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How is (Insect) population control through male sterilization effective

How is (Insect) population control through male sterilization effective


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This is no doubt a simple question.

I was reading about Sterile Insect Technique where sterile males out-compete non-sterile ones to mate with females and thus reduce the population.

My question is - why don't the females just re-mate after they don't reproduce?

I'm assuming that the number of sterile males will be much less than the existing male population.

Is it because my assumption is incorrect and there are more sterile males? Or my assumption is correct and given the life-cycle of the organism, the females have only once chance to mate? Or something else?


Sterile insects are typically produced by radiation. A sufficient dose is used to cause substantial DNA damage in the gametes of the males. However, this doesn't mean the sperm are completely non-functional.

In fact, it is important that the sperm are functional and simply contain dominant-lethal mutations at a sufficient probability (Robinson, 2005 describes this in detail as well as the dosing strategies to optimize the desired outcome).

The males still mate and fertilize the eggs of the female; it is only later that the eggs will eventually fail to develop, but there is no way for the females to be aware of this (nor much that can be done even if they were - once the eggs are fertilized they cannot be fertilized again).

It is also important to release a sufficient population of sterile males to impact the population. Depending on the species, it may or may not be necessary to completely overwhelm the existing population, but a typical goal seems to be 10-100x the native population. By raising males in laboratory conditions, it may be possible to produce a larger population than would typically survive to mating age in the wild.


References:

Cockburn, A. F., Howells, A. J., & Whitten, M. J. (1984). Recombinant DNA technology and genetic control of pest insects. Biotechnology and genetic engineering reviews, 2(1), 68-99.

Klassen, W., & Curtis, C. F. (2005). History of the sterile insect technique. In Sterile insect technique (pp. 3-36). Springer Netherlands.

Robinson, A. S. (2005). Genetic basis of the sterile insect technique. In Sterile Insect Technique (pp. 95-114). Springer, Dordrecht.


Sterile insect technique

The sterile insect technique (SIT) [1] [2] is a method of biological insect control, whereby overwhelming numbers of sterile insects are released into the wild. The released insects are preferably male, as this is more cost-effective and the females may in some situations cause damage by laying eggs in the crop, or, in the case of mosquitoes, taking blood from humans. The sterile males compete with wild males to mate with the females. Females that mate with a sterile male produce no offspring, thus reducing the next generation's population. Sterile insects are not self-replicating and, therefore, cannot become established in the environment. Repeated release of sterile males over low population densities can further reduce and in cases of isolation eliminate pest populations, although cost-effective control with dense target populations is subjected to population suppression prior to the release of the sterile males.

The technique has successfully been used to eradicate the screw-worm fly (Cochliomyia hominivorax) from North and Central America. Many successes have been achieved for control of fruit fly pests, most particularly the Mediterranean fruit fly (Ceratitis capitata) and the Mexican fruit fly (Anastrepha ludens). Active research is being conducted to determine this technique's effectiveness in combatting the Queensland fruit fly (Bactrocera tyroni).

Sterilization is induced through the effects of irradiation on the reproductive cells of the insects. SIT does not involve the release of insects modified through transgenic (genetic engineering) processes. [3] Moreover, SIT does not introduce non-native species into an ecosystem.


Mosquito Population Successfully Suppressed Through Pilot Study Using Nuclear Technique in China

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The Aedes albopictus is the world’s most invasive mosquito species. A successful pilot trial for controlling this insect pest recently concluded and the results were published in Nature on 17 July 2019. (Photo: N. Culbert/IAEA)

For the first time, a combination of the nuclear sterile insect technique (SIT) with the incompatible insect technique (IIT) has led to the successful suppression of mosquito populations, a promising step in the control of mosquitoes that carry dengue, the Zika virus and many other devastating diseases. The results of the recent pilot trial in Guangzhou, China, carried out with the support of the IAEA in cooperation with the Food and Agriculture Organization of the United Nations (FAO), were published in Nature on 17 July 2019.

SIT is an environmentally-friendly insect pest control method involving the mass-rearing and sterilization of a target pest using radiation, followed by the systematic area-wide release of sterile males by air over defined areas. The sterile males mate with wild females, resulting in no offspring and a declining pest population over time. IIT involves exposing the mosquitoes to the Wolbachia bacteria. The bacteria partially sterilizes the mosquitoes, which means less radiation is needed for complete sterilization. This in turn better preserves the sterilized males’ competitiveness for mating.

While SIT, as part of area-wide insect management strategies, has been successfully used to control a variety of plant and livestock pests such as fruit flies and moths, the control of mosquitoes still had to be demonstrated.

The main obstacle in scaling up the use of SIT against various species of mosquitoes has been overcoming several technical challenges with producing and releasing enough sterile males to overwhelm the wild population. Researchers at Sun Yat-sen University, and its partners, in China, have now successfully addressed these challenges, with the support of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, which is leading and coordinating global research in SIT.

For example, the researchers used racks to rear over 500 000 mosquitoes per week that were constructed based on models developed at the Joint FAO/IAEA Division’s laboratories near Vienna, Austria. A specialized irradiator for treating batches of 150 000 mosquito pupae was also developed and validated with close collaboration between the Joint FAO/IAEA Division and the researchers.

Mosquito larval rearing racks at a mosquito mass-rearing facility at the Wolbaki Biotech Company in Guangzhou, China, in May 2019. The company is using the most advanced mass-rearing technology for mosquitoes. These racks are based on models developed by the Joint FAO/IAEA Insect Pest Control Laboratory. Each has the capacity of producing about 500 000 males per week. (Photo: J. Bouyer/IAEA)

The results of this pilot trial, using SIT in combination with the IIT, demonstrate the successful near-elimination of field populations of the world’s most invasive mosquito species, Aedes albopictus (Asian tiger mosquito). The two-year trial (2016-2017) covered a 32.5-hectare area on two relatively isolated islands in the Pearl River in Guangzhou. It involved the release of about 200 million irradiated mass-reared adult male mosquitoes exposed to Wolbachia bacteria.

The study has also shown the importance of socioeconomic aspects for the successful use of the IIT/SIT approach. Social acceptance, for example, increased during the study as support of the local community went up following mosquito releases and the resulting decrease in nuisance biting for the IIT/SIT approach to be successful, the local community needs to be on board and work together to ensure consistent and integrated use of the approach over the entire area in order to effectively counteract and control the movement of the insects. Another aspect is the cost-effectiveness overall future costs of a fully-operational intervention are estimated at US$ 108-163 per hectare per year, which is considered cost-effective in comparison with other control strategies.

Experts in China plan to test the technology in larger urban areas in the near future using sterile male mosquitoes from a mass-rearing facility in Guangzhou, said Zhiyong Xi, Director of Sun Yat-sen University-Michigan State University’s Joint Center of Vector Control for Tropical Diseases and Professor at Michigan State University in the United States. The company operating the facility uses advanced mosquito mass-rearing and irradiation equipment that have been developed in collaboration with the Joint FAO/IAEA Division.

Global cooperation on the development of SIT to control mosquitoes intensified following the Zika epidemic in 2015 to 2016. The incidence of dengue is on the rise, with the number of cases reported to the World Health Organization (WHO) increasing from 2.2 million in 2010 to over 3.3 million in 2016. The actual incidence is much higher, and one estimate, according to the WHO, indicates 390 million new infections each year.


Genetic Sexing Techniques

In many applications of autocidal control, it would be efficient to separate the males and females before release. Possible reasons for such separation are to avoid assortative mating to avoid any increase in the size of the feral population during a genetic control procedure to eliminate females which may be disease vectors, extremely obnoxious, or which cause damage to produce or livestock. Another reason for using a genetic sexing procedure is to bring about cost savings in the rearing process if one sex could be eliminated in the egg stage (pre-zygotic sexing). In this case, twice as many insects (of one sex) could be produced with a given expenditure for diet and labor.

Thus, removal of females during the rearing process has been a goal in autocidal control research for many pest insects. The most successful efforts have been with a mosquito, Anopheles albimanus, the medfly, Ceratitis capitata, and the stable fly, Stomoxys calcitrans. Two of these approaches demonstrate what can be done. A combination of mechanical separation of the sexes and genetic manipulation has been employed in the medfly. Wild-type pupal color is brown. Mutants involving either black pupae or white pupae have been linked with the sex chromosomes by means of single or multiple chromosomal translocations. By this means strains are produced in which male pupae are black and female pupae are brown. The pupae are then separated by electronic color recognition circuitry in a mechanical or air-driven sorter. Some disadvantages of this technique are the possible breakdown of the translocation stocks due to crossing-over, contamination of the strain by wild-type individuals, and partial sterility of the strain due to the translocation.

If no use can be found for the reared females, then it would be worthwhile to eliminate them before they consume expensive diet. A number of schemes have been proposed to accomplish removal of females at the egg or early larval stage. In some species, alleles resistant to specific toxic chemicals, such as ethyl alcohol, endrin, purine, potassium sorbate, dieldrin, cyromazine, and propoxur have been selected. Then translocations between one of the sex-chromosomes (usually the Y-chromosome) and the chromosome bearing the resistance locus are induced. When the toxic substance is introduced into the colony, only the sex carrying the resistant allele survives. This method has been successful for three species of mosquitoes and is being actively investigated for the medfly.


Introduction

Mosquito-borne illnesses including dengue fever, lymphatic filariasis (elephantiasis), yellow fever, and malaria make up 16% of the global disease burden, particularly so in the developing world [1]. Of these, malaria accounts for 18% of childhood deaths in sub-Saharan Africa [2] and in 2010 afflicted 219 million people and resulted in 660,000 deaths [3]. Malaria has been successfully controlled in many regions through vector-targeted intervention such as insecticide-treated bed nets (ITNs) and indoor residual sprays (IRS). However, these interventions will fail to eliminate malaria in regions with extremely high rates of parasite transmission and in areas where mosquito vectors are not susceptible to existing control techniques (such as by exophily or insecticide resistance) [4].

Sterile Insect Technique (SIT) is one control strategy that is gaining renewed interest for the control of mosquito populations [5-7]. The technique involves the mass release of males sterilized through radiological or chemical means. These mate with the wild population by out-competing non-sterile wild males [8]. Females mosquitoes (generally) mate only once, thus a successful mating with a sterile male will prevent the development of any offspring from the inseminated female [9,10]. In some insect pests such as the tsetse fly [11], medfly [12], and melon fly [13] SIT has proved enormously successful in achieving local control or elimination, including eradication of the screwworm from all of North America [14]. In mosquitoes, over two dozen SIT trials have been reported however, issues such as poor competition with wild males, semi-sterility, or no ultimate adult population reduction - even despite successful sterile matings have been reported, reviewed in Benedict and Robinson 2003 [15].

Promising new advances in mosquito population control using the release of transgenic, instead of chemically or radioactively sterilized mosquitoes, are now garnering substantial interest [6]. These transgenic implementations are extensions of SIT, in that released males mate with wild-type females to abnormal results due to the males carrying a cell-lethal transgene. These implementations may allow for straightforward mass-rearing of a male-only population for release maintain larval competition with wild type mosquitoes extend the “lifetime" of the intervention via propagation of the transgene through the population and/or allow for the ability to induce or suppress the lethal trait through larval chemical exposure [16-19]. This paper broadly places these transgenic SIT implementations into one of four categories:

Early acting bisex (EBS) which is most similar to classical SIT whereby wild-type females mating with released males will produce no offspring. For modelling purposes, EBS is described as any implementation that involves the release of male mosquitoes modified such that no viable offspring (including larvae) are produced.

Early acting female-killing (EFK) whereby wild-type females mating with released males will produce no female offspring, but the transgene can be passed on through male progeny.

Late acting bisex (LBS) whereby wild-type females mating with released males will produce offspring that only survive through the aquatic stage and die shortly prior to or after emergence. Transgenic larvae that will eventually die prior to adulthood provide larval competition reducing wild-type larvae’s chances of survival.

Late acting female-killing (LFK) whereby wild-type females mating with released males will produce offspring, but only male offspring survive to adulthood where they may propagate the transgene to their progeny. Transgenic female larvae that will eventually die prior to adulthood provide larval competition to wild-type larvae.

Release of insects carrying a dominant lethal gene (RIDL) is a transgenic implementation that has received the most recent attention. Thomas et al. and Heinrich et al., [20,21] reported early success in the development of RIDL, generating strains of Drosophila with cell-lethal gene products under chemically repressible promoters expressed either in females only, or with female specific toxicity. Since then, the generation of two late acting RIDL strains of the dengue fever mosquito, Aedes aegypti, has been reported. This includes an EBS implementation with a repressible strain that kills all larvae, leaving no viable offspring [22]. This strain has been shown to compete reasonably well with the wild-type, with only a 5% reduction in survivability, a 4-day shortened average lifespan, and (perhaps beneficially) a one-day earlier emergence as an adult [23]. The other RIDL strain is a LFK implementation where adult females die immediately due to their inability to fly, whereas males remain to propagate the transgene [24]. RIDL strains are currently in trials, with early success reported in both large-cage [25] and field trials [26-28].

The success of SIT implementations is dependent on wild-type and mass-reared mosquitoes readily mating [29], although experience from mass-rearing campaigns of agricultural pests such as the screwworm has shown that significant loss of mating competitiveness can arise in mass-reared populations [30]. Some processes to sterilize mosquitoes (e.g. irradiation) can induce a reduction in mating competitiveness [31] but transgenic techniques can generate a line of sterile mosquitoes with no loss in mating competitiveness. This has been shown in Anopheles stephensi, Anopheles arabiensis, and Ae. aegypti when compared against the parent (non-transgenic) lab colonies [32-34]. However, when lab-reared transgenic Anopheles gambiae mosquitoes were compared in large-cage field trials against wild collected mosquitoes, reductions in mating competitiveness were indeed noted, although competitiveness was still better than those achieved and accepted for use in the medfly control programs [35]. Taking into account the mating competitiveness of transgenic-mass reared mosquitoes is therefore another important consideration to make when planning or considering the implementation of an SIT campaign, especially when determining the mosquito release size which may need to be increased to counteract reduced competitiveness.

Much work on the malaria mosquito, An. gambiae, remains to be done. However, there are early and promising successes. Recent work developing mosquitoes carrying homing endonucleases (HEG) has shown progress [17,36]. Various implementations of homing endonucleases work by selectively destroying the X chromosome– preventing female offspring (an EFK implementation) or genes vital to males and/or females. Additionally, Thailayil et al., [37], successfully demonstrated an EBS implementation using RNAi to knockdown the production of sperm.

This manuscript reports the first use of agent-based modelling to evaluate four implementations for the control of An. gambiae populations through the release of transgenic male mosquitoes at various release proportions and mating competitiveness rates. This work complements previous efforts to model SIT implementations which are summarized in Table 1. Agent-based modelling is used to simulate frequent releases of male transgenic mosquitoes homozygous for a cell-lethal transgene. The transgene exclusively kills the intended individuals 100% of the time (i.e. LFK will kill no males, kills all females, and no early larvae die). The results provide additional evidence that transgenic implementations of SIT could be used with success to eliminate An. gambiae vector populations, and estimate the relative success of various implementation strategies.


Author information

These authors contributed equally: Xiaoying Zheng, Dongjing Zhang, Yongjun Li, Cui Yang, Yu Wu

Affiliations

Key Laboratory of Tropical Disease Control of the Ministry of Education, Sun Yat-sen University–Michigan State University Joint Center of Vector Control for Tropical Diseases, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China

Xiaoying Zheng, Dongjing Zhang, Yongjun Li, Cui Yang, Yu Wu, Yongkang Liang, Linchao Hu, Qiang Sun, Jiajia Zhuang, Meichun Zhang, Zhongdao Wu & Zhiyong Xi

Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna International Centre, Vienna, Austria

Dongjing Zhang, Andrew G. Parker, Jeremie R. L. Gilles, Kostas Bourtzis & Jérémy Bouyer

Guangzhou Wolbaki Biotech Co., Ltd, Guangzhou, China

Yongjun Li, Cui Yang, Yongkang Liang, Xiaohua Wang, Yingyang Wei, Jian Zhu, Wei Qian, Julian Liu & Zhiyong Xi

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA

Xiao Liang, Xiaoling Pan, Qiang Sun, Luke Anthony Baton & Zhiyong Xi

School of Medicine, Hunan Normal University, Changsha, China

Guangzhou Center for Disease Control and Prevention, Guangzhou, China

Ziqiang Yan, Zhigang Hu & Zhoubing Zhang

Department of Mathematics, Michigan State University, East Lansing, MI, USA

Center for Applied Mathematics, College of Mathematics and Information Sciences, Guangzhou University, Guangzhou, China

Department of Entomology, Nanjing Agricultural University, Nanjing, China

Jun-Tao Gong & Xiao-Yue Hong

Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China

State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

Lingnan Statistical Science Research Institute, Guangzhou University, Guangzhou, China

Bio21 Institute, School of BioSciences, University of Melbourne, Melbourne, Victoria, Australia

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Contributions

Z.X., X.Z., D.Z., Y. Li, C.Y., Y. Wu, A.G.P., J.R.L.G., K.B., Z.W., L.A.B. and A.A.H. developed the concept and methodology D.Z. performed radiation and male mating-competitiveness assay Y. Liang and C.Y. performed population suppression and population replacement in laboratory cages Y. Li and X.Z. performed human-landing assay C.Y. performed mosquito quality control Y. Li, Y. Wu, X.L. and X.P. performed vector competence assays A.G.P designed the X-ray irradiator D.Z., K.B. and J.R.L.G. performed the population-suppression experiment in semi-field cages X.Z., Z.Y., Y. Wu and J. Zhuang performed community engagement X.L., X.P., Q.S., J.-T.G. and M.Z. performed cell culture, virus titration and Wolbachia density quantification Z.Y., Zhigang Hu, Z.Z., L.L. and Q.L. identified the field sites B.Z., L.H. M.T. and J.Y. developed the mathematical model and performed spatial analyses X.W. and J. Zhu performed mosquito mass rearing Y. Wei and W.Q. performed release and field surveillance J. Zhu, W.Q., X.-Y.H., Zhiyong Hu and Z.W. performed coordination for the project W.Q. obtained regulatory approvals for mosquito releases J.L. performed mosquito crosses and maintenance of mosquito lines J.B. and Z.X. performed cost-effectiveness analysis Z.X. provided oversight of the project and contributed to all experimental designs, data analysis and data interpretation Z.X., L.A.B., X.Z., D.Z., Y.L. and A.A.H. wrote the manuscript. All authors participated in manuscript editing and final approval.

Corresponding author


Methods

Ethics statement

Human ethics was sought through the CSIRO Social and Interdisciplinary Science Human Research and Ethics Committee (CSSHREC) and approved under project 026/16 named “Sterile insect technology development for Aedes aegypti “. As part of this approval all residents in release areas provided written consent for scientists to operate within their property, and were provided with an information sheet detailing how, why, where and when the research was to be performed and funding bodies. All residents were informed about the risks and benefits, including the potential for an increase in mosquito numbers during male releases. To enhance communication, brochures were distributed to homeowners, articles were posted in local newspapers, a website was setup for enquiries and residents were engaged through a project advisory group containing members of the local community.

Study sites

Six mark-release-recapture experiments were performed during two seasonal periods, representing dry and wet seasons, in North Queensland, Australia. Mark-release-recapture experiments 1–3 (season 1) occurred late dry season, between 18 November and 13 December 2016, while MRR experiments 4–6 (season 2) occurred during the wet season, between the 7 and 27 February 2017. The study site in South Innisfail (17.5435°S, 146.0529°E) was situated in a residential area, 0.18 km 2 in size to the south east of Innisfail, a rural town on the main highway 88 km south of Cairns. The site contained 95 residential premises bounded by the Johnson River to the West and by grass sports fields and forest to the east. The site also contained a primary school to the north and a number of small commercial buildings (Fig 1). The Innisfail region is one of the wettest in Australia, averaging 3,547 mm of rainfall annually with tropical cyclones occurring throughout Summer and Autumn [36]. The urban landscape of Innisfail is unusual for northern Australia, with dwellings in the town a mix of Queenslander (constructed of wood with tin rooves and typically raised off the ground by 1.5–2 m) single floor fibre board, modern brick single floor, and ‘art deco’ style single floor constructions. House block size were approximately 800 m 2 with simple fencing or hedge-like greenery on boundaries, with open space underneath raised buildings utilized for storage, laundry and recreation areas. Roads averaged 25 m wide (fence to fence).

Maps indicate landscape characteristics which include natural imagery (1A) and land use (1B). Rhodamine B marked Aedes aegypti were released at single point (blue triangle) and multi-point locations (purple triangles) and recaptured using Biogents Sentinel traps (red circles). Base layer imagery of South Innisfail (1A) provided by State of Queensland [2018] under licence [37] and landuse basemap (1B) digitized manually [38].

Rearing and release

Aedes aegypti colonies were newly established with wild type eggs collected from multiple ovitraps in Innisfail before each experimental period. Mosquito colonies were maintained using standard laboratory rearing protocols with 28°C ± 1°C, a 70% (± 10%) relative humidity, and a 12:12 hour light cycle and twilight period. F1 –F3 generation wild-type Ae. aegypti larvae were hatched into a solution of 0.2 g/L yeast in water, in which they were allowed to feed for 24 hours. Five-hundred first and second instar larvae were pipetted into a three-litre bucket to an approximate density of one larvae per 6 ml of water. Larvae in each bucket were fed ground Tetramin Tropical Fish Flakes (Tetra, Germany) provided at 0.45 g on day 2, 0.8 g on day 5 and again on day 6 if required. Ten minutes after the food settled, bucket water was stirred in a ‘side to side’ motion to distribute ground fish flakes. Male pupae were separated with a one ml bulb pipette based on size with 20 individuals placed into 300 ml Styrofoam rearing cups covered with mesh. After emergence, cups were visually inspected for the presence of females and if detected these were removed through aspiration. Adult males were fed a 0.4% rhodamine B (weight to volume) in a solution consisting of 160 mg rhodamine B dissolved in 40 ml of a 25% honey solution following the methods of Johnson et al. [31]. Males were maintained on the solution for four days to ensure adequate body and seminal fluid marking [31]. Males were transported to the study sites the day before release and released when five days of age.

Approximately 1,250 males were released during each of the six MRR experiments, with a delay of between seven and nine days between releases to separate recaptures. Releases occurred at 6am for day releases for MRR 1–5 and 7 pm for the night release (MRR 6). Release location varied depending on experimental design, with single point releases occurring at the southern end of the study site (MRR 1, 2 & 6 Fig 1B). For multi-point releases, males were divided evenly and released at five points along the eastern side of the central road (MRR 3 & 5 Fig 1A). Mark-release-recapture 4 (MRR 4) was a single linear release of males from a prototype mechanical device used in Crawford et al. [39], on the eastern side of the road, from north to south (Fig 1A).

Trapping arrays and recaptures

The study site contained 83 Biogents Sentinel traps without lures (BGS Biogents GmbH, Regensburg, Germany) set with the goal of distinguishing landscape characteristics that affected the movement of males through blocks and across movement barriers such as roads (Fig 1). To do this, one trap was placed close to each chosen dwelling, one in the backyard and, where possible, one near the forested area adjacent to the residential area. Additional traps were placed at dwellings across the road from the release sites to monitor movement across a known dispersal barrier. For MRR 1 and 2, traps were turned on after 24 hours to allow for mixing of marked males with the wild population. For MRRs 3–6, traps were turned on two hours post-release.

All traps were serviced daily throughout each MRR experiment until no marked males were present in the sample. Captured adult Ae. aegypti were stored at

4°C for transfer to the laboratory for identification, after which both males and females were processed for the presence of rhodamine B following the methods of Johnson et al. [31]. Females were considered to be inseminated by a released marked male if rhodamine B was observed in the bursa, spermathecae or both. Females were considered to have mated with a wild, unmarked male if sperm, visualised by DAPI staining, was present in the bursa, spermathecae or both in the absence of rhodamine B.

Determination of biological parameters, statistical analysis and dispersal kernel framework

For all experiments the probability of daily survival (PDS) was estimated by regressing log10 (x+1) the number of recaptured males against days since release, where the antilog10 of the regression slope was the PDS [40]. The ALE was calculated from the PDS as 1/-logePDS [41]. The Lincoln Peterson Index with Chapman modifier was used to estimate population size [42]. Where is the size of the population, n1 is the number of marked animals released into the population, n2 the total number of individuals captured (marked and unmarked), and m2 the total number of marked individuals recaptured:

To account for the assumption that marked insects become completely mixed within the local population, only males from the release block and from multi-point (MRR 3 & 5) or the linear releases were selected for analysis (MRR 4). To account for the low survival of released Ae. aegypti males, only marked males captured the days before the ALE (rounded to the nearest whole number) of each experiment were selected [42]. The remaining assumptions of the LPI [8] were met with reasonable certainty which include: 1) the mark should not affect insects, 2) sampling is random with respect to marked insects, 3) samples are measured at discrete time intervals in relation to total time, and 4) the population is not unduly influenced by immigration or emigration during the period of study.

Traditional methods of MDT were calculated using the methods of Lillie et al [29] and Morris et al [28] where annuli are drawn around the release point to estimate dispersal distances: and then a correction factor (CF) is applied to accommodate unequal trapping densities where:

Flight range (FR) of male movement was estimated from the linear regression of the cumulative ERs for each annulus (x axis) on the log10 (y axis) as the value of the y axis at 50% (FR50) and 90% (FR90), of the largest value of the x axis [13]. We introduce the concept of mean insemination distance (MID) by modifying the above methods of Lillie et al [29] and Morris et al [28] by estimating the mean distance over which rhodamine B inseminated females were captured during each experiment.

We then compared traditional MDT estimates with the spatially and temporally evolving isotropic kernel (STEIK) framework developed by Trewin et al. [19]. The STEIK framework uses an isotropic Gaussian diffusion model with kernels defined as a temporally-evolving probability density function (PDF) over two-dimensional space [19]. Here the probability of a mosquito being trapped per unit area is a function of the distance from the release location and the time since release [19]. Multi-point releases treat the trapping intensity at each site as a finite mixture of the dispersal kernels from each release point, so the unknown latent variable of release location for each trapped mosquito has been integrated out of the probability density function used for the likelihood equation. For multi-point releases we divided the total number of mosquitoes released evenly between release points. For STEIK estimates of 50% and 90% FR, quartiles of simulated kernel distributions were calculated from parameter estimates relevant to average lifetime and the standard deviation of the isotropic kernel for each experiment. To facilitate the use of our STEIK framework by experimentalists, we have stored male recapture data and R code at https://github.com/dpagendam/MRRk [43] and at the CSIRO Software Collections under an Open Source Software Licence [44]. Raw mosquito and trap data are available at CSIRO Data Collections under a Creative Commons 4.0 Licence [45].

Mating competitiveness was estimated using the methods of Reisen et al. [46] where the mating competitiveness (C) was calculated from the number of unmarked males among all males (w) and the number of unmarked male matings among all determined matings (f) where:

The variation and test for significance between experiments was via chi-squared test following Grover et al. [47]. To examine differences in the daily proportion of rhodamine B inseminated females with the total number of mated females between seasons, we used a mixed effects, logistic regression model with a binomial distribution and logit link function. Fixed effects included season and release type (multi-point vs point release) with a random effect of experiment number. The same model framework was used to examine differences in the total daily proportion of mated females (both rhodamine B and wild mated) between seasons and the daily proportion of wild-type mated females with total females captured. The Akaike information criterion (AIC) was used to selecte the most parsimonious model. Odds ratios (OR) were calculated for coefficients exhibiting significant differences in proportions. The R package ‘glmmTMB’ [48] was used for all mixed effects models and the packages ‘DHARMa’ [49] was used for model diagnostics and ‘ggplot2’ [50] for visualisations. To look for collinearity in predictors, correlations were examined using the R package ‘corrplot’ [51]. The Wall-Raff rank sum test of angular distance from the R package ‘circular’ was used to compare similarities between wind direction and male recapture angle [52]. To compare whether rhodamine B and wild males and wild female Ae. aegypti were more likely to be captured by BGS traps at certain locations (house, backyard or forest), we used contingency table analysis with odds ratios calculated via the R package ‘epitools’ [53]. All analyses were performed using R version 3.5.3 [54]. All landscape maps were digitized by outlining landscape features (houses, roads, blocks, river) in Google Earth [55] and modified in ArcGIS Desktop [56] and provided as a layer file [38]. Two-dimensional kernels were output as images from an R density function where the mean was equal to zero and the spread equal to the time dependent standard deviation of the kernel. Kernels were then overlaid on maps to scale in standard image editing software.


Invasive Fruit Flies

Study of Attraction of Nontarget Organisms to Fruit Fly Female Attractants and Male Lures in Hawaii.

Luc Leblanc, Daniel Rubinoff, and Mike San-Jose

&emsp&emspThe main purpose of this project was to assess environmental risks of using fruit fly (Tephritidae) attractants for their control or eradication in the Hawaiian environment. More specific focus was on the use of male lures (cue-lure, methyl eugenol) and female synthetic food attractants (BioLure, torula yeast and solulys).

Study Sites

&emsp&emspTraps baited with female attractants and male lures were setup in native, mixed native and non-native forests, farmlands, orchards and residential areas. Traps were maintained for 10-24 weeks and emptied weekly at 35 sites on Hawaii Island (2005) (Figure 1) and 46 sites on Maui (2006) (Figure 2). Trap catches were compared against catches from unbaited control traps. On Hawaii island, 9 sites formed a 20 km transect along the Stainback Highway (138-1,045 m above sea level), 15 sites were maintained in a 35 km transect along the Saddle Road (439-2,012 m), 6 sites were along the upper Hamakua Ditch Trail in wet native forest (North Kohala Forest Reserve) (906-1,019 m), and 5 sites were in the agricultural community of Waimea (744-872 m). On Maui, we maintained 14 sets of traps in 9 sites in the Kula agricultural community (517-1,138 m), primarily in persimmon and coffee orchards and their adjacent non-native forests, and 37 sites in mostly endemic forest on the northern slope of Haleakala Mountain (1,184-1,583 m) in the Makawao, Waikamoi and Koolau Forest reserves.

Fig 1: Trapping sites on Hawaii island

Fig 2: Trapping sites on Maui island

Attraction to male Lures

&emsp&emspMale lures, combined with insecticides, are commonly used in traps to monitor male Bactrocera fruit fly populations and detect incursion of exotic species. They also constitute a powerful tool for fruit fly suppression and eradication through male annihilation. The large-scale use of male lures for control and eradication has raised concern of possible nontarget impacts on insects other than fruit flies, especially beneficial species and the numerous endemic Hawaiian insects.

&emsp&emspPast attempts in Hawaii to characterize the range of nontargets attracted to male lures, by comparing captures in lure-baited traps with unbaited control traps, have resulted in an impressive list of 36 insect species, in 16 families of Diptera, Coleoptera, Hemiptera and Hymenoptera, assumed to be attracted to methyl eugenol. Among them were 11 species of endemic Hawaiian Drosophilidae (Figures 3 to 7), a very diverse group with 559 described species, of which 12 are recognized as endangered by the US Fish and Wildlife. However, at least 26 of these purportedly lure-attracted species are scavengers, and authors cautioned that some of them may have been actually secondarily attracted to decaying fruit flies that could not be excluded from entering male lure traps.

Fig 3 Fig 4 Fig 5 Fig 6 Fig 7
Figures 3 to 7: Various representatives of endemic Hawaiian Drosophilidae

&emsp&emspOur study aimed to further investigate male lure nontarget effects by carefully discriminating between actual attraction to male lures and secondary attraction to decaying insects. Trapping was carried out across a broad range of environments, including intact native forest, mixed native/invasive forest, invasive forest, agricultural and residential areas.

&emsp&emspBucket traps made from drinking cups and baited with cue-lure or methyl eugenol (ME) (Scentry lure plugs) were maintained and emptied weekly at every trapping sites on Hawaii and Maui islands. To control for the possible attraction to dead insects, bucket traps artificially baited with decaying oriental fruit flies were maintained at all sites, except in the Maui endemic forest. Unbaited bucket traps were also maintained at all sites to control for the random entry of insects into traps. Vapor tape and a 20% solution of propylene glycol were included in all traps to kill and preserve trapped insects, respectively. In addition to the bucket traps, MultiLure traps charged with the 3-component BioLure food attractant and, in Maui forest, bucket traps baited with fermented mushroom, were maintained to ensure that the apparent lack of attraction to male lures is not due to the absence of potential nontarget species at trapping sites.

&emsp&emspTraps with male lures and decaying flies captured 401 recognized arthropod species, in 17 orders and 93 families, dominated by Diptera (94.9% of all captures, 248 species), primarily in the families Drosophilidae, Phoridae and Milichiidae.

&emsp&emspCue-lure did not significantly attract any nontarget insects, and melon flies were usually not numerous enough in traps to cause secondary attract scavengers. These results are fully consistent with conclusions from previous studies.

&emsp&emspSeven nontarget species in five insect orders were significantly attracted to ME-baited traps, regardless of the presence or absence of decaying fruit flies. Five of these are closely associated with flowers, feeding on pollen or nectar. Honeybees (Apis mellifera L.) (Apidae) and the flower fly Allograpta obliqua (Say) (Syrphidae) were attracted in rather small numbers (0.04-0.09 per trap per day). Honeybee attraction to ME in Hawaii was previously documented in literature, and orchid bees (Apidae: Euglossinae) are similarly drawn to ME in South America. Two endemic species of Crambid moths [Mestolobes minuscula (Butler) (Figure 8) and Orthomecyna exigua (Butler) (Figure 9)] were also attracted to ME in Kula (Maui) orchards. Although endemic, these two species are common on Maui in non-native habitats at lower altitude. The introduced sap beetle Carpophilus marginellus Motsch. (Nitidulidae), a common flower visitor that contributes to fruit tree pollination in Japan, is attracted to decaying fruit flies, but also to ME in traps. The attraction of flower insects is no surprise, since ME or some of its related compounds have been detected in the flower blossoms of a diversity of plant families.

&emsp&emspAlthough our traps captured limited numbers of green lacewings (Chrysopidae), they were previously reported as attracted to ME in Hawaii, the Philippines, and Taiwan. Adult chrysopids, depending on species, feed either on live insects, or on flower pollen and nectar and honeydew from Hemiptera. At least of two of the three species attracted to ME are flower feeders, strongly suggesting an attraction to ME as emulation of floral compounds, further supported by the lacewing attraction to the natural flower fragrance compound eugenol in Malaysian rainforest.

&emsp&emspThe endemic plant bug Orthotylus coprosmae Polhemus (Hemiptera: Miridae) (Figure 10) is attracted to ME in Maui endemic forests, consistent with previous ME attraction records of three other endemic mirids on Kauai. At least one of these feeds on a host plant known to contain ME. A similar plant kairomone relationship may also explain published records of endemic anobiid beetles attraction to ME traps on Kauai.

&emsp&emspMethyl eugenol attracts females of the endemic fungus gnat Bradysia setigera (Hardy) (Sciaridae) (Figure 11). Conspecific males and other common sciarids are not attracted. In this case it likely acts as a pheromone analogue rather than a kairomone.

Fig 8 Fig 9 Fig 10 Fig 11
Figures 8 to 11: Endemic Hawaiian nontarget insects attracted to methyl eugenol

&emsp&emspAt least 56 species in 21 families of Diptera, Hymenoptera and Coleoptera were significantly drawn to decaying fruit flies rather than male lure. They were abundant in traps with decaying flies, and were collected in ME traps only when enough dead trapped flies had accumulated. We demonstrated that 8 of the 36 species previously reported as attracted to ME are actually drawn to dead flies, and that most other species belonged to families attracted to decaying flies, rather than ME, most commonly the Drosophilidae, Phoridae, Chloropidae, Lonchaeidae, Milichiidae, Neriidae, Otitidae, Psychodidae, Sphaeroceridae, Calliphoridae, Muscidae, and Sarcophagidae. Most species in these families are indeed scavengers. Aside from the ME-attracted C. marginellus, other endemic and introduced Nitidulidae were attracted only to decaying flies.

&emsp&emspDrosophilidae were the most numerous and diverse nontargets attracted to decaying flies at all sites. Our results confirm or strongly support that most or all of the 11 endemic and 5 introduced drosophilids reported in literature as ME-attracted were actually attracted to dead flies. Nearly half (143 of 306) of the drosophilids expected to occur at the trapping sites were collected using BioLure and mushroom bait traps, but not in male lure traps. Invaluable data was generated on drosophilid distribution in endemic and disturbed ecosystems in Hawaii. It is an unforeseen extra benefit from this research project that is the focus of a publication soon to be submitted to an invertebrate conservation-oriented journal. Fragrant leaves of Cheirodendron trigynum (Gaud.) Heller (Araliaceae), the most common larval host species for endemic drosophilids, were demonstrated through Solid Phase Microextraction (SPME) analysis not to contain ME.

&emsp&emspA few species of predators and parasitoids were attracted to decaying flies. Almost all are associated with decaying matter (Staphylinidae) or parasitoids of houseflies (Encyrtidae, Braconidae, Pteromalidae).

&emsp&emspPrecautionary suggestions are provided to minimize the undesirable nontarget impact of the use of ME for control or eradication on flower-associated insects, endemic plant bugs and fungus gnats and scavenger insects. Flower insects were attracted in small numbers (0.03-0.15 per trap per day) in our study, consistent with previously published data, suggesting that attraction is likely to be short-ranged, and can be further minimized if a ME trap or dispenser is hung to a tree past its flowering stage. This was confirmed by the much lower honeybee and moth captures in non-flowering persimmon trees that in adjacent flowering coffee trees in Kula. A comparison of captures of endemic saprophagous insects in decaying fly and BioLure traps in orchards and backyards, native forest and ecotone forest adjacent (< 100 m) to native forest shows that very small numbers of a few endemic species are captured in non-native sites.

Fig 12: Endemic Hawaiian viviparous blowflies (Dyscritomyia)

&emsp&emspThis is not the case in endemic forest and its adjacent ecotones, where a broad diversity of endemic drosophilids and calliphorids (the larviparous Dyscritomyia) (Figure 12) are trapped. Based on studies of range of dispersal of 14 common North American Drosophilid species by Donald McInnis (USDA-ARS), who estimated their maximal dispersal distance to be 300 meters at most, we concluded that using traps at least 300 meters from native forest will minimize possible nontarget effects, if dead target flies accumulate inside traps.

Attraction to BioLure

&emsp&emspThe dry food lure BioLure in the MultiLure trap (Figure 13) was developed as an alternative to the traditionally used liquid protein lures in glass McPhail traps (Figure 14) by identifying and using the individual volatiles from the bacterial breakdown of protein hydrolysate that were most attractive to fruit flies. The 3-component BioLure, used in our study, is composed of ammonium acetate, trimethylamine hydrochloride and putrescine. Although originally developed as a monitoring tool, BioLure has become a common tool to control the Mediterranean fruit fly [Ceratitis capitata (Wiedemann)], through mass-trapping, in fruit tree orchards in Spain and Israel. In Hawaii it is used, in combination with protein bait sprays, to suppress C. capitata in Maui orchards.

&emsp&emspNontarget attraction to BioLure, mainly saprophagous Diptera, has been reported in literature, but not studied systematically in a diversity of habitats. We have therefore investigated nontarget attraction to BioLure across a range of endemic and agricultural habitats in Hawaii, compared nontarget attraction of the individual BioLure components, and determined whether the omission of the putrescine ingredient results in decreased nontarget catches without reducing captures of target C. capitata.

&emsp&emspMultiLure traps baited with 3-component BioLure, as well as unbaited controls, were maintained continuously, or intermittently in endemic forest, at every trapping site on Hawaii and Maui islands. A solution of 20% propylene glycol was added to each trap to preserve and facilitate identification of captured insects. Trap contents was emptied weekly. These traps served the dual purpose of characterizing nontarget attraction to BioLure and confirming the presence of nontargets that may potentially be attracted to the male lure traps also present at each site. Additionally, three sites were selected, one in endemic forest and two in orchards, to study fruit fly and nontarget attraction to the separated individual components of BioLure. A third trap, with only two components, was also maintained at each site in Kula (Maui), to determine if the omission of the putrescine ingredient results in a decrease in nontarget captures without compromising captures of C. capitata.

&emsp&emspCaptures in BioLure traps were numerically dominated by Diptera (94.3%). The endemic forest site samples were dominated by endemic and introduced Drosophilidae and endemic Calliphoridae, while most of the captured nontargets in the nonnative sites were of introduced species. The majority of nontarget species belonged to families whose larvae are scavengers on decaying plant or animal matter (Drosophilidae, Chloropidae, Lonchaeidae, Neriidae, Otitidae, Phoridae, Anthomyiidae, Calliphoridae, Muscidae, Sarcophagidae and Nitidulidae). These same families and species of were also strongly attracted to the bucket traps baited with decaying fruit flies.

&emsp&emspBioLure attracted few beneficial predators or parasitoids, except for a few parasitoids of house flies and moderate numbers of tachinids flies. Pollinators were not attracted to BioLure. Although it attracted very few green lacewings (Chrysopidae), there is literature evidence that protein hydrolysate and BioLure can attract the pollen and nectar-feeding species.

&emsp&emspA comparison of attraction to the components in separate traps and the three components together inside a trap in a randomized block design has shown that ammonium acetate or, to a lesser extent, putrescine, are the main components attractive to nontargets, depending on the species. It was also demonstrated, as is the case for target fruit flies, that the three components act in synergy, attracting larger numbers of nontargets together in a trap than in three traps baited with the separate components.

&emsp&emspThe elimination of the putrescine ingredient from BioLure traps resulted in significant capture reduction for five nontarget species and an overall 20% reduction in the number of nontargets attracted, with no reduction in target C. capitata captures. I can therefore be omitted from BioLure when used for Mediterranean fruit fly monitoring or control in Hawaii.

Publications

&emsp&emspLeblanc, L., Rubinoff, D., and R.I. Vargas. 2009. Attraction of nontarget species to fruit fly (Diptera: Tephritidae) male lures and decaying fruit flies in Hawaii. Environ. Entomol. 38: 1446-1461.

&emsp&emspLeblanc, L., O&rsquoGrady, P.M., Rubinoff, D., and S.L. Montgomery. 2009. New immigrant Drosophilidae in Hawaii, and a checklist of the established immigrant species. Proceedings of the Hawaiian Entomological Society. 41: 121-127.

&emsp&emspLeblanc, L., Vargas, R.I., and D. Rubinoff. 2010. Attraction of Ceratitis capitata (Diptera: Tephritidae) and endemic and introduced nontarget insects to BioLure bait and its individual components in Hawaii. Environ. Entomol. IN PRESS.

&emsp&emspLeblanc, L., Vargas, R.I., and D. Rubinoff. 2010. A comparison of nontarget attraction to BioLure and liquid protein food lures in Hawaii. IN PREPARATION.

&emsp&emspVargas, R.I., Shelly, T.E., Leblanc, L. and J.C. Piñero. 2010. Recent advances in methyl eugenol and cue-lure technologies for fruit fly detection, monitoring, and control. Vitamins and Hormones. Section: Pheromones, vol. 83. Academic Press. SUBMITTED.

There are four species of pest fruit flies (Tephritidae) in Hawai'i. All are invasive, and most of them are from tropical Asia.
For more information on fruit fly diversity and management, consult the Pacific Fruit Fly Website and the Hawaii Area-Wide Fruit Fly Pest management Program site.

Melon Fly (Bactrocera cucurbitae) was the first pest fruit fly detected in Hawai'i in 1895. Its larvae breed on cultivated and wild Cucurbitaceae, as well as papaya.

Mediterranean Fruit Fly (Ceratitis capitata) has been the dominant polyphagous fruit pest since from its discovery in 1907, until the introduction of Oriental fruit fly.

Oriental Fruit Fly (Bactrocera dorsalis) was detected in Hawai'i in 1945, and has become the main fruit pest of edible and wild fruits in Hawai'i. Following its introduction, it has displaced the Mediterranean fly to become the dominant species, limiting the Mediterranean fly to attack coffee and fruit at higher elevations.

Malaysian fruit fly (Bactrocera latifrons) is the most recent migrant, known in Hawai'i since 1983. Its host range is restricted to cultivated and wild solanaceous plants. It is not a serious crop pest.

Photos are courtesy of Jari Sugano (University of Hawai'i at Manoa)


Joint FAO/IAEA Programme - NAFA

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Tsetse flies

&bull Guidelines for Blood Collection, Processing and Quality Control for Tsetse Rearing Insectaries, Version 2.0 [pdf]. Vienna, Austria (2019)[.pdf]. This document is intended for use in laboratories and institutions that maintain colonies of tsetse flies. It describes the procedures for the collection of animal blood in the abattoir, decontamination through ionizing radiation, preservation and storage, quality control assurance and processing of the blood into diet for feeding tsetse flies.
&bull Standard Operating Procedures for Detection and Identification of Trypanosome Species in Tsetse Flies Version 1.0 [.pdf]. The manual provides an easy-to-use work flow that combines several different PCR-based methods that can be used for trypanosome detection and identification in field-caught tsetse flies and/or host blood samples collected from livestock and wildlife animals.
&bull Standard Operating Procedures for Identification of Tsetse Species from Wild Populations and Laboratory Colonies [pdf]. This document provides useful information to correctly identify specimens of nine tsetse flies species/subspecies derived from field collections or laboratory colonies using molecular techniques. Vienna, Austria (October 2018).
&bull Guidelines for Mature Tsetse Sterile Male Pupae Packaging for Long Distance Shipment [pdf]. Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria (May 2017).
&bull Standard operating procedure manual for sterile Tsetse release. This manual describes the standard procedures involved in preparing tsetse flies reared in a breeding facility for release in the field for the sterile insect technique (SIT). Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria (November 2016).
&bull Standard Operational Procedures to Detect and Manage Glossina pallidipes Salivary Gland Hypertrophy Virus (GpSGHV) in Tsetse Fly 'Factories' [pdf]. Vienna, Austria (2015).
&bull Collection of Entomological Baseline Data for Tsetse Area-Wide Integrated Pest Management Programmes [pdf]. FAO Animal and Heaalth Guidelines and Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. FAO, Roma, Italy (2008). ISBN 978-92-5-106158-9.
&bull Standard Operating Procedures for Mass-Rearing Tsetse Flies [pdf]. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. IAEA, Vienna, Austria (2006).
&bull Integrating the Sterile Insect Technique as a Key Component of Area-Wide Tsetse and Trypanosomiasis Intervention [pdf]. PAAT Technical and Scientific Series. FAO Agriculture and Consumer Protection. FAO, Roma, Italy (2001). ISBN 92-5-104646-8.
&bull A field guide for the Diagnosis, Treatment and Prevention of African Animal Trypanosomosis [pdf]. Food and Agriculture Organization of the United Nations. FAO, Rome, Italy (1998). ISBN 92-5-104238-1.

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Moths

&bull Rearing codling moth for the sterile insect technique [pdf]. FAO Plant Production and Protection and Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, FAO, Roma, Italy (2010). ISBN 978-92-5-106548-8.
&bull Cactoblastis cactorum. The biology, History, Threat, Surveillance and Control of the Cactus Moth [pdf]. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. IAEA, Vienna, Austria (2007).

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Screwworm

&bull Manual for the Control of the Screwworm Fly Cochliomyia hominivorax, Coquerel [pdf]. Food and Agriculture Organization of the United Nations. FAO, Rome (1990).
&bull Manuel de Lutte Contre la Lucilie Bouchére Cochliomyia hominivorax, Coquerel [pdf]. Food and Agriculture Organization of the United Nations. FAO, Rome (1990).

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Mosquitoes

&bull Guidelines for Irradiation of Mosquito Pupae in Sterile Insect Technique Programmes [pdf]. This publication is intended as guidance for the irradiation of the pupal stage of Aedes aegypti, Aedes albopictus and Anopheles arabiensis, for routine studies on the biological effects of radiation exposures, in particular, irradiation induced sterility in male (and female) mosquitoes.
&bull Guidance framework for testing the sterile insect technique as a vector control tool against Aedes-borne diseases [pdf]. This document is intended to be a comprehensive guide for programme managers tasked with recommending a “go/no-go” decision on testing, full deployment and scale-up of the sterile insect technique (SIT) in regions of the world affected by diseases transmitted by Aedes mosquitoes. However, the authors hope that the material presented herein will be used more widely—by scientists, decision makers, review groups and others.
&bull Guidelines For Mark-Release-Recapture Procedures of Aedes Mosquitoes_v1.0. [pdf]. The success of any Area-Wide Integrated Pest Management (AW-IPM) programme including a Sterile Insect Technique (SIT) component relies on the ability of the irradiated sterile males to survive, disperse and compete sexually with their wild counterparts to induce sterility in wild females. In a phased conditional approach for such a programme, it is considered essential to estimate these quality control parameters (survival, dispersal and competitiveness) in field conditions using mark-release-recapture (MRR) trials during the baseline data collection phase before shifting to a pilot trial. The protocols presented in this guideline are the results of lessons from collaborations with Member States preparing SIT pilot trials against Aedes species.
&bull Guidelines for mass rearing of Aedes mosquitoes_v1.0. [pdf]. The number and production capacity of mass-rearing insectaries for Aedes mosquitoes is increasing as the SIT technology is being validated in field pilot projects. These guidelines have been developed to provide a description of procedures required for Aedes aegypti and Aedes albopictus mass-rearing. It is a summary of necessary steps of larval and adult mass-rearing as used at the FAO/IAEA. This guide will be continuously updated considering the improvements brought to the mass-rearing of mosquitoes.
&bull Spreadsheet for Designing Aedes Mosquito Mass-Rearing and Release Facilities Version 1.0.[pdf]. The number and production capacity of mass rearing insectaries for mosquitos is expected to increase in the coming years. This FAO/IAEA interactive Excel Spreadsheet has been developed to assists in technical and economic decision making associated with design, costing, construction, equipping, and facility operation. The spreadsheet is user friendly and thus largely self-explanatory. Nevertheless, it includes a basic instruction manual that has been prepared to guide the user, and thus should be used together with the software.
&bull Guidelines for Colonization of Aedes Mosquito Species - Version 1.0. This document aims to provide a description of procedures required for the establishment of Aedes aegypti and Ae. albopictus colonies in your insectary or laboratory. It presents a summary of the necessary steps such as collecting material from the field, species identification, and adapting your wild colony to laboratory conditions and artificial rearing procedures. Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria, (May 2018).
&bull Mosquitoes SIT trifold [pdf]. The document contains in a nutshell a description of SIT use against mosquitoes from mass rearing to sterilization and release. It also contains basic elements necessary for the cost-effective application of area-wide SIT technology against mosquitoes vectors of disease. Vienna, Austria (April 2018).
&bull Guidelines for routine Colony Maintenance of Aedes Mosquito Species Version 1.0. [pdf]. This guideline aims to provide a description of procedures required for Ae. aegypti and Ae. albopictus colony routine rearing. It is a summary of necessary steps such as optimizing climatic conditions in the insectary, egg hatching, larval rearing, pupal and larval sorting, sugar and blood feeding, egg collection, handling and storage, used at the FAO/IAEA Insect Pest Control Laboratory (IPCL) to build and maintain a lab colony. Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria, (November 2017).
&bull Guidelines for standardised mass rearing of Anopheles mosquitoes Version 1.0 [pdf]. The guideline provides a description of all procedures required for the mass-rearing of An. arabiensis using these newly designed adult cage and larval rearing unit, with a step-by-step guide to establishing, up-scaling and maintaining a large Anopheles colony, including all stages of the mosquito’s life cycle. Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria, (October 2017).

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Miscellaneous

&bull Dose mapping by scanning Gafchromic® film to measure the absorbed dose of insects during their sterilization. The development of better system for dose distribution within an irradiation container and the development of an accurate dose-response curve for the target insect using precise dosimetry is a prerequisite of any programmes releasing sterile insects. This manual describes the operational procedures to develop dose maps by scanning Gafchromic film and the calibration of the system, to be used in the insect irradiation process for SIT programmes. The manual is also available in Spanish/Español.
&bull Technical Specification for an X-Ray System for the Irradiation of Insects for the Sterile Insect Technique and Other Related Technologies [pdf]. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. IAEA, Vienna, Austria (August 2017).
&bull Manual for the Use of Stable Isotopes in Entomology [pdf]. IAEA-SI. IAEA, Vienna (2009). ISBN 978-92-0-102209-7.
&bull Model Business Plan for a Sterile Insect Production Facility. IAEA-MPB. IAEA, Insect Pest Control Section of the Joint FAO/IAEA Centre, International Atomic Energy Agency. Vienna, Austria (2008). ISBN 978-92-0-110007-8.
&bull Designing and Implementing a Geographical Information System: A Guide for Managers of Area-Wide Pest Management Programmes [pdf]. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. IAEA, Vienna, Austria (2006).
&bull Gafchromic® dosimetry system for SIT - Standard operating procedure. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. IAEA, Vienna, Austria (2000).
&bull Laboratory Training Manual on the Use of Nuclear Techniques in Insect Research and Control - Third Edition [pdf]. IAEA Technical Reports Series No. 336. IAEA, Vienna (1992). ISBN 92-0-101792-8.
&bull Laboratory Training Manual on the Use of Isotopes and Radiation in Entomology [pdf]. Second Edition. IAEA Technical Reports Series No. 61. STI/DOC/10/61/2, IAEA, Vienna (1977). ISBN 92-0-115177-2.
&bull Laboratory Training Manual on the Use of Isotopes and Radiation in Entomology [pdf]. IAEA Technical Reports Series No. 61. STI/DOC/10/61, IAEA, Vienna (1966).

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Results

Sterility Curve

Male Ae. albopictus from the Reunion strain were exposed to gamma radiation at various doses and crossed with non treated females. The control fertility was 97%. It was reduced to 7% and 4% by irradiation with 35 and 40 Gy, respectively (Fig. 1). As no significant difference existed between the sterility levels induced by these two radiation doses, the 35 Gy dose was used for the competitiveness experiments.

Mean fertility (%) as a function of radiation dose. Unlike letters indicate significant difference between the points (P<0.05).

Recovery of Fertility

The persistence of male sterility after irradiation was tested. In this experiment, females mated with 40 Gy irradiated males aged 1 to 5 days had a mean fertility of 3.0±0.1%, which was not different from the result of the sterility curve. Each male inseminated 1 to 4 females during this first mating period. During the second period of mating, the same males then aged 10 to 15 days old were able to inseminate 1 to 6 females the fertility of all of these females (N = 17) was zero, which was significantly lower than during the first period (two-tailed paired Student’s t-test, t = 6.32, df = 21, P<0.001). During these two 5-days periods an individual male was able to inseminate a total of 2 to 8 females.

Effect of Irradiation on Male Sexual Maturation

The temporal sexual maturation process of freshly emerged untreated and sterile males was assessed by examining the terminalia rotation and the insemination ability.

Terminalia rotation could be detected 4 h post-emergence for untreated males. After 10 h, all males had started the rotation and the first males with the terminalia fully rotated were observed after 11 h (25%). After 17 h, all males were in stage 3 or 4. In groups aged 18 to 25 h old, 80 to 100% of the males had completed rotation.

The first irradiated males recorded in stage 1 were observed 3 h after their emergence, which was significantly earlier than for untreated males (proportion test with Yates correction, X 2 = 4.02, df = 1, P<0.05). Similarly to the untreated males, after 10 h every sterile male had started the rotation. However, a delay in the rotation speed was observed in sterile males aged 15 to 19 h compared with untreated ones. The first fully rotated sterile males were observed after 13 h (11%) and the rotation process was completed 20 h post-emergence. Significant differences between untreated and sterile males were observed at the age of 12 h (X 2 = 1.56, df = 1, P<0.01), 15 h (X 2 = 9.77, df = 1, P<0.01), 16 h (X 2 = 12.8, df = 1, P<0.001), and 18 h old (X 2 = 6.89, df = 1, P<0.01).

Very few males were able to inseminate females during the first 15 h of their adult life as only 1 cage out of 5 contained a female inseminated for both untreated and sterile groups. After 20 h post-emergence, untreated males had inseminated twice as many females as sterile males (one-way ANOVA, F(1, 23) = 5.66, P<0.05 Fig. 2). However after 25 h, all the cages contained inseminated females and both untreated and sterile males had a comparable insemination capacity with respectively 43 and 40% of females being inseminated: 87% of these females had 2 spermathecae out of three filled with sperm.

Percentage (± SEM) of females inseminated in small cages (10 females and 10 males) for different durations after male emergence (3 replicates). NS indicates a non-significant difference and * stands for a significant difference (P<0.05) between sterile and untreated males.

Insemination Rates

The insemination rate of a group of 50 females caged for 48 h with 1- or 5-day old untreated or sterile males, at a 1∶1:1 or 5∶1:1 ratio was assessed. No statistical difference in insemination rates was observed in relation to age, irradiation treatment or sex ratio (Table 1): on average 93% females were inseminated after 48 h, and 92% of them had 2 filled spermathecae.

Daily Mating Success

In order to assess male mating performance, a new group of 10 females was offered daily to one sterile or untreated male for 15 days.

Over the 15-day period, sterile males inseminated significantly fewer females (one-way ANOVA, F(1, 104) = 4.89, P<0.05) per day compared with untreated males (Table 2). On the first day, a fertile male inseminated on average twice as many females than a sterile one, but this difference was not statistically significant (two-tailed paired Student’s t-test, t = 2.78, df = 4, P = 0.07). The number of females inseminated per day decreased until the 5 th day and increased again during the next 5 days this cyclic pattern was observed for both untreated and sterile males. The mean number of females inseminated by a sterile male was always slightly lower than for an untreated male, but this difference was significant only after the 9 th day (t = 2.36, df = 7, P<0.05). Most of the inseminated females had only one spermatheca filled, only 18 and 6% of them had two spermathecae filled when mated by untreated and sterile males respectively this difference was not significant (proportion test with Yates correction, X 2 = 2.98, df = 1, P = 0.084). Female wing size was not correlated to the insemination status (logistic regression, z = 1.56, df = 659, P = 0.16 for untreated males z = −0.176, df = 509, P = 0.86 for sterile males).

Effect of the Age on Mating Success of Sterile Males

In order to compare the mating performance of 1- and 5-day old sterile males, the number of females inseminated by one male after 5 days was assessed.

There was no significant difference between young and older sterile males for the number of inseminated females (one-way ANOVA, F(1,17) = 0.95, P = 0.34) or spermathecae filled per female (F(1,17) = 1.82, P = 0.2). Sterile males aged 1 to 5 days old inseminated on average 3.3±0.5 females of which 15% and 67% had respectively one and two spermathecae filled. When aged 5 to 10 days old they inseminated on average 2.7±0.4 females of which 17% and 79% had respectively one and two spermathecae filled.

Competitiveness of Sterile Males against Wild Males

Experiments were carried out under semi-field condition to assess the fertility of the caged population and the competitiveness index of sterile males when they where competing with wild males for inseminating wild females adults were released at 1- or 5-day old and in a 1∶1:1 or 5∶1:1 ratio.

The mean fertility of the wild males used for these experiments was 93±1% and 5±0.9% for males irradiated with 35 Gy. Preliminary tests in non-competitive situations were conducted to ensure that both wild and irradiated males were able to survive and to inseminate wild females in this experimental set-up under semi-field conditions.

In competitive situations, when males were released in the cage on the day following their emergence, with an equal ratio of wild and sterile males, the mean fertility was reduced to 82.7% (Table 3), which was significantly higher than for the two other experiments (one-way ANOVA, F(2,14) = 17.7, P<0.001) this resulted in competitiveness indexes (C) ranging between 0.06 and 0.35. When males and females were kept 5 days under laboratory conditions before the release in the semi-field cages, the mean fertility was reduced to 62.2%, C values ranged between 0.44 and 0.76, and were significantly different from the two other experiments (F(2,14) = 9.71, P<0.01). When increasing the ratio of 1-day-old sterile to wild males, the mean fertility was twice as low as the one of a wild population as it averaged 46.4±7.6%. High variations of fertility and thus C were observed between the five replicates in experiment 3, C varying between 0.10 and 0.62.

The mean fecundity differed significantly between each experiment (F(2,14) = 17.37, P<0.001). According to the previous experiment, a female oviposited an average of 50 eggs. If this assumption is correct, the mean number of females participating in the egg laying was 108, 154, and 47 respectively for the experiments 1 to 3. In experiment 1 and 2, 200 females were released whereas only 100 were used for the third test therefore half of the females would have laid eggs in experiments 1 and 3, and 75% of them in experiment 2. The age of the females during the whole test differed between these experiments, as they were aged 1 to 7 days old in experiments 1 and 3, and 5 to 12 days old in experiment 2.


Supporting Information

Figure S1

The effect of male mating competitiveness (from 1 to 0.1) and additional mortality incurred by sterile males over wild-type males (expressed as different values of a constant mortality factor in the Gompertz-Makeham survivorship function) on the suppression of the female population achieved after 20 weeks of sterile male releases, when released males are not completely sterile (is =𠂠.03).

Figure S2

The effect of mosquito size and assortative mating on population size and vectorial capacity during and after the release of sterile males. Solid lines indicate simulations where 1000 large males are released weekly, dashed lines indicate simulations where a mixture of 500 large and 500 small males are released. The degree of assortative mating, Ca, is either 0.5 or 0.9. A) Population sizes of small (left panel) and large females (right panel). B) Vectorial capacity, a measure of disease transmission potential, of mosquito populations comprising small and large females. The shaded area represents the period during which sterile males are released.