Likewise, a corresponding reduction in the percentage of injured fruit was observed. These data indicate the compatibility of these techniques and suggest the possibility of using Adress coupled with SIT to reduce C. capitata populations in locations with high population densities, where SIT alone is not sufficiently effective to suppress fruit fly populations to below damaging levels.     

Spatial patterns of sterile Mediterranean fruit fly dispersal

The success of the sterile insect technique (SIT) for the control of the Mediterranean fruit fly or medfly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), depends largely on the ability of sterile flies to spread in the target area and compete with the wild males for wild females. Our objectives in the present study were three‐fold: (1) to evaluate the dispersal ability of sterile male medflies and compare their spatial dispersion patterns with that of wild males, (2) to evaluate how different release methods affect subsequent spatial dispersal, and (3) to determine whether manipulating the pre‐release diet of sterile males affects their dispersal. To achieve these objectives, we conducted three experiments in the field where we quantified and analyzed the spatial and temporal dispersal patterns of sterile medflies and the dispersion of resident wild males. Overall, ca. 5% of the released sterile flies were recaptured 100 m from the release point, and ca. 2% were recaptured 200 m from the release point. The released flies rarely survived longer than 5–7 days. We repeatedly found that the spatial dispersion patterns of sterile males significantly correlated with those of wild males. Release methods strongly affected subsequent fly dispersal in the field as significantly more flies were recaptured following a scattered release vs. a central one. Finally, we show that enriching sterile fly pre‐release diet with protein did not affect subsequent dispersal in the field. We conclude that sterile males are able to match the dispersion patterns of wild males, an outcome that is highly important for SIT success. Large releases from central points distant from each other may leave many areas uncovered. Accordingly, scattered releases, repeated twice a week, will provide better coverage of all available aggregations sites. The spatial performance of protein‐fed males suggests that pre‐release diet amendments may be used without detriment as a sexual stimulant in SIT programs.     

Mortality of olive fruit fly pupae in California

Efforts to control the olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), in California have focused on insecticidal baits and biological control by parasitoids, which primarily target the adult and larval stages, respectively. The pupal stage, which occurs in the soil, has largely been overlooked. This study investigated mortality factors for olive fruit fly pupae in California olive orchards, using a combination of exclusion experiments and observation and trapping of potential predators. Results show predation and climatic factors contribute to pupal mortality. Ants (Formicidae) were the most numerous predators observed. Soil-borne pathogens caused no mortality in this study. Potential applications of these results in the development of a sustainable management program are discussed.     

Chilled packing systems for fruit flies (Diptera: Tephritidae) in the sterile insect technique

We evaluated three packing systems (PARC boxes, "GT" screen towers and "MX" screen towers) for the emergence and sexual maturation of sterile fruit flies, at three adult fly densities (1, 1.2 and 1.3 fly/cm2) and three food types. At the lowest density, results showed no significant differences in the longevity and flight ability of adult Anastrepha ludens (Loew) and Anastrepha obliqua Macquart among the three packing systems. Higher densities resulted in a decrease in these parameters. In the evaluation of the three food types, no significant differences were found either on longevity or flight ability of A. ludens. However, the greatest longevity for both sexes A. obliqua was obtained with commercial powdered Mb? and the mix of sugar, protein and corn starch on paper (SPCP) food types. The highest value for flight ability in A. obliqua males was obtained with powdered Mb? and SPCP food types, and for females with Mb? powdered food. Our data indicated that GT and MX screen tower packing systems are an alternative to the PARC boxes, since they were suitable for adult fly sexual maturation without any harm to their longevity or flight ability. The tested foods were equivalent in both fruit fly species, with the exception of the agar type for A. obliqua, which yielded the lowest biological parameters evaluated. Our results contribute to the application of new methods for the packing and release of sterile flies in large-scale programs.     

Medfly promoters relevant to the sterile insect technique

This review summarizes structural and functional studies on medfly promoters and regulatory elements that can be used for driving sex-specific, conditional and constitutive gene expression in this species. Sex-specific and conditional promoters are important for generating transgenic sexing strains that could increase the performance of the Sterile Insect Technique while strong constitutive promoters are necessary for developing sensitive transgenic marker systems. The review focuses on the functional analysis of the promoters of two male-specific and heat shock medfly genes. A special emphasis is put on the potential utility of these promoters for developing transgenic sexing strains.     

Developing transgenic Anopheles mosquitoes for the sterile insect technique

In the last 10 years the availability of the genome sequence of Anopheles gambiae and the development of a transgenic technology for several species of Anopheles mosquitoes have, in combination, helped in enabling us to gain several insights into the biology of these mosquitoes that is relevant to their capacity as vectors of the malaria parasite. While this information is anticipated to inform many novel vector control strategies, the technique most likely to benefit in the near future from the availability of a reliable transgenic technology is the sterile insect technique (SIT), which relies on releasing large numbers of sterile insects to compete for mates in the wild, leading to population suppression. Although SIT has been proven to work reliably for many insects, the construction of suitable strains, and induction of sterility, has until now been a laborious process, combining classical genetics with radiation-induced sterility. Using transgenesis to create strains of Anopheles suitable for SIT could potentially offer several advantages over current approaches, in that the basic design of transgenic constructs designed for other insects should be rapidly transferable to mosquitoes, and induction of sterility as a product of the transgenic modification could obviate the requirement for radiation and its associated deleterious effects. In this paper the progress of different transgenic approaches in constructing tools for SIT will be reviewed.     

Age-dependent variation in mating success of sterile male Mediterranean fruit flies (Diptera: Tephritidae): implications for sterile insect technique

The sterile insect technique (SIT) is widely used in integrated programs against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in the mating ability of the released males. To counter this deficiency, SIT programs rely upon the production and release of large numbers of sterile males to achieve high overflooding (sterile:wild male) ratios. To ensure a high release volume, emergence facilities release adult males at a young age (2 d old in some cases). The primary objective of this study was to describe age-dependent variation in the mating propensity and competitiveness of sterile males of C. capitata. Males that were 2 or 3 d old had lower mating propensity than males that were > or =4 d old, and 3-d-old males had lower mating competitiveness than males that were > or =4 d old. Given these results, we measured the effect of a longer holding period on male mortality in storage boxes. With delayed food placement, males held in storage boxes for 4 d after emergence showed no higher mortality than males held for only 2 d (the standard interval). Using large field enclosures, we compared the levels of egg sterility attained via releases of 2- versus 4-d-old sterile males at two overflooding ratios (5:1 and 100:1). At the lower ratio, the proportion of unhatched eggs observed for trials involving 2-d-old sterile males was not, on average, significantly higher than that observed for matings between wild flies (33 versus 25%, respectively), whereas the level of egg sterility observed for releases of 4 d old sterile males was 62%. At the 100:1 overflooding ratio, the proportion of unhatched eggs associated with the 2-d-old sterile males was 58%, a level not significantly different from that induced by 4-d-old sterile males at the 5:1 ratio and significantly lower than the level (79%) observed for 4-d-old sterile males at 100:1 overflooding ratio. The implications of these results for SIT are discussed.     

Generic insect repellent detector from the fruit fly Drosophila melanogaster

Background

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Methodology

Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.

Conclusions

For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.     

Control of the mediterranean fruit fly (Ceratitis capitata) using sterile males: Effects of nitrogen and chilling during gamma-irradiation of puparia

Nitrogen has proved effective in protecting puparia of Ceratitis capitata (Wiedemann) from gamma-irradiation induced damage as evidenced by increased adult emergence from early irradiated puparia, adult male survival, and male fertility. Chilling to 4° during irradiation had the opposite effect and it is concluded that the extent of damage which can be altered by these treatments is due to oxygen-dependent effects of irradiation. Increased fertility after irradiation in nitrogen may be due to increased sexual vigour rather than to reduction in damage of genetic material but this has not been proved. Effects on percentage adult emergence, survival and fertility are probably manifestations of the same phenomenon of irradiation-induced lethargy. It is considered that the effect of treatment with nitrogen at 25° or chilling to 4° in air during irradiation may be of value in increasing the flexibility of a large-scale campaign to control C. capitata by the sterile-insect release method.Adults from puparia of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), subjected to sterilizing doses of gamma radiation, survive best when treated late in puparial life (Arroyo et al. 1965). This fact greatly affects the logistics of any large-scale sterilization programme (Knipling 1960) for the control of this or other insects in the field (see Smith 1963). Hence we have tried to separate the genetic and somatic effects of gamma-irradiation upon puparia of C. capitata. We hoped that if somatic effects could be lessened without affecting the degree of sterility induced by a given dose, the age-range of puparia which could be successfully sterilized might be increased, thus allowing greater flexibility in the development of a control programme.The lessening of radiation-induced damage under conditions of low oxygen tension has been well established in both plant and animal tissues (Patt & Brues 1954; O'Brien & Wolfe 1964). Nitrogen increases the radiation resistance of the wasp Habrobracon during development (Clark & Herr 1955), and Baldwin and Salthouse (1959) showed that oxygen deficiency protected the bug Rhodnius from the dual effect of delayed moulting and burning of the epidermis following administration of X-rays.Present results involving irradiation of C. capitata puparia in nitrogen at 25° or in air at 4° show that some protection from lethal somatic effects is possible.

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Zusammenfassung Es konnte gezeigt werden, dass bei der Bestrahlung der Puparien von Ceratitis capitata (Wiedemann) mit 10 k-rad Gammastrahlen in einer Stickstoffatmosphäre der erreichte Prozentsatz schlüpfender Imagines—im Vergleich mit der Bestrahlung in Luft bei 25° oder 4°C—vergrössert ist, wenn die Puparien am 4. Tag der Entwicklung bei 25°C behandelt werden. Die Schutzwirkung des Stickstoffs ist noch grösser bei einer Dosis von 30 k-rad. Nach Bestrahlung mit 10 k-rad in einem späteren Stadium der Puparien-Entwicklung blieb das Überleben der geschlüpften Männchen von der Behandlung mit Stickstoff oder von der Abkühlung unbeeinflusst. Jedoch ergaben am 4. Tag der Entwicklung behandelte Puparien nach Bestrahlung in Stickstoff länger lebende Männchen und das Überleben war nach Bestrahlung in Luft bei 4°C am geringsten. Diese Ergebnisse weisen auf die Existenz einer, sekundären Letalwirkung der Bestrahlung hin, die von Sauerstoff abhängig ist, da Stickstoff die Sauerstoff-spannung in den Geweben herabsetzt, während Abkühlung auf 4°C die Löslichkeit von Sauerstoff in den Gewebeflüssigkeiten erhöht. Der primäre Letaleffekt der Bestrahlung ist der, welcher die Weiterentwicklung durch Schädigung der sich differenzierenden Gewebe verhindert. Der Sekundäreffekt verhindert das Schlüpfen der voll entwickelten pharaten Imago aus ihrem Puparium, und es wird angenommen, dass die auf Bestrahlung während früher Entwicklungsstadien folgende Einschränkung der Überlebensfähigkeit der Erwachsenen — wie sie von anderen Untersuchern bei anderen Insekten gefunden wurde — eine Manifestation der gleichen Erscheinung darstellt. Es wird vermutet, dass diese sauerstoffabhängige sekundäre Letalität irgendwie mit einem Eingreifen in den normalen Atmungsstoffwechsel des Insekts zusammenhängt.Es wird im Vergleich mit der Bestrahlung in Luft bei 25°C eine Steigerung der Fertilität der in Stickstoff bei 25°C bestrahlten Männchen und eine Minderung der Fertilität nach Bestrahlung in Luft bei 4°C festgestellt. Das könnte eher eine Folge von Veränderungen in der sexuellen Vitalität als von Veränderungen in der Häufigkeit der Induktion dominanter Letalfaktoren in den Spermatozoen sein. Weitere Untersuchungen zur Bestimmung dieses Punktes sind notwendig.Es wird angenommen, dass die Wirkungen einer Behandlung mit Stickstoff oder Abkühlung auf 4°C während der Bestrahlung von Nutzen für die Steigerung der Flexibilität bei einer weitgespannten Bekämpfungsaktion von C. capitata durch die S.I.R.M. sein dürften.

     

Molecular technologies to improve the effectiveness of the sterile insect technique

The application of the Sterile Insect Technique (SIT) in area-wide integrated pest management (AW-IPM) programmes continues to increase. However, programme efficiency can still be considerably enhanced when certain components of the technology are improved, such as the development of improved strains for mass rearing and release. These include strains that (1) produce only male insects for sterilization and release and (2) carry easily identifiable markers to identify released sterile insects in the field. Using both classical and modern biotechnology techniques, key insect pests are targeted, where SIT programmes are being implemented. The pests include mosquitoes, the Mexican fruit fly, the codling moth, the oriental fruit fly and the pink bollworm. This special issue summarizes the results of research efforts aimed at the development and evaluation of new strains to a level where a decision can be made as to their suitability for use in large scale SIT programmes. Major beneficiaries will be operational AW-IPM programmes that apply the SIT against major insect pests.