Sex peptide receptor is not required for refractoriness to remating or induction of egg laying in Aedes aegypti.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating-processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

Accelerating research and development of new vaccines against tuberculosis: a global roadmap.

To eliminate tuberculosis globally, a new, effective, and affordable vaccine is urgently needed, particularly for use in adults and adolescents in low-income and middle-income countries. We have created a roadmap that lists the actions needed to accelerate tuberculosis vaccine research and development using a participatory process. The vaccine pipeline needs more diverse immunological approaches, antigens, and platforms. Clinical development can be accelerated by validated preclinical models, agreed laboratory correlates of protection, efficient trial designs, and validated endpoints. Determining the public health impact of new tuberculosis vaccines requires understanding of a country's demand for a new tuberculosis vaccine, how to integrate vaccine implementation with ongoing tuberculosis prevention efforts, cost, and national and global demand to stimulate vaccine production. Investments in tuberculosis vaccine research and development need to be increased, with more diversity of funding sources and coordination between these funders. Open science is important to enhance the efficiency of tuberculosis vaccine research and development including early and freely available publication of study findings and effective mechanisms for sharing datasets and specimens. There is a need for increased engagement of industry vaccine developers, for increased political commitment for new tuberculosis vaccines, and to address stigma and vaccine hesitancy. The unprecedented speed by which COVID-19 vaccines have been developed and introduced provides important insight for tuberculosis vaccine research and development.

Correction to: Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.

Please be advised that one of the author names is incorrectly spelled in the published article: 'Irene Kyomuhagi' should be 'Irene Kyomuhangi'.

Male contributions during mating increase female survival in the disease vector mosquito Aedes aegypti.

Aedes aegypti is a vector of medically important viruses including those causing Zika, dengue, and chikungunya. During mating, males transfer a number of proteins and other molecules to the female and these components of the male ejaculate are essential in shifting female post-mating behaviors in a number of insect species. Because these molecules are highly variable by species, and female post-mating behavior by species is also varied, behavioral assays testing the function of the ejaculate are necessary before we can develop control strategies targeting the mating system to reduce mosquito populations. Because increased survival in mosquitoes strongly increases vectorial capacity and can influence population sizes and potential risk we tested the effect of mating on female survival. The ejaculate can either promote or reduce female survival, as both have been shown in multiple insect species, yet this effect has not been directly assessed in mosquitoes. We compared survival of females in four treatment groups: mated females, virgin females, and virgin females injected with either an extract from the male reproductive glands or a saline control. Survival, blood feeding frequency, fecundity and cumulative net reproductive rate (R0) were determined after multiple feedings from a human host. Our results confirm that male reproductive gland substances increase female fecundity and blood feeding frequency, resulting in dramatic increases in fitness (R0). We also demonstrate, for the first time, an effect of male reproductive gland extracts alone on female survival, regardless of whether or not the female ingested a vertebrate blood meal. Thus, the effects of MAG extract on survival are not secondary effects from altered blood feeding. Collectively, we demonstrate a direct role for Ae. aegypti male-derived molecules on increasing female fitness, reproductive success and, ultimately, transmission potential for vector borne pathogens.

Insecticide resistance patterns in Uganda and the effect of indoor residual spraying with bendiocarb on kdr L1014S frequencies in Anopheles gambiae s.s.

BACKGROUND: Resistance of malaria vectors to pyrethroid insecticides has been attributed to selection pressure from long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and the use of chemicals in agriculture. The use of different classes of insecticides in combination or by rotation has been recommended for resistance management. The aim of this study was to understand the role of IRS with a carbamate insecticide in management of pyrethroid resistance. METHODS: Anopheles mosquitoes were collected from multiple sites in nine districts of Uganda (up to five sites per district). Three districts had been sprayed with bendiocarb. Phenotypic resistance was determined using standard susceptibility tests. Molecular assays were used to determine the frequency of resistance mutations. The kdr L1014S homozygote frequency in Anopheles gambiae s.s. was used as the outcome measure to test the effects of various factors using a logistic regression model. Bendiocarb coverage, annual rainfall, altitude, mosquito collection method, LLIN use, LLINs distributed in the previous 5 years, household use of agricultural pesticides, and malaria prevalence in children 2-9 years old were entered as explanatory variables. RESULTS: Tests with pyrethroid insecticides showed resistance and suspected resistance levels in all districts except Apac (a sprayed district). Bendiocarb resistance was not detected in sprayed sites, but was confirmed in one unsprayed site (Soroti). Anopheles gambiae s.s. collected from areas sprayed with bendiocarb had significantly less kdr homozygosity than those collected from unsprayed areas. Mosquitoes collected indoors as adults had significantly higher frequency of kdr homozygotes than mosquitoes collected as larvae, possibly indicating selective sampling of resistant adults, presumably due to exposure to insecticides inside houses that would disproportionately affect susceptible mosquitoes. The effect of LLIN use on kdr homozygosity was significantly modified by annual rainfall. In areas receiving high rainfall, LLIN use was associated with increased kdr homozygosity and this association weakened as rainfall decreased, indicating more frequency of exposure to pyrethroids in relatively wet areas with high vector density. CONCLUSION: This study suggests that using a carbamate insecticide for IRS in areas with high levels of pyrethroid resistance may reduce kdr frequencies in An. gambiae s.s.

What drives the consistent use of long-lasting insecticidal nets over time? A multi-method qualitative study in mid-western Uganda.

BACKGROUND: The distribution of long-lasting insecticidal nets (LLINs) through universal coverage campaigns is a widely adopted approach for the prevention of malaria at scale. While post-distribution surveys play a valuable role in determining cross-sectional levels of LLIN retention and use, as well as frequently cited reasons for non-use, few studies have explored the consistency of LLIN use over time, within the expected lifespan of the net, and the factors which may drive this. METHODS: In this qualitative study, 74 in-depth interviews were conducted with (male) household heads and (female) caregivers of children in LLIN recipient households, as well as community health workers, in Buliisa, Hoima and Kiboga districts in Uganda, 25-29 months following a LLIN mass campaign distribution. A triangulation approach to data analysis was taken, incorporating thematic analysis, most significant change and positive deviance. RESULTS: The factors found to be most influential in encouraging long-term LLIN use were positive experience of net use prior to the distribution, and appreciation or awareness of a range of benefits arising from their use, including protection from malaria as well as importantly, other health, lifestyle, social and economic benefits. Social support from within the community was also identified as an important factor in determining continued use of LLINs. Net use appeared to be more consistent amongst settled urban and rural communities, compared with fishing, pastoralist, refugee and immigrant communities. CONCLUSIONS: A multitude of interplaying factors encouraged consistent LLIN use in this setting. Whilst the protection of malaria remains a powerful motivator, social and behaviour change (SBC) strategies should also capitalize on the non-malaria benefits of net use that provide a long-term rationale for consistent use. Where supplies are available, SBC campaigns should promote replacement options, emphasizing ongoing net care and replacement as a household responsibility, thus reducing dependence on free distributions. The triangulation approach to qualitative data analysis enabled increased confidence in the validity of findings and an enhanced contextual understanding of the factors promoting consistent net use in mid-western Uganda. The approach should be considered when designing future studies to explore factors driving net retention and use trends.

Impact of a behaviour change communication programme on net durability in eastern Uganda.

BACKGROUND: The importance of net durability and the average useful life of a net is increasingly recognized as one of the critical factors that determine how often nets need to be replaced. A study to assess the effect of a net care and repair behaviour change communication (BCC) programme on net durability was conducted in one district in Eastern Uganda with a district in a neighbouring region serving as a comparison. Both districts had received LLINs in September of 2012. METHODS: The intervention was comprised of radio programmes, school and community events. Two-stage cluster sampling household surveys to assess net condition, exposure to BCC messages, and attitudes towards net care and repair were conducted in both districts at baseline (2-3 months post net distribution) and endline (20-21 months post distribution). Net condition was assessed using the proportionate hole index, with nets being classified as either serviceable or too torn. RESULTS: The intervention led to an additional 31.2 % increased exposure to net care and repair messages in the intervention district. Respondents in the intervention district had a more positive attitude towards net care and repair (32 % of respondents were classified as having a very positive attitude compared to 10 % in the comparison district), which was positively associated with the number of channels through which messages had been received (P < 0.001). Nets belonging to respondents with a very positive attitude were more often categorized as serviceable (80.2 %) compared to respondents with a poor/average attitude (66.4 %; odds ratio: 2.05, P = 0.028); however, this was only observed for the net brand with the greater physical integrity. Additionally, socio-economic status was a significant predictor of net condition. Although nets in the intervention district had significantly more repairs done per net, the act of repairing alone did not improve net condition. CONCLUSIONS: In conclusion, the evaluation showed that the BCC programme resulted in improved knowledge and attitudes towards care and repair, which impacted positively on net condition. Repairs alone were not sufficient to improve net condition. Additional research on which care behaviours and attitudes are most associated with improved net condition would help BCC planners hone their campaigns.

Monitoring changes in malaria epidemiology and effectiveness of interventions in Ethiopia and Uganda: Beyond Garki Project baseline survey.

BACKGROUND: Scale-up of malaria interventions seems to have contributed to a decline in the disease but other factors may also have had some role. Understanding changes in transmission and determinant factors will help to adapt control strategies accordingly. METHODS: Four sites in Ethiopia and Uganda were set up to monitor epidemiological changes and effectiveness of interventions over time. Here, results of a survey during the peak transmission season of 2012 are reported, which will be used as baseline for subsequent surveys and may support adaptation of control strategies. Data on malariometric and entomological variables, socio-economic status (SES) and control coverage were collected. RESULTS: Malaria prevalence varied from 1.4 % in Guba (Ethiopia) to 9.9 % in Butemba (Uganda). The most dominant species was Plasmodium vivax in Ethiopia and Plasmodium falciparum in Uganda. The majority of human-vector contact occurred indoors in Uganda, ranging from 83 % (Anopheles funestus sensu lato) to 93 % (Anopheles gambiae s.l.), which is an important factor for the effectiveness of insecticide-treated nets (ITNs) or indoor residual spraying (IRS). High kdr-L1014S (resistance genotype) frequency was observed in A. gambiae sensu stricto in Uganda. Too few mosquitoes were collected in Ethiopia, so it was not possible to assess vector habits and insecticide resistance levels. ITN ownership did not vary by SES and 56-98 % and 68-78 % of households owned at least one ITN in Ethiopia and Uganda, respectively. In Uganda, 7 % of nets were purchased by households, but the nets were untreated. In three of the four sites, 69-76 % of people with access to ITNs used them. IRS coverage ranged from 84 to 96 % in the three sprayed sites. Half of febrile children in Uganda and three-quarters in Ethiopia for whom treatment was sought received diagnostic tests. High levels of child undernutrition were detected in both countries carrying important implications on child development. In Uganda, 7-8 % of pregnant women took the recommended minimum three doses of intermittent preventive treatment. CONCLUSION: Malaria epidemiology seems to be changing compared to earlier published data, and it is essential to have more data to understand how much of the changes are attributable to interventions and other factors. Regular monitoring will help to better interpret changes, identify determinants, modify strategies and improve targeting to address transmission heterogeneity.

Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus and Ae. aegypti mosquitoes.

Male mosquitoes transfer seminal fluid proteins (hereafter 'SFPs') during mating. These proteins can have profound effects on female behavior in the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. SFPs are thought to be responsible for female refractoriness to mating in both species. However, only limited information is available about the duration of induced refractoriness or the quantity of SFPs required to be effective in Ae. albopictus. Here, we tested the duration of the effect of SFPs on female refractory behavior for both Aedes species. Additionally, we determined the lowest SFP dose required to induce female refractory behavior in Ae. aegypti. Virgin females were injected intra-thoracically with doses ranging from 0.25 to 0.008 equivalents of one male's SFP amount. Our results demonstrate high sensitivity of female Ae. aegypti and Ae. albopictus to SFPs of their own species, with the majority of females becoming refractory at doses ≥ 0.031 male-equivalents after injection into the hemocoel. This effect was long-lasting in both species; none of the injected females were inseminated when presented with males of their own species 30 to 34 days post-injection, whereas most saline-injected control females mated at this time point. These results will aid future work to characterize individual SFPs involved in post-mating refractoriness in these two species. Moreover, they show that as is the situation in the mosquito Anopheles gambiae, and unlike Drosophila melanogaster, sperm are not required for the maintenance of a sexual refractoriness response in Ae. aegypti and Ae. albopictus.

Evidence of polyandry for Aedes aegypti in semifield enclosures.

Female Aedes aegypti are assumed to be primarily monandrous (i.e., mate only once in their lifetime), but true estimates of mating frequency have not been determined outside the laboratory. To assess polyandry in Ae. aegypti with first-generation progeny from wild mosquitoes, stable isotope semen-labeled males ((15)N or (13)C) were allowed to mate with unlabeled females in semifield enclosures (22.5 m(3)) in a dengue-endemic area in southern Mexico. On average, 14% of females were positive for both labels, indicating that they received semen from more than one male. Our results provide evidence of a small but potentially significant rate of multiple mating within a 48-hour period and provide an approach for future open-field studies of polyandry in this species. Polyandry has implications for understanding mosquito ecology, evolution, and reproductive behavior as well as genetic strategies for mosquito control.

The role of male harassment on female fitness for the dengue vector mosquito Aedes aegypti.

Sexual harassment studies in insects suggest that females can incur several kinds of costs from male harassment and mating. Here, we examined direct and indirect costs of male harassment on components of female fitness in the predominantly monandrous mosquito Aedes aegypti. To disentangle the costs of harassment versus the costs of mating, we held females at a low or high density with males whose claspers were modified to prevent insemination, and compared these to females held with normal males and to those held with females or alone. A reduced longevity was observed when females were held under high density conditions with males or females, regardless if male claspers had been modified. There was no consistent effect of harassment on female fecundity. Net reproductive rate (R0) was higher in females held at low density with normal males compared to females held with males in the other treatments, even though only a small number of females showed direct evidence of remating. Indirect costs and benefits that were not due to harassment alone were observed. Daughters of females held with normal males at high density had reduced longevity compared to daughters from females held without conspecifics. However, their fitness (R0) was higher compared to females in all other treatments. Overall, our results indicate that A. aegypti females do not suffer a fitness cost from harassment of males when kept at moderate densities, and they suggest the potential for benefits obtained from ejaculate components.

Male mating history and body size influence female fecundity and longevity of the dengue vector Aedes aegypti.

Male reproductive success is dependent on insemination success and reproductive output. During mating, male mosquitoes transfer not just sperm, but also seminal fluid proteins that may have profound effects on mated female biology and behavior. In this study, we investigated the role of male body size and mating history on semen depletion, female longevity, and reproductive success in Aedes aegypti L. Small and large males were mated in rapid succession with up to five females. Our results indicate that large males had greater mating capacity than small males. A reduction in fecundity by >50% was observed in females that were fourth to mate with small males in comparison with females that mated earlier in sequence. For females mated to large males, this reduction became evident for females that mated fifth in sequence. No loss of fertility (measured as hatch rate) was observed in females that were third-fifth in mating sequence compared with females mated to virgin males. When females were maintained on a low-quality (5% sucrose) diet, those mated to virgin males had a greater longevity compared with females mated third in sequence. We conclude that small males experience more rapid seminal depletion than large males, and discuss the role of semen depletion in the mated female. Our results contribute toward a better understanding of the complexity of Ae. aegypti mating biology and provide refined estimates of mating capacity for genetic control efforts.

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions.

BACKGROUND: No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating. METHODOLOGY AND PRINCIPAL FINDINGS: Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera. CONCLUSION AND SIGNIFICANCE: This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.

Radiation biology of mosquitoes.

There is currently renewed interest in assessing the feasibility of the sterile insect technique (SIT) to control African malaria vectors in designated areas. The SIT relies on the sterilization of males before mass release, with sterilization currently being achieved through the use of ionizing radiation. This paper reviews previous work on radiation sterilization of Anopheles mosquitoes. In general, the pupal stage was irradiated due to ease of handling compared to the adult stage. The dose-response curve between the induced sterility and log (dose) was shown to be sigmoid, and there was a marked species difference in radiation sensitivity. Mating competitiveness studies have generally been performed under laboratory conditions. The competitiveness of males irradiated at high doses was relatively poor, but with increasing ratios of sterile males, egg hatch could be lowered effectively. Males irradiated as pupae had a lower competitiveness compared to males irradiated as adults, but the use of partially-sterilizing doses has not been studied extensively. Methods to reduce somatic damage during the irradiation process as well as the use of other agents or techniques to induce sterility are discussed. It is concluded that the optimal radiation dose chosen for insects that are to be released during an SIT programme should ensure a balance between induced sterility of males and their field competitiveness, with competitiveness being determined under (semi-) field conditions. Self-contained 60Co research irradiators remain the most practical irradiators but these are likely to be replaced in the future by a new generation of high output X ray irradiators.

Sperm quantity and size variation in un-irradiated and irradiated males of the malaria mosquito Anopheles arabiensis Patton.

Anopheles mosquitoes are important candidates for genetic control strategies. However, little is known about sperm quality and quantity as determinants of male reproductive success. In this study, sperm quantity and length variation were assessed in testes of un-irradiated and irradiated Anopheles arabiensis. Male reproductive organs were dissected for sperm and an estimate of the total number of spermatozoa was made. Sperm lengths were measured using imaging software. The effects of irradiation were evaluated for males exposed in the pupal or adult stage to a full (120 Gy) or partially sterilising dose (70 Gy). Sperm length variation in the laboratory strain was compared to the distribution observed in wild males. We also determined the size distribution of sperm lengths in spermathecae of inseminated females compared to those observed in male testes. Sperm quantity increased with age, and 12-day-old males had significantly more sperm in their testes (8214+/-467) than males aged 3 days (5022+/-375). Mosquitoes irradiated in the pupal stage had significantly fewer sperm (2982+/-125) than un-irradiated males (4950+/-848) although for adult stage irradiation similar amounts of sperm were observed compared to un-irradiated males. Sperm length variation was detected with sperm lengths ranging between <50 and 500 microm. There were no differences in sperm length distribution compared to wild males. Sperm length distributions were similar to those reported for the closely related sibling species An. gambiae s.s. There was no major effect of irradiation on the distribution of sperm lengths in the testes, with the exception that pupal irradiation resulted in a significant increase in sperm numbers in the category of 100-200 microm. Sperm length distributions in spermathecae were different to those measured directly from sperm in the testes and harboured less cells of the smaller (<100-200 microm), and more cells of the larger category (300-400 microm). The finding that testes of pupal irradiated males produce fewer and smaller sperm in comparison to un-irradiated and adult irradiated testes are discussed in the context of genetic control strategies, in particular the Sterile Insect Technique (SIT).

A 15N stable isotope semen label to detect mating in the malaria mosquito Anopheles arabiensis Patton.

In previous studies it was determined that the stable isotope 13-carbon can be used as a semen label to detect mating events in the malaria mosquito Anopheles arabiensis. In this paper we describe the use of an additional stable isotope, 15-nitrogen (15N), for that same purpose. Both stable isotopes can be analysed simultaneously in a mass spectrometer, offering the possibility to detect both labels in one sample in order to study complex and difficult-to-detect mating events, such as multiple mating. 15N-glycine was added to larval rearing water and the target enrichment was 5 atom% 15N. Males from these trays were mated with unlabelled virgin females, and spiked spermathecae were analysed for isotopic composition after mating using mass spectrometry. Results showed that spermathecae positive for semen could be distinguished from uninseminated or control samples using the raw delta15N per thousand values. The label persisted in spermathecae for up to 5 days after insemination, and males aged 10 days transferred similar amounts of label as males aged 4 days. There were no negative effects of the label on larval survival and male longevity. Enrichment of teneral mosquitoes after emergence was 4.85 +/- 0.10 atom% 15N. A threshold value defined as 3 standard deviations above the mean of virgin (i.e. uninseminated spermathecae) samples was successful in classifying a large proportion of samples correctly (i.e. on average 95%). We conclude that alongside 13C, 15N can be used to detect mating in Anopheles and the suitability of both labels is briefly discussed.

Towards a sterile insect technique field release of Anopheles arabiensis mosquitoes in Sudan: irradiation, transportation, and field cage experimentation.

BACKGROUND: The work described in this article forms part of a study to suppress a population of the malaria vector Anopheles arabiensis in Northern State, Sudan, with the Sterile Insect Technique. No data have previously been collected on the irradiation and transportation of anopheline mosquitoes in Africa, and the first series of attempts to do this in Sudan are reported here. In addition, experiments in a large field cage under near-natural conditions are described. METHODS: Mosquitoes were irradiated in Khartoum and transported as adults by air to the field site earmarked for future releases (400 km from the laboratory). The field cage was prepared for experiments by creating resting sites with favourable conditions. The mating and survival of (irradiated) laboratory males and field-collected males was studied in the field cage, and two small-scale competition experiments were performed. RESULTS: Minor problems were experienced with the irradiation of insects, mostly associated with the absence of a rearing facility in close proximity to the irradiation source. The small-scale transportation of adult mosquitoes to the release site resulted in minimal mortality (< 6%). Experiments in the field cage showed that mating occurred in high frequencies (i.e. an average of 60% insemination of females after one or two nights of mating), and laboratory reared males (i.e. sixty generations) were able to inseminate wild females at rates comparable to wild males. Based on wing length data, there was no size preference of males for mates. Survival of mosquitoes from the cage, based on recapture after mating, was satisfactory and approximately 60% of the insects were recaptured after one night. Only limited information on male competitiveness was obtained due to problems associated with individual egg laying of small numbers of wild females. CONCLUSION: It is concluded that although conditions are challenging, there are no major obstacles associated with the small-scale irradiation and transportation of insects in the current setting. The field cage is suitable for experiments and studies to test the competitiveness of irradiated males can be pursued. The scaling up of procedures to accommodate much larger numbers of insects needed for a release is the next challenge and recommendations to further implementation of this genetic control strategy are presented.

A stable isotope dual-labelling approach to detect multiple insemination in un-irradiated and irradiated Anopheles arabiensis mosquitoes.

BACKGROUND: In the context of a Sterile Insect Technique programme, the occurrence of multiple insemination in the malaria mosquito Anopheles arabiensis Patton was studied using a novel labelling system with the stable isotopes 15N and 13C. The incidence of multiple insemination in the absence of radiation, and when males were irradiated in the pupal stage and competed against un-irradiated males were assessed. Males used in the experiments were labelled with either 15N or 13C and the label was applied to the larval rearing water. Males with either label and virgin females were caged at a 1:1:1 ratio. Males used in the radiation treatments were irradiated in the pupal stage with a partially or fully-sterilizing dose of 70 or 120 Gy, respectively. After mating, females were dissected and inseminated spermathecae analysed using mass spectrometry. RESULTS: The data indicate that about 25% of inseminated females had been inseminated multiply. The presence of irradiated males in the experiments did not affect the incidence of multiple insemination. In line with previous research, irradiated males were generally less competitive than un-irradiated males. CONCLUSION: The implications of these findings for the Sterile Insect Technique are discussed, and further experiments recommended. The dual-labelling system used to determine paternity gave good results for 13C, however, for 15N it is recommended to increase the amount of label in future studies.

Stable isotope-mass spectrometric determination of semen transfer in malaria mosquitoes.

The potential use of stable isotopes to study mosquito mating was investigated by tracing the fate of labelled semen into spermathecae. [(13)C]glucose was incorporated in the diet of the malaria mosquito Anopheles arabiensis. Treatments included labelling of either the larval water or adult sugar water, or a combination of both. After mating, ;spiked' spermathecae were analysed for isotope ratios using mass spectrometry. Results demonstrated that spermathecae positive for semen could successfully be distinguished from empty ones or controls (i.e. filled with unlabelled semen) using the raw delta(13)C values. Labelling during larval development and combined labelling of larvae and adults resulted in detectable values. The label persisted in spermathecae for up to 7 days after mating, and unlabelled sugar feeding of males labelled in the larval stage did not result in a detectable turnover of the semen label. There were no detrimental effects of the addition of labelled glucose on larval development and survival, adult size, male longevity and mating performance. We have proved that it is possible to label male mosquitoes and detect the semen label in females after insemination. This method offers great potential to study mating in mosquitoes and other insects and could prove useful in genetic control studies of medical or agricultural pest insects, with male mating success in the field as a critical verifiable indicator for a positive outcome of the intervention.