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BIOLOGY OF MALE MOSQUITOES IN RELATION TO GENETIC CONTROL PROGRAMMES

Report of the Second Research Coordination Meeting of an FAO/IAEA Coordinated Research Project, held in Vienna, Austria, from 1 to 5 February 2010

Reproduced by the IAEA Vienna, Austria 2010

NOTE

The material in this document has been supplied by the authors and has not been edited by the IAEA. The views expressed remain the responsibility of the named authors and do not necessarily reflect those of the government(s) of the designating Member State(s). In particular, neither the IAEA not any other organization or body sponsoring this meeting can be held responsible for any material reproduced in this document.

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TABLE OF CONTENTS BACKGROUND.....................................................................................................................................1 COORDINATED RESEARCH PROJECT (CRP) & RESEARCH COORDINATION MEETING (RCM)..................................................................................................................................3 SPECIFIC R&D ACTIVITIES.............................................................................................................4

1. MOSQUITO MALE PRE MATING CONDITIONS ..............................................................7 1.1 GENERAL FOCUS OF THE R&D ...........................................................................................7 1.1.1 Male Physiology and Behaviour..................................................................................7 1.1.2 Resources acquisition and allocation..........................................................................7 1.1.3 Male dispersal & sampling tools.................................................................................8

1.2 SPECIFIC OBJECTIVES (INCLUDING PROGRESS TO DATE) ......................................................8 1.2.1 How rearing conditions affect fitness of the males......................................................8 1.2.2 Male nutrition in the field............................................................................................9 1.2.3 Develop purpose specific trapping systems .................................................................9 1.2.4 Develop appropriate olfactometers and related guidelines to investigate species specific behaviours ...............................................................................................................9 1.2.5 Developing release device systems for adults and pupae............................................9

1.3 R&D PROPOSED BY PARTICIPANTS......................................................................................9 2. MOSQUITO MATING SYSTEMS .........................................................................................14

2.1 GENERAL FOCUS OF THE R&D .........................................................................................14 2.2 SPECIFIC OBJECTIVES (INCLUDING PROGRESS TO DATE) ....................................................15 2.2.1 Determine the temporal and spatial characteristics of mating encounter sites of Aedine and Anopheline mosquitoes. ...................................................................................15 2.2.2 Investigate the behaviors involved in courtship.........................................................16 2.2.3 Copulation and Insemination ....................................................................................17 2.2.4 Determine patterns of female remating, and how male performance affects this behavior. .............................................................................................................................18

2.3 R&D PROPOSED BY PARTICIPANTS....................................................................................18 3. CONTRIBUTION OF MOLECULAR/CHEMICAL APPROACHES TO THE UNDERSTANDING OF MALE MOSQUITO MATING BEHAVIOR...................................19

3.1 GENERAL FOCUS OF THE R&D .........................................................................................19 3.2 SPECIFIC OBJECTIVES (INCLUDING PROGRESS TO DATE) ....................................................20 3.2.1 Extract and identify potential swarming/aggregation volatiles from mosquitoes. ....20 3.2.2 Extract and identify volatiles from release-site plants as potential attractants for males. ..................................................................................................................................20 3.2.3 Extract and identify compounds in known feeding sources of nectar, fruit and perhaps honeydew for male mosquitoes. Use as a model for a dietary formulation for sterile males. .......................................................................................................................20 3.3.4 Use olfactometers to test potential attractive volatile compounds using wild males versus mass-reared, irradiated males.................................................................................21 3.3.5 Use PCR-based analysis of Y chromosome markers in An. gambiae to assess M and S molecular form distribution and male dispersal characteristics in Ghana. ....................21

3.3 R&D PROPOSED BY PARTICIPANTS....................................................................................21 REFERENCES .....................................................................................................................................23 APPENDIX 1 - LOGICAL FRAMEWORK APPENDIX 2 - AGENDA APPENDIX 3 - PARTICIPANTS ABSTRACTS APPENDIX 4 - LIST OF PARTICIPANTS

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BACKGROUND

Mosquito-borne diseases threaten the lives and livelihoods of millions of people worldwide 1. Malaria alone affects over 500 million Africans and exerts such a huge public health burden that it has been blamed for the continued underdevelopment of the continent as a whole1a,2. It kills between one and three million people every year, mostly children and pregnant mothers in Africa1,3. Its burden is now estimated at 45.6 million DALYs *. Most of sub-Saharan Africa suffers from stable endemic malaria because climatic conditions ideal for transmission coincide with the ranges of Anopheles gambiae, An. arabiensis and An funestus, the most efficient vector mosquitoes in the world.4,5 In eastern and southern Africa, the proportion of deaths caused by malaria has increased from 18 % in the 1980s to 37% in the 1990s6. Endemic malaria cripples economies and is estimated to slow economic growth by approximately 1.3 % per year2 Malaria has been identified as a key contributor to weak economic growth and investment in Africa because it experiences the most intense malaria transmission in the world.5,7 It is commonplace in tropical Africa for more than half the population to be infected with Plasmodium falciparum, by far the most dangerous of the four parasite species that infect humans5. Not only does malaria place a huge burden directly upon the healthcare systems of African nations, it has also been shown that malaria control can have huge macroeconomic impacts and greatly facilitate economic development at national levels2,8,9.

Dengue fever (DF) and dengue haemorrhagic fever (DHF), transmitted by Aedes vectors, are probably the fastest spreading mosquito-borne diseases at the moment. With no vaccine or efficient drugs, reliance on vector control is heavy. Until recently, there has been no promising solution for sustainable control of dengue vectors. The trend for dengue vector control in most tropical regions has shifted from relying solely on insecticides to biological control, source reduction and environmental management through community participation9a. Several South-East Asian countries have recently carried out integration of vector control approaches.

Two proven vector control strategies are currently advocated for reducing transmission of malarial disease in Africa, namely indoor residual spraying (IRS)10-14 and insecticide-treated bednets (ITNs)15-18. Both methods are based on the use of residual insecticides in the intra-domiciliary domain and target mosquito vectors either before (ITNs) or after host-feeding (IRS). Impressive reductions in childhood morbidity and mortality have been demonstrated in a variety of epidemiological settings15, and it can be expected that IRS/ITNs will remain on the forefront of malaria vector control for at least the remainder of this decade. However, in spite of their proven efficacy and effectiveness14-18 both methods have some drawbacks and limitations such as insecticide resistance19-22, environmental or human health concerns23,24 and socio-economic or cultural acceptance by communities. It is also clear that, effective as these powerful tools are, they are not sufficient on their own to eliminate or drastically reduce the malaria burden from the most intensely endemic regions of the tropics, notably sub-Saharan Africa25,26. An expansion of this limited arsenal of vector control tools, with new strategies that can reduce human exposure, the density of mosquito populations27, or transmissibility of infection, is therefore

** DALYs = Disability-adjusted life years

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needed, and should preferably be appropriate for use in an integrated fashion with IRS/ITNs28-30.

The control of dengue vectors is highly dependent on either source reduction or the use of adulticides. However, breeding of Aedes mosquitoes often occurs in a large variety of small water bodies that may be difficult to trace. Nevertheless, recent successes with the release of Mesocyclops in breeding containers in Vietnam, have been impressive30a. Such methods may be appropriate for certain settings only, and may not be useful in high population density settings like Singapore (which experiences > 10.000 cases of dengue in 2005 (Prof. P. Reiter, pers. comm.)

These considerations have led to renewed interest in the potential of the Sterile Insect Technique (SIT) for suppressing mosquito vectors in suitable areas. The remarkable success of area-wide programmes integrating the SIT against screwworm, tsetse, and fruit flies provides a sound basis for contemplating the prospects for SIT interventions for suppressing the mosquito vectors. It is envisaged that SIT would be used under specific conditions as an adjunct to other technologies. This would conform with the World Health Organization’s current vector-borne disease control strategy which emphasizes not relying on any single intervention approach.

Compared to the initial successful pilot trials of using SIT for mosquito elimination in the early 1970s, the technology has developed enormously for other pest insects in terms of delivery mechanisms of sterile males, the molecular basis to develop sexing strains and in sterilising the males themselves 31. In addition, the regulatory framework of SIT has been developed, quality assurance mechanisms have become more refined and links with the Anopheles genome project have also been established improving development of transgenic mosquitoes. Technology such as GIS/GPS has facilitated the planning, implementation and evaluation of area-wide programmes integrating the SIT.

Unlike female mosquitoes, that frequently imbibe blood (every 2-3 days), male mosquitoes are not blood feeders and thus do not transmit disease. Because mosquitoes undergo sexual reproduction, males are necessary for reproduction and are therefore logical agents for genetic control. Paradoxically, although female biology and behaviour have been studied intensively, relatively little is known about males. In particular, the specific factors that contribute to male reproductive success are virtually unknown32. Nevertheless, the success of any genetic control programme will hinge on the degree to which mass-reared released males are able to confer sterility in the target population.

Any genetic control programme is a process, beginning with colonization and mass rearing of males, followed by shipping and finally release of these males in the target population. An existing CRP is focusing on developing methods of mosquito mass rearing, leading to the point of release. As a logical extension, this CRP proposes to study the factors occurring following release that may have an impact on the success of the programme.

Accordingly, the objectives of the CRP are to study laboratory and field populations of adult male mosquitoes to establish the specific biological and behavioural determinants that contribute to male sexual competitiveness.

We define male competitiveness as the degree to which one will be able to copulate with wild females in the field relative to the rate of wild males, and subsequently prevent females from laying viable eggs. The factors that contribute to

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this competitiveness are myriad and are determined both by natural and sexual selection. Accordingly this CRP will focus on male physiology and sexual behaviour, male bionomics, and male influences on the females they inseminate Considering the current absence of sampling methods for male mosquitoes, and appropriate marking methods to study the fate of released males, research in these two areas has also been included.

COORDINATED RESEARCH PROJECT (CRP) & RESEARCH COORDINATION MEETING (RCM)

The CRP serves to bring together a large research consortium that through collaborative research and knowledge exchange (through four RCMs) on the above aspects will significantly enhance the development of successful mosquito programmes integrating the SIT in selected Member States.

The proposed CRP will provide a unique opportunity for the scientific community engaged in mosquito research, to collaborate on the above-identified issues through a consortium network. The framework of a CRP will be new to most researchers working on mosquitoes yet will create a great many opportunities to further research into adult (male) mosquito biology. This will greatly benefit scientists working on the development of mosquito SIT, but equally be of use to those developing alternative genetic control strategies like genetically-engineered refractory mosquitoes. RCMs will provide a platform for information and data exchange, and publication of research outputs will be highly encouraged. Ultimately, the CRP is also intended to bridge the gap between established Northern laboratories (agreement holders) and laboratories in Member States that require capacity strengthening (contract holders).

It was recognised that although scientific communities in various Member States actively engage in this field of research, none do so specifically for developing the SIT for mosquitoes. It will thus be of importance to actively seek collaboration and announce the forthcoming CRP widely. The research topics proposed should therefore not only be of direct benefit to the SIT development effort, but also serve to augment current vector control strategies and/or those under development. Pro-active engagement of various research groups through the Agency’s efforts will therefore be required.

The Overall Objective of the CRP is to assist Member States in achieving sustainable control of mosquito transmitted diseases through development and integration of the SIT into area-wide programmes against major mosquito species. The Specific Objectives of the CRP (see next paragraph in detail) are to advance the understanding of critical (behavioural) ecological components of male mosquito biology so as to be capable to produce high quality and sexually competitive sterile males in mass-rearing facilities. Basic and applied research contributes to the foreseen implementation of mosquito SIT by studying field populations of adult male mosquitoes in order to establish the specific biological and behavioural determinants that contribute to male sexual competitiveness.

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SPECIFIC R&D ACTIVITIES

The CRP is focussing on the biology and competitiveness of male mosquitoes in the field once they have been mass-reared and released. Thus, we are striving to acquire detailed, specific laboratory and field-based information on all events in the life of adult male mosquitoes that impact their sexual competitiveness.

Male Physiology and Behaviour Nutrition

Male mosquitoes do not ingest blood, but frequently ingest plant derived substances33-36. Although the sources of these substances for several new world aedine and culicine mosquitoes have been established, where anophelines acquire their nutrients remains largely unknown. Empirical studies have shown that frequent sugar meals are significantly associated with reproductive success of males, as one bout of swarming activity consumes ~50% of the energetic reserves available37. Furthermore, other nutrients, such as bacteria or amino acids present in aphid honeydew, may contribute to male reproductive success by fuelling spermatogenesis and incorporation into accessory gland proteins, or cuticular volatiles. Accordingly, the objective of this part of the project will be to determine what male mosquitoes feed on, where these nutrients are acquired, what their energetic status in the field is and how they contribute to reproductive success. This information will contribute to SIT operations by allowing us to formulate and feed males on an optimal diet before release. Furthermore, once sources of nutrition in the field are identified, release strategies can be tailored (in space and time), to optimize male performance.

Spatial and temporal partitioning of sexual encounter sites The most commonly described sexual encounter site for anopheline mosquitoes

is in crepuscular swarms that form in response to prominent visual markers38-40. However, some anopheline species apparently do not swarm41, and others may combine typical swarming behaviour with an alternative tactic, such as copulating at emergence, blood feeding or resting sites. For mass-reared males to succeed in their quest for virgin females, it is imperative that they show up at the right time and place. At present, it is impossible to say with any precision what that time and place is for males belonging to species in the Anopheles gambiae complex. These males have proved to be rather difficult to study, as swarms are rather small and inconspicuous, suggesting that an alternative tactic may be employed42,43. Furthermore, we know very little about how encounter sites are located and how sympatric pre-copulatory mechanisms operate. In particular, the nature of the cues – mechanical, visual, auditory or olfactory- used in these processes must be identified. Thus, the objective in this section will be to determine precisely where and when, in the field, copulations of anopheline mosquitoes take place. This knowledge is important for the success of a programme with an SIT component as released males must have the ability to show up at the correct time and place.

Aedes mosquitoes display a different mating strategy, whereby the males also respond to human host cues, and the encounter site moves from the outdoor to the indoor environment. As more species employ this strategy, precopulatory isolation

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mechanisms in the form of contact pheromones are used to distinguish between species43a.

Male copulatory success Males that swarm or otherwise seek females invest energy and expose

themselves to predators. The reward for this expensive and risky behaviour is reproduction. This reward, however, is not evenly distributed among all investors. The high ratio of males in swarms results in intense competition for the relatively few females, and some males mate several times, while most never mate at all40, 44-46. Male size has been significantly associated with copulatory success in An. freeborni40, as has female size in An. gambiae47. However, such an association has not been found for males of An. gambiae42 or An. funestus43. Another factor significantly associated with copulatory success of An. freeborni is sugar feeding- participation in a swarm is predicated on a successful foraging bout during the preceding night37. The correlates of male mating success in other mosquitoes are unknown. Quantitative models of swarming and copulatory success have been useful in identifying such characteristics48, and if extended will enable us to endow mass reared males with qualities associated with enhanced reproductive success.

Male effects on females As a consequence of their mating, male mosquitoes significantly affect many

aspects of female biology. In Aedes mosquitoes, males modulate a number of physiological functions including subsequent mating behaviour, host-seeking and oviposition behaviours, and reproductive metabolism49-50. Although in these species, the events are specifically modified by peptides produced by the male accessory gland, in An. gambiae mosquitoes, which are major vectors of human malaria, these male substances are not involved51. Because it has long been incorrectly assumed that this mechanism of male products affects very critical behaviours in all female mosquitoes, it is important to specifically establish the role of mating on the behaviours of female An. arabiensis and other vectors.

Males of several species in the An. gambiae complex produce polymorphic sperm of detectably varying lengths whose frequency in spermathecae differ52. The biological significance of this polymorphism has yet to be established but may be critical for an understanding of the role that sperm play in regulating female behaviour. Only a few studies have examined the development of spermatozoa and their storage and distribution in the female. An understanding of the maturation of polymorphic sperm within the testes and the storage of these sperm prior to copulation may be important, both for the male and for the possible effects on the female. The number of sperm transferred and their survival in the female spermatheca influence the number of eggs that can potentially be fertilized and possibly the time until the female re-mates. For the success of an SIT operation targeting mosquitoes, we need to determine the effect of mass rearing and irradiation of males on the post-copulatory behaviour of the females they inseminate.

Male dispersal

Dispersal is a critical issue for all living organisms, ensuring optimal genetic interchange and exploitation of habitat53. In mosquitoes, dispersal is restricted to the adult stage. Mark-recapture studies of several species have demonstrated movement

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ranging from a few hundred meters to 90 kilometers54. Nearly all such studies, however, have focused on female dispersal55. Thus, although it is generally assumed that males remain close to their emergence sites, there are virtually no published supporting studies.

Delivery of radiation-sterilized males will be a critical parameter in ensuring maximal interaction of the treated insects with the wild population. Fundamental knowledge of the natural behaviour associated with this parameter, dispersal of treated and untreated insects, will be a key component of implementation models that will form the basis of release strategies. In addition, knowledge of dispersal of females will be required to assess the significance of immigration of fertile insects from untreated areas. Specific knowledge on dispersal capabilities will enable the programme to define the area to be treated with sterile males and to establish effective barriers against immigration of fertile males and females.

Male Survival Rate

The life span of a female mosquito is a key parameter in its ability to transmit pathogens. For this reason, as with dispersal, nearly all studies have been devoted to this sex56-58. Thus, although it is generally assumed that males do not live as long as females, no usable data exists; such data will be imperative for practical application of implementation models.

As these activities proceed quantitative tools will be developed for assessing each of these components, both in the laboratory and the field. In particular, the effect of predation, nutrition, copulation and age need to be determined. Results will enable the planned SIT operation to schedule releases in a manner that will optimize the effectiveness of the control programme.

Male sampling tools

Development of sampling methods for male mosquitoes The ability to study the (behavioural) ecology of male mosquitoes depends

heavily on the availability of efficient sampling tools. At present either non-attractant traps (such as the Malaise trap) or sound traps can be used for this59. This latter group of traps is particularly interesting. Laboratory and field experiments have shown that males of Culex tarsalis60, Cx. tritaeniorhynchus61, Cx. quinquefasciatus62, Aedes albopictus63-65, Anopheles minimus and An. maculatus66 can all be attracted to sound traps. Although interest in such sampling devices has been limited (for the prime reason that these mainly catch males!), in the context of mosquito SIT this certainly deserves further research. The CRP will not be restricted to the evaluation and improvement of sound traps. Any other potential sampling tool for adult male mosquitoes will be considered.

Marking methods for male mosquitoes

Insect marking methods are plentiful and were recently reviewed by Hagler67. In a programme including the SIT, sterile males earmarked for release can be marked to study their fate under field conditions. Commonly used marking methods, notably stains, dusts and/or powders frequently have negative effects on insects thus altering true longevity, dispersal potential etc.59. Other methods like the use of trace elements (like rubidium)68-69 need further development. The use of radioactive labelling is no

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longer advocated, but the uses of stable isotopes are currently being investigated70. As for sampling methods, it is imperative that marking protocols are developed and field tested, hence their inclusion in the current CRP project.

During this second RCM, participants were divided into three subgroups according to their research activities:

- Mosquito male pre mating conditions (Dispersal, survival & adult nutrition) - Mating systems - Molecular / Chemical approaches for a better understanding of male mosquito mating behavior

1. MOSQUITO MALE PRE MATING CONDITIONS

1.1 GENERAL FOCUS OF THE R&D Data on mosquito male biology are crucially lacking and such knowledge is a

prerequisite for “biologically lucid” approaches to mosquito control that incorporate large-scale releases of sterile (or otherwise genetically modified) insects for population suppression/replacement.

This working group focused on pre-mating male conditions including resource acquisition, allocation and use in order to i) identify ecologically relevant behavioural and physiological traits that shape male fitness in the field and ii) use this knowledge to improve fitness and mating competitiveness of mass-reared mosquitoes to be released in the field. Sugar is the only source of energy for male mosquitoes, and different sugar sources might be of different value for male survival and mating ability in the wild. Furthermore, rearing mosquitoes under different diets, at their larval and/or adult stage prior to their release might differentially impact on their ability to compete with their wild counterparts. Sugar delivery during rearing might also impact on the ability of males to adequately locate, select and feed upon the most rewarding energy sources once released.

Because the ultimate outcome of male mass-releases is to succeed in the mating competition with wild males (that are adapted to their environment), one of the most relevant indicators of males competitiveness/sexual performance should be their mating potential. Work along this line of research will require the development of standard protocols and guidelines to explore and compare male competitiveness / sexual performance in the field and in the laboratory.

1.2 SPECIFIC OBJECTIVES (INCLUDING PROGRESS TO DATE)

1.2.1 How rearing conditions affect the “quality” of the males. Establishment of laboratory colony for the “most common species”.

Quality control parameters such as development/survival, adult longevity, flight

ability and mating competitiveness will be studied by the group.

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In detail, participant will answer how their rearing conditions affect the development/survival of the immature stages and the associated longevity of the adults and efforts will be done to develop standardized protocols to investigate life history parameters and to explore the relationship between longevity and mating success. A SOP to measure flight ability (e.g. IAEA flight tube) will be provided to the participants and flight profiles of lab reared vs. wild mosquitoes will be compared. We will also look at the eventual correlation between measured flight ability and dispersal. Finally, mating competitiveness studies needs to be standardized to assess the assays in conditions that trigger ‘natural’ mating behaviour

Progress:

- BANGLADESH (Saidul Islam), a new facility for mosquito rearing was completed. Protocols for Ae. aegypti rearing were developed in the laboratory. Preliminary radiosensitivity study was carried out at larval and adult stages. - SYRIA (Hayat Makee): Construction of mosquito laboratory. Field Collection of mosquitoes for classification. Studying the optimum rearing conditions (partially completed, effect of temperature on egg hatch). Training course at the Insect Pest Control Laboratories (IPCL, IAEA) on rearing system (completed). - FRENCH POLYNESIA (Hervé Bossin): a standard male mating competition assay using field cages has been developed and validated. Comparison of the relative mating competitiveness of incompatible vs wild Ae polynesiensis males. The results of this work will soon be submitted for publication and the article will be provided to the working group when available. - CUBA (Rene Gato Armas): Comparison of geographically distinct recently established colonies of Ae. aegypti in oviposition, hatching, and survival (completed). The minimal dose of exposition to chemosterilants was determined to produce sterility in males (completed).

1.2.2 Resources acquisition and allocation (Male nutrition in the field)

Work was conducted to determine what male An. gambiae mosquitoes feed on in the wild, where and when these nutrients are acquired. This work should be extended to other mosquito species (An. arabiensis, Ae. aegypti, Ae polynesiensis, An. sacharovi) in a range of eco-epidemiological settings (endemic and epidemic regions). Much work remains to be done to determine the energetic status of male mosquitoes in the field and how they contribute to reproductive success. Once sources of nutrition in the field are identified, release strategies can be tailored (in space and time), to optimize male performance. Important parameters that were identified by the group for follow-up: (i) Quantify average energy reserves (glycogen, lipids, proteins) of wild males at critical time points and select best protocol to measure energy reserves; (ii) What energy reserves are mobilized during specific behaviours (flight, swarming, etc.); (iii) Compare energy reserves of laboratory reared or sterile males with wild specimens and (iv) Identify and characterize nutritional resources in the field, their distribution, seasonality

Progress:

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- BURKINA FASO (Frederic SIMARD): (i) identification of the main sources of energy for male anophelines in the field along with sources quality and patterns of intake (Partially completed):

- Males (and females) mosquitoes were collected in the VK7 village, Bama rice cultivation area (Burkina Faso) using a range of entomological collection techniques (sweeping in swarms, aspiration while resting indoors and outdoors, clay pots in various flowering plant species and underneath vegetation) and stored at -20°C. - Recent sugar feeding was assessed through the cold anthrone test (detection of carbohydrates in the crop of field collected males): qualitative estimate (+/-). - Validity of the anthrone test was assessed under lab conditions, using contained males fed with mango shoots. Complete degradation (eg, negative anthrone test) was observed 16hrs after feeding. - Numbers of mosquitoes collected using the various methods. The % Anthrone test positive was compared between techniques, in space and time. No differences were observed between zones in the village. However, significant differences were observed at the micro-geographic scale, depending on the location where mosquitoes were caught (e.g., indoor vs. outdoor resting sites, and in different plant species). Furthermore, differences in the % positive males were found depending on the time of collection. The highest %+ was observed at dawn (5-7am) > dusk (6-8pm) > night (10pm-5am). These data need further investigation & analyses (ongoing). - Based on these data and on visual inspection, the predominant flowering plant species were tested for differential attractiveness to male An. gambiae mosquitoes (collected as immature stages in the field and reared to adults under lab conditions): - The ability of males to feed upon M. indica (mango), D. regia (flamboyant), T. neriifolia, S. siamea and C. sieberiana was assessed under experimental conditions. Batches of newly emerged males, starved for 6hrs after emergence were exposed to shoots of plants and allowed to feed for 30 minutes � there are measurable differences in the proportion of males that successfully feed on these plants in the order M. indica = D. regia >=T. neriifolia > S. siamea >= C. sieberiana. - Attractiveness of these different plants for males An. gambiae was assessed experimentally using customized home-made Y-shaped olfactometer. Single or dual choices were conducted. Significant differences were found in the responses of males to the different blossoms, suggesting host preference in males. Preferences were in the same direction than feeding ability. - Major achievements: 1 master (MIE) student report presenting these results; and a paper published: Gouagna et al., J Vector Ecol 2010 (in press).

(ii) Quantify male reserve status (amount of lipids, glycogen, proteins) that can be gained from different adult diets (glucose, natural sugar sources). For the moment, only the (qualitative) cold anthrone test was used to assess the feeding status of males. We now need to apply quantitative assays to measure glycogen, lipids and proteins content in single specimens. Protocols need to be transferred to Burkina Faso (ongoing). (iii) Assess male fitness when fed on various sugar sources: Preliminary work on the longevity of males fed on different diets (e.g., nothing, water, glucose, M. indica, T. neriifolia, S. Siamea) has been done. Batches of newly emerged males were offered

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different nectar sources for 6d (4 reps/treatment for a total of 90 males/treatment). Results are as follows:

- All males died within 2d when nothing was provided; - All males died within 3d when water only was provided; - 5% males survived 6d when exposed to S. siamea; - 45% males survived 6d when exposed to T. neriifolia; - 80% males survived 6d when glucose/M indica was provided. - All males died within 3d when sugar sources were removed.

Specific and standardized protocols need to be developed in order to assess fitness of males using relevant indicators (Longevity? Flying ability? Mating performance?).

1.2.3 Male dispersal & sampling tools (Develop purpose specific trapping systems)

Mark-recapture studies are required to gain knowledge about the size of natural target populations and dispersal and survival capacity of released male material. Adequate marking techniques need to be devised to minimize the interference with subsequent survival and behaviour in the field. Dispersal of radiation-sterilized males will be a critical parameter to ensure maximal interaction of the treated insects with the wild population.

The ability to study the (behavioural) ecology of male mosquitoes depends heavily on the availability of efficient sampling tools. Efficient male mosquito sampling devices (sweeping nets, resting pots, male attractants, sound trap, other) need to be developed and field validated for several species. The ability to catch males involved in sexual activity is of particular importance in order to gather specific and relevant knowledge for subsequent applications.

1.3 R&D Proposed by Participants Under 1.2.1 How rearing conditions affect the “quality” of the males. Establishment of laboratory colony for the “most common mosquito species”.

- BANGLADESH (Saidul Islam): Adult longevity study in laboratory. Field cage trials between laboratory-reared and wild mosquitoes. Sterilization and lab-based mating competitiveness studies between mass reared and wild mosquitoes. Radiosensitivity study using Cobalt 60 irradiation needs to be continued on both pupae and adults. - SUDAN (Mo’awia M. Hassan) will continue to work on (i) the determination of swarming sites and time for An. arabiensis in the field and in a contained semi-field system, (ii) the effect of male age on mating success using isolated and competition assays in large field cages using males dusted with fluorescent particles, (iii) the mating compatibility of laboratory strains and field specimens from Dongola and Kariema areas.

- SYRIA (Hayat Makee): (i) Determine the effect of larval density on mortality, development time, sex ratio, adult size, longevity and fertility using IAEA standard larval diet; (ii) Determine the effect of adult density in a standard lab mating cage on the survival, mating ability, and fertility and fecundity and (iii) Evaluate the effect of

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various irradiation dose (Gammacell Co60) on longevity, flight activity and reproduction - ITALY (Romeo Bellini): (i) Acquire useful data on the effects of larval (IAEA) diets components on adult male flight ability, survival, mating capacity in lab cages: male flight ability will be measured by the IAEA flight tube and by the flight mills (Briegel); male survival will be measured in lab cage without any sugar provision, and mating capacity in relation to male age studied in lab cage; (ii) The optimal radiation dose to be administered to aged male pupae will be determined to obtain the best performing males for the purpose of population suppression (not elimination). In large field enclosures, 30-35-40 Gy doses will be evaluated with 100:100:100 irradiated males vs. normal males vs. virgin females (lab and wild specimens). - FRENCH POLYNESIA (Hervé Bossin): (i) Investigate the biological parameters from field-caught male mosquito that are likely to influence longevity and mating competitiveness; (ii) Collect wild specimens during the two tropical seasons, to conduct mating assays and measure wing length, energy reserves and longevity, and (iii) Investigate frequency of female remating in Ae. polynesiensis under laboratory and natural conditions. - CUBA (Rene Gato Armas): (i) Determine the persistence of the sterility by Thio-TEPA in treated males and in females crossed with treated males (ongoing); (ii) Evaluate the influence of volatile substances and females on mosquito male behaviour. (iii) Determine the mating competitiveness of males exposed to Thio-TEPA in large lab cages, and (iv) How rearing conditions affect fitness of the males (Mating competitiveness: Large Lab-cage experiments. Longevity: Explore the relationship between longevity and mating success. Develop standardized protocols to investigate life history parameters as death probability, survivorship, survival probability, fecundity, hatching rate, life expectancy, net reproduction rate, Mean generational time, finite rate of natural increase, intrinsic rate of natural increase, reproductive value, stable age distribution). Under 1.2.2 Resources acquisition and allocation (Male nutrition in the field) - BURKINA FASO (Frederic SIMARD): (i) Determine the main sources of energy for male anophelines in the field along with sources quality and patterns of intake (Data analysis and publication); (ii) Quantify male reserve status (Amount of lipids, glycogen, and protein) that can be gained from different diets (Glucose, natural sugar sources). Assess the amount of glycogen, proteins and lipids in males: at emergence (teneral reserves), when starved to death (minimal), after feeding on various sugar sources (Glucose, plants). Comparative approach between species (An. gambiae M, S, An. arabiensis), between lab-reared and field-collected mosquitoes; (iii) Assess male fitness when fed on various sugar sources (survival/longevity, flying ability, mating ability). Devise protocols to assess these parameters and their relevance for mating success and (iv) Explore resources utilization in males: Quantify glycogen, lipids and proteins use during flight, swarming and determine the dynamics of “energy” usage in the lab (experimental: before/after flying; before/after mating) and in the field (mosquitoes sampled while resting during the day/night, in early/late swarms).

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- ITALY (Romeo Bellini): Evaluate the efficacy of specially designed devices to be included in the pupae field release station, in order to supply energetic sources (and possibly other useful substances??) to improve male performances:

- sponge with 10% sucrose, 20% sucrose, 10% sucrose+10% honey - other substances ?? probiotics? - influence of ants - time of drying - % of male feeding (anthrone test) - dispersal & survival of the fed males (marked with stable isotopes?)

Under 1.2.3 Male dispersal & sampling tools (Develop purpose specific trapping systems) - FRENCH POLYNESIA (Hervé Bossin): (i) Include flight tube developed by the IAEA as a mean to measure and compare mass-reared vs. wild mosquito specimens; (ii) Perform mark-release recapture studies to assess population structure and size and evaluate dispersal capacity of Ae. polynesiensis. - ITALY (Romeo Bellini): Male flight ability will be measured by the IAEA flight tube and by the flight mills. The group has identified additional topics that need to be answered. The development of an appropriate olfactometers and related guidelines to investigate species specific behaviours and the development of release device systems for adults and pupae are required. Rene Gatos Armas will work of the olfactometer aspect and Romeo Bellini will continue to work on the release device.

2. MOSQUITO MATING SYSTEMS

2.1 GENERAL FOCUS OF THE R&D Mating systems encompass all the species specific ecological, behavioural

physiological and behavioural attributes that lead to encounter, copulation, insemination and fertilization. Many lacunae remain in our understanding of mosquito mating systems. The objective of the R&D is to understand the structure of the mating systems of various mosquito species, and establish correlates to male success within these systems, which can be applied to rearing, handling and release of mass reared mosquitoes in SIT operations.

2.2 SPECIFIC OBJECTIVES (INCLUDING PROGRESS TO DATE)

2.2.1 Determine the temporal and spatial characteristics of mating encounter sites of Aedine and Anopheline mosquitoes.

Anopheles and Aedes mosquitoes have different strategies to encounter females. Whilst Aedes males once sexually mature are capable of mating throughout the diel mating in anophelines only occurs at specific times and, perhaps, in specific sites. In anophelines mating activity occurs at specific sites and is largely limited to a short

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period at dusk and is largely under the control of the circadian clock modulated by light activity at sunset. The location of species specific mating sites is perhaps the most efficient way of maintaining isolation. Little is however known about the mating behaviour of many species, including An. arabiensis, the target species for SIT in Sudan and Reunion. Where they have been studied, males (of species that form over identifiable markers) will follow artificial markers for limited distances before returning to their natural markers. What cues produce the switch back to the original marker remains unknown and is probably important in general site selection.

Mating strategies may change at different population densities and the relationship between numbers of females mating in swarms at different densities is required. It is important that colonisation does not affect either timing of the clock or response to light at sunset.

• Understanding the specific nature of cues that are used by males to congregate

is required for a variety of species (so that general principles can be derived) – specifically the nature of markers.

• Can a ‘super-marker’ be produced that draws males in and ‘captures’ them? • Determination of the characteristics of swarm sites (and times/light intensities

at which this occurs) for An. arabiensis is required. • In aedines, the role of the blood meal host as a mating encounter site should be

investigated. Progress: The swarming and mating system in natural populations of An.

gambiae M and S molecular forms were investigated across longitudinal surveys from July 2006 to October 2009 in Soumousso and Vallée du Kou (VK7), two areas of western Burkina Faso where these forms are sympatric. In Soumousso, a wooded savannah, overall 205 swarms and 109 pairs caught in copula were sampled whereas in VK7 more than 250 swarms with 491 couples were sampled and effectively PCR analysed. In both sites no spatial segregation has been observed between the swarming places of M and S forms which shared the same type of visuals markers. Even though some mixed swarms were annually collected in varying frequencies from one site to other, the major swarming behaviour was that the two forms swarm separately in more than 90% of cases. Analysing the mating feature within the swarms, although collected within mixed swarms the frequencies of pure form pairings was higher and the mixed ones did not reach up 4%. The insemination rate estimated within natural swarming populations of M form averaged 68% without any mixed insemination corroborating the existence of low hybrids rate in the field. The relation between environmental factors and swarming behaviour showed that some parameters such as temperature and relative humidity were not associated to the swarming formation. But the time point of swarms’ apparition seemed to be fairly conserved in An. gambiae s.s. natural populations and did not vary significantly between the two forms. However the swarms of the S form (Soumousso) appeared more precociously than those of the M form (VK7). They followed the seasonal fluctuation of photoperiodicity suggesting that the apparition of swarms was tightly regulated by circadian rhythms and/or biological horologe. The mechanisms regulating the recognition between inter or intra forms partners during the swarming and mating process are discussed.

In Sudan, An. arabiensis swarm was observed in the vicinity of breeding sites.

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In Trinidad, it was determined that Ae. aegypti mating occurs during 2 distinct peaks (morning and afternoon). The location of mating was in the vicinity of breeding sites, but also within houses.

In Italy, Ae. albopictus mating was observed in the late afternoon, both in small

swarms that formed close to the ground in shaded areas or close to tree trunks. Copulation was also observed in the vicinity of the host.

2.2.2 Investigate the behaviours involved in courtship.

As far as we know, most vector species mate on the wing and it is generally assumed that most mating occurs in station-keeping swarms which consist mainly of males. Little is known, however, about the short-range interactions that lead to mating (courtship), or what the complete range of behaviours are involved with species specific mating, but clearly these behaviours are likely to be vulnerable to selection pressures during colonisation where mating occurs in a limited space and it is not known how courtship behaviour is affected.

Olfactory cues

Sex pheromones play a key role in the mating behaviour of many dipteran species, serving as a long-range attractant to bring males and females together and as a means of species identification. Pheromones have not been shown to occur in vector species, but little research has been done. Clearly, if sex pheromones are produced in vector species, this would be of direct relevance to the likelihood of success of SIT.

Progress: Preliminary studies have shown that at least three compounds

increase during flight behaviour of male An. gambiae (species arabiensis). The next step has been to identify the origin if these compounds in the mosquito by labelling molecules in the diet of larvae and adults. The fate of the labelled compounds will be tracked. Nothing is known about the response of males and females (virgin or mated) to the compounds that are released by flying males. In the research on male and female volatiles affecting mosquito swarming behaviour larvae and adult An. arabiensis, Dongola strain, were fed on U- 13C6 labelled glucose. Chemical analyses with SPME-GC-MS were made of the emissions from live and recently freeze killed mosquitoes from the two treatments. Different volatiles were labelled when fed on 13C-glucose as larvae compared to the adult 13C-glucose fed. Some of these compounds have recently been identified from gut microbes in An. gambiae.

Auditory cues

It has been known for decades that male mosquitoes hear and respond to female flight tones by flying toward the source of the sound. The range of female flight tones is so wide that this characteristic cannot be used as a species-specific identifying trait. Until recently it has been assumed that the only role of auditory cues in mating behaviour was for male location of females.

Progress: It has been shown in An. gambiae M & S form and Ae. aegypti that

male-female pairs enter into auditory interactions, harmonising their wing-beat frequencies when they are within ~ 10 cm of each other. These have been laboratory-

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based studies of tethered insects. The response of individual mosquitoes depends on the identity of the other mosquito, suggesting that this behaviour enables sex- and species-recognition. Nothing is known about the next stage in mating behaviour; i.e., in free-flight how long do pairs of mosquitoes continue to harmonise? Does this bring them closer together spatially and allow them to form a copula? Do harmonising couples go on to mate successfully? What happens to mixed-form non-harmonising pairs in free-flight?

Tactile cues

Many dipteran species use cuticular hydrocarbons to detect conspecific individuals, but this has not been fully explored in mosquitoes.

Progress: Some basic research has been instigated to identify the cuticular

hydrocarbons of An. gambiae spp, suggesting that the profile of components alters over the course of the lifetime of the mosquito. Further work is required to follow-up these preliminary studies.

2.2.3 Copulation and Insemination

Copulation The mechanics of copulation have been described in detail in the past (reviewed

by Clements). The operational sex ratio is highly skewed towards males. As copulatory success is non random, it is important to identify the characteristics that determine which males copulate.

Progress: Under 2.2.3.1 Male copulatory success: It was determined, for A.

gambiae that male size is correlated with copulatory success in laboratory cages and the field.

Sperm transfer and storage

Insect seminal fluid proteins are powerful modulators of many aspects of female physiology and behaviour including longevity, egg production, sperm storage, and remating. The crucial role of these proteins in reproduction makes them promising targets for developing tools aimed at reducing the population sizes of vectors of disease.

Progress: Under 2.2.3.2 Sperm transfer and storage: In the malaria mosquito

An. gambiae, seminal secretions produced by the male accessory glands (MAGs) are transferred to females in the form of a coagulated mass called the mating plug. The potential of seminal fluid proteins as tools for mosquito control demands that we improve our limited understanding of the composition and function of the plug. Here, we show that the plug is a key determinant of An. gambiae reproductive success. We uncover the composition of the plug and demonstrate it is formed through the cross-linking of seminal proteins mediated by a MAG-specific transglutaminase (TGase), a mechanism remarkably similar to mammalian semen coagulation. Interfering with TGase expression in males inhibits plug formation and transfer, and prevents females from storing sperm with obvious consequences for fertility. Moreover, we show that the MAG-specific TGase is restricted to the anopheline lineage, where it functions to

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promote sperm storage rather than as a mechanical barrier to re-insemination. Taken together, these data represent a major advance in our understanding of the factors shaping Anopheles reproductive biology.

2.2.4 Determine patterns of female remating, and how male performance affects this behavior.

Although females of most insect species remate, mosquito females in general are considered monandrous. This has been demonstrated conclusively (with genetic evidence from field populations), for several anopheline species. However, there is no field data on aedine species. The presence of 3 spermathecae in aedine females suggests that remating may be common. Furthermore in all species, sterile males may not be able to inhibit receptivity as effectively as wild males.

Progress: For Ae. aegypti, in collections of mating pairs, as many as 15% of the

copulating females were parous. This suggests that some females do remate in the field.

2.3 R&D PROPOSED BY PARTICIPANTS

Under 2.2.2 Investigate the behaviours involved in courtship. Olfactory cues

- SWEDEN (Anna-Karin Borg-Karlson) & ILE DE LA REUNION (Guy Lemperiere): Will pursue compound D. Determine its suitability as an attractant and its potential for manipulating the formation of swarms.

Auditory cues

- UNITED KINGDOM (Gabriella Gibson): Will investigate the auditory interactions between mosquitoes in free flight, using Culex quinquefasciatus in the laboratory as a model, to test our findings in tethered flight. - BURKINA FASO (Roch Dabire) & UNITED KINGDOM (Gabriella Gibson) collaboration: Repeat findings of M and S form behaviour in the field in different seasons, different sites and with strains of M and S from other areas.

Under 2.2.3 Copulation and insemination Copulation - BURKINA FASO (Roch Dabire), GHANA (Derek Charlwood): For An. gambiae M and S forms, and An. arabiensis, determine the relationship between size, age, nutrition and copulatory success in the field. - ILE DE LA REUNION (Louis Clement Gouagna): For An. arabiensis, in the laboratory and the field, determine the relationship between size, age, nutrition and copulatory success.

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- BURKINA FASO (Frederic Simard): Develop protocols for measuring fitness based on copulatory success in An. gambiae. - TRINIDAD (Chadee): For Ae. aegypti, determine the relationship between size, age, nutrition and copulatory success.

Sperm transfer and storage:

- BURKINA FASO (Roch Dabire) (in collaboration with Catteruccia): For An. gambiae M and S forms, determine the attributes of mating plug formation and disappearance following copulation. Determine if the mating plug or other components of the ejaculate are involved in incipient speciation of these forms. Determine the relationship between the mating plug and sperm storage. - ILE DE LA REUNION (Guy Lemperiere) & TRINIDAD (Chadee): Determine the functional significance of multiple spermathecae in relation to male quality and number of available mates for Ae. aegypti and Ae. albopictus.

Under 2.2.4 Determine patterns of female remating, and how male performance affects this behavior.

- FRENCH POLYNESIA (Hervé Bossin) & TRINIDAD (Chadee): For Ae. aegypti, Ae. polynesiensis, expand the novel observation that parous females were found copulating in the field. Characterize this behavior according to mating site, and patterns of sperm storage in the 3 spermathecae.

3. CONTRIBUTION OF MOLECULAR/CHEMICAL APPROACHES TO THE UNDERSTANDING OF MALE MOSQUITO MATING BEHAVIOR

3.1 GENERAL FOCUS OF THE R&D In the world of insects, a wide range of studies have then been carried out

during the past forty years in chemical ecology, leading to the production of a vast literature and also to the use of products (pheromones, kairomones…) in order to operate in the control of pests in agriculture and forestry. In the world of mosquitoes, it is not until 1922 with preliminary Willem Rudolfs’s work on the chemotropism of mosquitoes that the role of chemicals in the biology of those insect has been explored. In between, work on mosquitoes was mostly concentrated on the control of populations despite some studies carried out in the sixties (Sandhlom and Price 1962, Thorsteinson and Brust 1962…) until the eighties (Hancock and Forster 1982, Healy and Jepson 1988). Later, Clements’s general textbook on mosquitoes (1999) covered and synthesized several aspects of olfaction, sensory reception and behaviour giving the general basis of chemoreception in mosquitoes. Since, the odor-mediated host interactions have been widely investigated but it is only recently that other

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behavioural aspects like mosquito-plant interactions have been studied. Chemical Ecology of mosquitoes requires better knowledge at different levels: insect-host preferences (attraction to humans or other vertebrates), insect-plant relationships (attractants and repellents in host feeding of adults and larvae), insect-insect (chemical communication between adults, swarms…), insect-environment (attraction to oviposition sites…) using new tools for the investigation of mosquito biology like the molecular biology of mosquito olfaction, electrophysiology and covering the various aspects of emission, perception, odor coding…The results of this type of research have implications in present and future integrated vector management strategies using SIT or GM mosquitoes.

3.2 Specific Objectives (including progress to date) The expert group agreed that two kinds of approaches would be the most

important contributions to the program in general: a chemical approach and a molecular approach.

3.2.1. A chemical approach or chemical ecology of mosquitoes

Under the chemical approach the group decided to focus on (i) the extraction and identification of potential swarming/aggregation volatiles from mosquitoes; (ii) the extraction and identification of volatiles from release-site plants as potential attractants for males and the extraction and identification of compounds in known feeding sources of nectar, fruit and perhaps honeydew for male mosquitoes and (iii) the use of olfactometers to test potential attractive volatile compounds using wild males versus mass-reared, irradiated males..

The identification of the chemical nature of volatiles associated with plants that are known to be most attractive to male mosquitoes and identification of the chemical content of preferred natural diets of male mosquitoes. This knowledge could potentially enhance the ability of sterile males to locate and feed on natural sources and optimize dietary formulations for sterile males with the goal of improving survivorship, mating success or both. Follow-up studies would be needed to assess the impact of volatiles on male behaviour in olfactometers as well as the impact of dietary formulations on male survivorship and mating success. Of critical importance will be comparative studies of sterilized males versus wild-caught males.

Progress: Collection of volatiles from swarming males and/or aggregating males has been initiated for some mosquito species including An. arabiensis (KGB, Dongola and La Reunion strains) and Ae. albopictus. Volatiles emitted from male An. arabiensis (KGB strain), during swarming and during forced flight in daytime have been compared. Cuticular hydrocarbons and other compounds with putative behavioral effect have been identified in An. arabiensis by GC-MS. In addition, compounds involved in the behaviour of both An. arabiensis and Ae. albopictus have been identified from mosquitoes collected on La Reunion.

3.3.2 A molecular approach

Use PCR-based analysis of Y chromosome markers in An. gambiae to assess M and S molecular form distribution and male dispersal characteristics. Molecular studies based on Y chromosome molecular markers may provide important information about male population structure and male dispersal dynamics, particularly

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with respect to An. gambiae M and S forms. While several markers currently exist for An. gambiae, more markers are forthcoming. Perhaps markers can also be developed for other species.

Progress: Sample sites have been identified in 3 ecological zones in Ghana:

coastal savanna, mangrove strand, and forest zones. Samples have been collected from 8 sites in the coastal savanna zone. DNA has been extracted from all samples and PCR analysis has been completed, providing the identification of species and molecular forms.

3.3 R&D Proposed by Participants

Under 3.2.1. A chemical approach or Chemical ecology of mosquitoes

Extraction and identification of potential swarming/aggregation volatiles - SWEDEN (Anna-Karin Borg-Karlson): Continue collecting volatile emissions from An. arabiensis (KGB and Dongola) to increase the number of replicates. - ILE DE LA REUNION (Guy Lemperiere): Duplicate collection experiments in swarming/aggregating An arabiensis.

Extraction and identification of volatiles from release-site plants as potential attractants for males and the extraction and identification of compounds in known feeding sources of nectar, fruit and perhaps honeydew for male mosquitoes.

- SWEDEN (Anna-Karin Borg-Karlson): Analyze samples from the most, and least, preferred nectar plants to identify potentially important compounds for mosquito attraction or repulsion. If chiral compounds are present, identify the proportions of the enantiomers. - ILE DE LA REUNION (Guy Lemperiere): Assess male An. arabiensis and Ae. albopictus attraction to flower and fruit volatiles in olfactometers.

Use of olfactometers to test potential attractive volatile compounds using wild males versus mass-reared, irradiated males

- ILE DE LA REUNION (Guy Lemperiere): Initiate bioassays - olfactometers with male and female An. arabiensis and Ae. albopictus using identified mosquito-emitted volatiles. Assess male An. arabiensis and Ae. albopictus attraction to flower and fruit volatiles in olfactometers. Under 3.3.2 A molecular approach - GHANA (Alex Egyir-Yawson): Optimize PCR protocol with newly designed primers for Y chromosome markers. Use PCR products from Y chromosome markers to optimize for PAGE conditions. Sample at least 6 sites in the next ecological zone, mangrove strand zone.

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Use newly identified Y chromosome markers for PCR analysis when/if they become available (from J. Krzywinski). Additional studies are ongoing in the group such as: - SWEDEN (Anna-Karin Borg-Karlson): Continue experiments with feeding stable isotopes to larvae and adults, comparing the different patterns in male and female. Test egg laying preferences of An. gambiae using the bacterial related odors (NIH project). - ILE DE LA REUNION (Guy Lemperiere): Assess behavioural sequences in Ae. Albopictus and Ae. arabiensis using IR video cameras.

Several points were of general concern to the group. Molecular biological

studies often require more funding than non-molecular studies, based on reagent and equipment costs. The need to identify and perhaps share common resources, where possible, within the SIT group was discussed. This may help alleviate duplicating studies. For example, it might be strategically beneficial for a single lab to be charged with performing the chemical ecology, which could receive materials from other groups who need such support. The MR4 facility in the Unites States may be able to offer support for participants, especially to endemic nations, in terms of mosquito lines, PCR primers, DNA samples, etc. Often this can be offered at no cost to the participant. MR4 could also serve as a central repository for SIT groups by receiving wild material or recently-reared lines from around the world for distribution to other groups as needed.

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30. Killeen GF, McKenzie FE, Foy BD, Schieffelin C, Billingsley PF, Beier JC: The potential impacts of integrated malaria transmission control on entomologic inoculation rate in highly endemic areas. Am J Trop Med Hyg 2000, 62: 545-551. 30a. Kay B, Vu SN: New strategy against Aedes aegypti in Vietnam. Lancet. 2005, 365: 613-617. 31. Dyck A, Hendrichs J, Robinson AS: The Sterile Insect Technique: Principles and Practice in Area-Wide Integrated Pest Management. Springer, Heidelberg, Germany (2005) 32. Ferguson FM, John B, Ng'habi K, Knols BGJ: Adressing the sex imbalance in knowledge of vector biology. Trends Evol Ecol 2005, 20, 202-209. 33. Foster WA: Mosquito sugar feeding and reproductive energetics. Annu Rev Entomol 1995, 40: 443-474. 34. Yuval B: The other habit- sugar feeding by mosquitoes. Bull Soc Vector Ecol 1992, 17: 150-156. 35. Impoinvil DE, Kongere JO, Foster WA, Njiru BN, Killeen GF, Githure JI, Beier JC, Hassanali A, Knols BGJ: Feeding and survival of the malaria mosquito Anopheles gambiae on plants growing in Kenya. Med Vet Entomol 2004, 18: 108-115. 36. Gary RE, Foster WA: Anopheles gambiae feeding and survival on honeydew and extra-floral nectar of peridomestic plants. Med Vet Entomol 2004, 18:102-107. 37. Yuval B, Holliday-Hanson M, Washino RK: Energy budget of swarming male mosquitoes. Ecol Entomol 1994, 19: 74-78. 38. Downes JA: The swarming and mating flight of Diptera. Annu Rev Entomol 1969, 14: 271-298. 39. Sullivan RT: Insect swarming and mating. Florida Entomologist 1981, 64: 44-65. 40. Yuval B, Wekesa JW, Washino RK: Effects of body size on swarming behavior and mating success of male Anopheles freeborni (Diptera: Culicidae). J Insect Behav 1993, 6: 333-342. 41. Lounibos LP, Lima DC, Lourenco de Oliveira R : Prompt mating of released Anopheles darlingi in western Amazonian Brazil. J Am Mosq Control Assoc 1998, 14: 210-213. 42. Charlwood JD, Pinto J, Sousa CA, Madsen H, Ferreira C, do Rosario VE: The swarming and mating behaviour of Anopheles gambiae s.s. (Diptera : Culicidae) from Sao Tome Island. J Vector Ecol 2002, 27: 178-183. 43. Charlwood JD, Thompson R, Madsen H: Observations on the swarming and mating behaviour of Anopheles funestus from southern Mozambique. Malar J 2002, 2: 2. 43ª. Nijhout HF, and Craig Jr. GB: Reproductive isolation in Stegomyia mosquitoes. III. Evidence for a sexual pheromone. Ent Exp Appl 1971, 14: 399-412. 44. Reisen WK, Sakai RK, Baker RH, Azra K, Niaz S: Anopheles culicifacies: observations on population ecology and reproductive behavior. Mosq News 1982, 42, 93-101.

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45. Bock ME, Reisen WK, Milby MM: Lifetime mating patterns of laboratory adapted Culex tarsalis males. Mosq News 1983, 43: 350-354. 46. Slooten E, Lambert DM: Evolutionary studies of the New Zealand coastal mosquito Opifex fuscus (Hutton) II. Competition for mates. Behaviour 1984, 85: 1-12. 47. Okanda FM, Dao A, Njiru B, Arija J, Akelo HA, Touré Y, Odulaja A, Beier JC, Githure JI, Yan G, Gouagna LC, Knols BGJ, Killeen GF: Behavioural determinants of gene flow in malaria vector populations: Anopheles gambiae males mate only with large females. Malar J 2002, 1: 10. 48. Gibson G: Swarming behavior of the mosquito Culex pipiens quinquefasciatus: a quantitative analysis. Physiol Entomol 1985, 10: 283-296. 49. Craig Jr. GB: Mosquitoes: Female monogamy induced by male accessory gland substance. Science 1967, 156: 1499–1501. 50. Klowden MJ: The check is in the male: Male mosquitoes affect female physiology and behavior. J Am Mosq Control Assoc 1999, 15: 213-220. 51. Klowden MJ: Sexual receptivity in Anopheles gambiae mosquitoes: absence of control by male accessory gland substances. J Insect Physiol 2001, 47: 661–666. 52. Klowden MJ, Chambers GM: Production of polymorphic sperm by anopheline mosquitoes and their fate within the female genital tract. J Insect Physiol 2004, 50: 1163-1170. 53. Kennedy , J. S. Insect dispersal. In: Insects Science & Societ. (Pimentel D, Ed.). pp. 103-119. New York Academic Press, 1975. 54. Service MW: Mosquito (Diptera: Culicidae) dispersal--the long and short of it. J Med Entomol 1997, 34: 579-588. 55. Takken W, Charlwood JD, Billingsley PF, Gort G: Dispersal and survival of Anopheles funestus and A. gambiae s.l. (Diptera: Culicidae) during the rainy season in southeast Tanzania Bull Entomol Res 1998, 88: 561-566. 56. Gillies MT: Studies on the dispersion and survival of Anopheles gambiae Giles in East Africa by means of marking and release experiments. Bull Entomol Res 1961, 52: 99-127. 57. Charlwood JD, Birley MH, Dagoro H, Paru R, Holmes PR: Assessing survival rates of Anopheles farauti (Diptera: Culicidae) from Papua New Guinea. J Animal Ecol 1985, 54: 1003-1016. 58. Costantini C, Li SG, Della Torre A, Sagnon N, Coluzzi M, Taylor CE: Density, survival and dispersal of Anopheles gambiae complex mosquitoes in a west African Sudan savanna village. Med Vet Entomol 1996, 10, 203-219. 59. Service MW: Mosquito Ecology: Field sampling methods. Chapman & Hall, 1993, 988 pp. 60. Ikeshoji T, Sakakibara M, Reisen WK: Removal sampling of male mosquitoes from field populations by sound trapping. Jap J Sanit Zool 1985, 36: 197-203. 61. Ikeshoji T: Ditribution of the mosquitoes Culex tritaeniorhynchus, in relation to disposition of sound traps in a paddy field. Jap J Sanit Zool 1985, 36: 95-101.

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62. Ikeshoji T, Yap HH: Monitoring and chemosterilization of a mosquito population, Culex quinquefasciatus (Diptera: Culicidae) by sound traps. Appl Ent Zool 1987, 22: 474-481. 63. Ikeshoji T, Ogawa K: Field catching of mosquitoes with various types of sound traps. Jap J Sanit Zool 1985, 39: 119-124. 64. Ikeshoji T, Yap HH: Impact of the insecticide-treated sound traps on an Aedes albopictus population. Jap J Sanit Zool 1985, 41: 213-217. 65. Kanda T, Loong KP, Chaing GL, Cheong WH, Lim TW: Field study on sound trapping and the development of trapping method for both sexes of Mansonia in Malaysia. Trop Biomed 1988, 5: 161-166. 66. Leemingsawat S: Field trials of different traps for malaria vectors and epidemiological investigations at a foot-hill basin in Kanchanaburi, Thailand. Jap J Sanit Zool 1989, 40: 171-179. 67. Hagler JR, Jackson CG: Methods of marking insects: Current techniques and future prospects. Annu Rev Entomol 2001, 46: 511-543. 68. Maciel-de-Freitas R, Goncalves JM, Lourenco-de-Oliveira R.: Efficiency of rubidium marking in Aedes albopictus (Diptera: Culicidae): preliminary evaluation on persistence of egg labeling, survival, and fecundity of marked female. Mem Inst Oswaldo Cruz 2004, 99: 823-827. 69. Liew C, Curtis CF: Horizontal and vertical dispersal of dengue vector mosquitoes, Aedes aegypti and Aedes albopictus, in Singapore. Med Vet Entomol 2004, 18: 351-360. 70. Hood-Nowotny R, Mayr L, Knols BGJ: Use carbon-13 as a population marker for Anopheles arabiensis in a Sterile Insect Technique (SIT) context. Malar J 2006, 5: 6. 71. Benedict M, Robinson AS: The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol 2003, 19: 349-355.

APPENDIX 1 - LOGICAL FRAMEWORK

NARRATIVE SUMMARY

OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OVERALL OBJECTIVE

To assist Member States in achieving sustainable control of mosquito transmitted diseases through development and integration of the SIT into area-wide programmes against major mosquito species.

N/A N/A Mosquito-borne disease due to increased to insecticide and drug resistance continues to pose a serious threat to human well-being and economic development. Short-term absence of suitable replacement strategies for sustainable vector control will increase mosquito-borne disease risk and reduce herd immunity. Intensive chemical-based anti-malaria interventions as with IRS or ITNs are inherently time-limited because of insecticide resistance. Scaled-up use of SIT will result in reduced insecticide contamination of the environment.

NARRATIVE SUMMARY

OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

SPECIFIC RESEARCH OBJECTIVE

To study field populations of adult male mosquitoes in order to establish the specific biological and behavioural determinants that contribute to male sexual competitiveness

Determinants of male mosquito reproductive success identified to contribute to the foreseen implementation of mosquito SIT.

Research and development outputs disseminated and published.

- Mass production of high quality male mosquitoes is a given prerequisite for a control programme with an SIT component. - The success of any genetic control programme will hinge on the sexual competitiveness of the mass reared released males. - The biology of adult mosquitoes is poorly understood. In particular little is known of the factors which contribute to male competitiveness.

NARRATIVE SUMMARY

OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 1: Male physiology and sexual behaviour

1.1. Nutrition - Nutrient sources identified in the field. - Establish relationship between diet, dispersal, longevity and competitiveness - Improved diets for males identified.

Achievements to Date:

Sugar feeding patterns of An. gambiae studied in Burkina Faso (West Africa) Plant-host preference studied in field and lab (olfactometer) for An. gambiae in Burkina Faso (West Africa), Ae. aegypti in Cuba, and Ae. albopictus in La Réunion (ongoing) SPME analysis of plant and insect volatiles in La Réunion (ongoing) Fish diet formulation found to be suitable to rear Ae aegypti larvae in Bangladesh Longevity of Ae. aegypti males in relation to sugar sources studied in the lab in Bangladesh

- Research published in peer-reviewed journals and/or presented in professional meetings. - Male competitiveness improved by providing, prior to release, a diet that will enhance their performance in the field.

Achievements:

1 publication in J. Vector Ecology 1 Master thesis completed (MIE) 1 Master thesis underway in Bangladesh 1 scientific communication at MIM 2009, Nairobi, Kenya

Three customized olfactometers developed in Burkina Faso, Cuba and La Réunion

Male nutrition is a significant component of competitiveness

1.2. Sperm production and accessory gland development

Factors affecting ejaculate quality established.

Achievements to Date:

Composition and function of mating plug of Anopheles gambiae deciphered.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Mass rearing procedures assure production of males with effective ejaculates capable of preventing female reproduction.

Achievements to Date:

Paper published in "PLOS Biology"

Biotic and abiotic factors affect production of sperm and accessory gland secretions.

NARRATIVE SUMMARY

OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 1: Male physiology and sexual behaviour

1.3. Spatial and temporal characteristics of sexual encounter sites

Spatial and temporal determinants of male and female encounter sites determined.

Achievements to Date:

Swarming sites of An. gambiae M and S forms studied in West Africa. Markers that elicit swarming identified. Role of swarms in incipient speciation determined. Swarming sites of An. arabiensis identified in Sudan. Diel periodicity (early morning and late afternoon) of sexual encounters of Ae. aegypti established. Encounters observed near breeding sites and human habitation. For Ae. albopictus, in Italy mating was observed outdoors, in the late afternoon, in minor swarms that form close to the ground in shaded areas, close to tree trunks. Copulation was also observed in the vicinity of the host.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Mass reared males conform to mating strategies of field population. Achievements to Date:

Manuscript submitted to "Medical and Veterinary Entomology" for peer review. Paper presented at International Meeting of Society of Vector Ecologists. Ms. Accepted for publication in "Journal of Medical Entomology"

Most anophelines copulate in swarms, however alternative tactics may exist.

1.4. Male copulatory success

Factors affecting how copulations are apportioned among cohorts of males, and which factors (age, physiological status, size etc') affect this inequality established.

Achievements to Date:

Established, for A. gambiae, that male size is correlated with copulatory success in laboratory cages and the field.

Shown in An. gambiae M & S form and Ae. aegypti that male-female pairs enter into auditory interactions, harmonising their wing-beat frequencies when they are within ~ 10 cm of each other.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Mass reared males endowed with qualities associated with copulatory success. Achievements to Date:

Manuscript submitted to "Medical and Veterinary Entomology" for peer review. Paper published in "Current Biology"

A small proportion of males in the population achieve a large proportion of copulations.

1.5. Sperm transfer and storage.

Factors affecting amounts of sperm transferred and their fate in the female reproductive tract determined.

Achievements to Date:

Composition and function of mating plug of Anopheles gambiae deciphered.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Mass reared males capable of transferring an effective ejaculate to wild females. Achievements to Date:

Paper published in "PLOS Biology"

The female reproductive tract influences sperm storage and use.

NARRATIVE SUMMARY

OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 2: Male bionomics

2.1. Male Dispersal

Determine how far male mosquitoes disperse from emergence sites, the temporal pattern of this behaviour and its regulation.

Achievements to Date:

Mark-release recapture studies: Ae. polynesiensis incompatible males released at a study site in French Polynesia (ongoing) Ae. albopictus males in Italy, and La Réunion An. arabiensis males in Dongola, Sudan

- Research published in peer-reviewed journals and/or presented in professional meetings. - Control programmme able to define the area to be treated by SIT. - Parameters for effective barriers against immigration of fertile males and females established

Achievements:

Ae. polynesiensis elimination feasibility study (NIH funded, 2006-2011) Publication in Journal of Medical Entomology (in press) Communication at AMCA conference, Reno, Nevada, 2008 Manuscripts under preparation

Male mosquitoes have innate patterns of dispersal which are regulated by biotic and abiotic factors.

2.2. Longevity Biotic and abiotic factors affecting male longevity in the field established.

Achievements to Date:

Mark release recapture studies underway in French Polynesia, La Réunion, Italy, and Sudan in the framework of male release studies

- Research published in peer-reviewed journals and/or presented in professional meetings. - Release schedules will be optimized. Achievements to Date:

Publication of La Réunion work by Lacroix et al. in Bulletin de la Société Française d’Entomologie (2007)

Increased male longevity will enhance the effectiveness of sterile male releases.

NARRATIVE SUMMARY OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 3. Male Effects on females

3.1. Inhibition of female receptivity

Establish how copulatory stimuli, sperm and accessory gland products contribute to the control of female sexual receptivity in mosquitoes.

Achievements to date:

In An. gambiae, established that the mating plug does not prevent remating. Evidence of remating for Aedes aegypti, in the field. In collections of mating pairs, as many as 15% of the females were parous.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Programmes produce mass reared sterile males capable of inhibiting the sexual receptivity of the females they copulate.

Achievements to date:

Paper published in "PLOS Biology".

The ability of mass reared released male mosquitoes to inhibit the subsequent receptivity of the females they copulate will greatly contribute to the success of genetic control operations.

3.2. Modulation of blood feeding and oviposition

Determine whether copulation (and storage of ejaculate), affects subsequent blood feeding and oviposition.

- Research published in peer-reviewed journals and/or presented in professional meetings. - Females mated by mass reared males lay infertile eggs.

Mating affects female blood feeding and oviposition behaviour.

NARRATIVE SUMMARY OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 4. Field research

4.1. Development of field methods

Effective methods for marking, trapping and assessing male competitiveness in the field developed, to support the objectives outlined in 1-3.

Achievements to Date:

An. gambiae male trapping systems used in trees Burkina Faso Marking techniques developed using stable isotopes (IAEA, Seibersdorf) Development of a field cage male mating competitiveness assay for Ae. polynesiensis developed and used to assess incompatible male competitiveness Mating competitiveness studies done in field cage in Dongola Sudan to compare irradiated vs wild An. arabiensis males

- Research published in peer-reviewed journals and/or presented in professional meetings. - New methods for marking, trapping and assessing male mosquito competitiveness developed, standardized and in use.

Achievements:

1 publication in J. Vector Ecology 1 publication in preparation

Currently available field study methods to study male competitiveness are inadequate.

OUTPUT 5. Synthesis 5.1. Defining male competitiveness.

The various factors contributing to male competitiveness ranked in order of importance. Achievements to Date:

Established, for A. gambiae, that male size is correlated with copulatory success in laboratory cages and the field.

- Information formalized in process and quality management manuals made available to action agencies. - Standard rearing and sterilization procedures result in highly competitive mass reared males. Achievements to Date:

Manuscript submitted to "Medical and Veterinary Entomology" for peer review.

Male competitiveness can be increased.

NARRATIVE SUMMARY OBJECTIVE VERIFIABLE INDICATORS

MEANS OF VERIFICATION IMPORTANT ASSUMPTIONS

OUTPUT 6. Member States Relationship Management

Basic and applied research contributes to the foreseen implementation of mosquito SIT through collaborative research.

Research consortium of selected agreement and contract holders facilitated and established; collaborative workplans and research activities drafted and undertaken, respectively. Research Coordination Meetings (RCM) organised and conducted; results published and publicised internationally.

Logical succession of research outputs and integration of various aspects of activities listed under OUTPUTS 1-5 will enable successful development and implementation in selected Member States

APPENDIX 2 – AGENDA

Monday, 1 February, 2010

OPENING SESSION

09.00-09.30 Opening and presentation of the participants

09.30-10.00 J. Gilles / M. Vreysen: Administrative details and introduction

10.00-10.30 COFFEE BREAK

SESSION 1 (Marc Vreysen) 10.30-11.00 B. Yuval : Mosquito control – Lessons from fruit fly Sterile Insect Technique

11.00-11.30 H. Bossin, L. Hapairai, B. Peel, S. Dobson: Wolbachia-mediated SIT yields strategy for South Pacific filariasis vector elimination

11.30-12.00 C. Oliva: Feasibility of control of emergent vector diseases by the SIT in La Reunion Island. Sexual competitiveness of sterile males and wild males in the two mosquito species, Aedes albopictus and Anopheles arabiensis

LUNCH

SESSION 2 (Herve Bossin) 14.00-14.30 R. Bellini, A. Medici: Eco-bio-ethology of Aedes albopictus (sterile) males

and options to improve field mating performances

14.30-15.00 S. Boyer, D. Fontenille, G. Lempérière: Behaviour of Aedes albopictus population from La Reunion Island

15.00-15.30 D. D. Chadee: Copulation vigour of Aedes aegypti males in the laboratory and the field in Trinidad

15.30-16.00 S.A. Khan, H. Akter, M. Momen, M.S. Islam: Management of mosquitoes using Sterile Insect Technique (SIT)

16.00-16.30 H. Makee: Biological and reproduction studies of mosquitoes in relation to SIT program in Syria.

Tuesday, 2 February, 2010

SESSION 3 (Boaz Yuval) 08.00-08.30 J.D. Charlwood, E.V.E. Tómas, P. Salgueiro, A. Egyir-Yawson: Studies

on the ecology and behaviour of Anopheles gambiae s-l-from the village of Okyreko, Ghana, with a special emphasis on males and newly emerged females

08.30-09.00 K.R. Dabire, S. Sawadodgo, A. Diabate, H. Toe, L.C. Gouagna, F. Simard, G. Gibson: Analysis of swarming and mating characteristics of the M and S molecular forms of Anopheles gambiae in areas of sympatry, Burkina Faso (West Africa)

09.00-09.30 F. Simard, H. Maiga, K.R. Dabire, A. Diabate, L.C. Gouagna: The ecology of sugar feeding in males Anopheles gambiae from Burkina Faso.

09.30-10.00 C. Pennetier, B. Warren, R. Dabiré, I. Russell, G. Gibson: Singing on the wing as a mechanism for species recognition in the malarial mosquito Anopheles gambiae

10.00-10.30 COFFEE BREAK

10.30-11.00 C.E. Annoh, A. Egyir-Yawson, M. Osae, M.J. Donnelly: PCR-based single-strand conformation polymorphism (PCR-SSCP) analysis of male Anopheles populations

11.00-11.30 J. Krzywinski: Analysis of genes expressed in the Anopheles gambiae testis.

11.30-12.00 D. W. Rogers., F. Baldini., F. Battaglia, M. Panico, A Dell, H.R. Morris, F. Catteruccia: Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito

12.00-12.30 R. Hood. Use of stable isotopes for understanding male mosquito behavior. LUNCH

SESSION 4 (Grabriella Gibson)

14.30-15.00 A-K. Borg-Karlson: Male and female volatiles affecting mosquito swarming behaviour

15.00-15.30 LJ. Zwiebel, J. Pitts: Molecular biology and functional biochemistry of olfaction in Anopheles gambiae and Aedes aegypti

15.30-16.00 R.G. Armas: Behavioural responses of males of Aedes aegypti (Diptera: Culicidae) as determined by an olfactometer

16.00-16.30 P.I. Howell. The role of the male Anopheles mosquito in mating. 16.30-17.00 M. Hassan. Studies on mating behavior of male Anopheles arabiensis Patton

(Diptera: Culicidae) in Dongola, northern Sudan 19h30 Diner Salm Brau (Traditional Austrian food)

Please indicate to Jeremie if you want to come in order to book it in advance Wednesday, 3 February, 2010

09.00-10.30 General discussions and composition of the working groups

10.00-10.30 COFFEE BREAK

10.30-12.00 Working group discussions and drafting of report

LUNCH

14.00-15.30 Working group discussions and drafting of report

COFFEE BREAK

16.00-17.00 Working group discussions and drafting of report

Thursday, 4 February, 2010

09.00-10.00 Working group discussions and drafting of report

10.00-10.30 COFFEE BREAK

10.30-12.00 Working group discussions and drafting of report

LUNCH

14.00-15.30 Working group discussions and drafting of report

COFFEE BREAK

16.00-17.00 Working group discussions and drafting of report

Friday, 5 February, 2010 RECOMMENDATIONS AND FINAL DOCUMENT

09.00-10.30 Working group discussions and drafting of report

COFFEE BREAK

11.00-12.00 Presentation of the working groups

LUNCH

13.30-14.30 Presentation of the working groups

COFFEE BREAK

15.00-17.00 Final session and review of the RCM Report Planning activities for the next RCM

APPENDIX 3 - PARTICIPANT ABSTRACTS

Eco-bio-etology of Aedes albopictus (sterile) males and options

to improve field mating performances

Romeo Bellini, Anna Medici Centro Agricoltura Ambiente “G.Nicoli”

In studies conducted in Italy by our group during past years, it has been possible to prove that

Aedes albopictus radiated sterile males are able to mate with wild females in the field inducing a significant reduction in the local population fertility and density levels. In competitiveness studies in green houses sterile males showed to have a competitiveness ratio in the range 0.65-0.75 against both wild and reared normal males for virgin wild and reared females. In mark-recapture studies conducted in urban Italian condition the dispersal and longevity of reared normal males were investigated using two mark/recapture methods: 1.Wolbachia free strain; 2.fluorescent pigments. The experiment output showed that males released as pupae (Wolbachia free strain) or adults (fluorescent pigments) were able to disperse an average of 130-180 m from the release point, and their longevity was strongly dependent from the climatic condition (RH in particular). The mating behaviour (participating in swarms and flying on human host) of reared males were found to be normal. Released males suffer high mortalities in the harsh summer condition, if they don’t find sugar source in the 24-48 h post-eclosion they die. During the summer, in the urban environment, the possibility to find energetic source are scarce, so it is crucial to develop a device to be included in the release station to supply helpful substances to the newly emerged males. The device under development to supply substances to the males in the release station is designed to keep the solution available to the males for 24-48 h, to allow easy escape of the males from the station without causing stress, to protect them from possible predators (e.g. birds, ants).

PCR-based Single-strand Conformation Polymorphism (PCR-SSCP) Analysis of Male Anopheles Populations

Charles E. Annoh, Alexander Egyir-Yawson, Michael Osae, Martin J. Donnelly Biotechnology & Nuclear Agriculture Research Institute

Anopheles larvae were collected using standard methods from 8 different breeding sites in the

Greater Accra Region separated by at least 5 Km distance. The Greater Accra Region is characterized by coastal savannah vegetation type. DNA was extracted from both male and female individuals per breeding site. A total of 250 individual male and 265 female mosquitoes have been processed for DNA from all of these sites. These DNA have been genotyped to the species and molecular form levels. With the exception of two sites, both molecular forms of Anopheles gambiae are represented in all the sites and more molecular hybrids were recorded in male populations from these sites than in females. Female DNA amplification and analysis has been included for comparison. Optimization of PCR conditions for amplification of the four Y-chromosome markers is on-going. These markers will be used to amplify male DNA. Primers for female DNA amplification and subsequent PAGE analysis are being designed in conjunction with the Liverpool School of Tropical Medicine.

Behavioural responses of males of Aedes aegypti (Diptera: Culicidae) as determined by an olfactometer

René Gato Armas

Institute of Tropical Medicine Pedro Kourí. Havana, Cuba.

A three way olfactometer was assembled with cardboard panels and PVC. A central triangle shape chamber connected with three extreme boxes (for attractants) by mean of pipes of 50 cm of large. A release device with remote controlled door was settled in the bottom side of the central chamber. Collection traps by mean of a transparent funnel settled on the distal extreme of each pipe. A slow air flow was obtained from the three extreme boxes to the outside of the room, passing through central chamber. Experiments were conducted with Aedes aegypti males, at higher activity horary (crepuscular). Sucrose, Guinea pigs and Ae. aegypti females were used as attractants. Most of the mosquitoes in all experiments left the central chamber (mean between 89 - 90.89%) apparently attracted by stimulus. When the males mosquitoes were prior fed with sucrose they preferred look for an animal. In the traps near Guinea pig were found 77.33%. No difference was obtained between sucrose source and females as stimulus. However, in the experiment with non-fed-males was recorded statistically difference for three groups (ANOVA; Tukey; F=85.14; P < 0.05). Females apparently were no stimulus for males neither in this assay (2.22%). But the 34.4% of males were attracted for sucrose and the 49.22% for animals. Our experiments confirm that Ae. aegypti males uses the attractive of host for blood feeding as a way for localize de females. But interestingly, a lot of non-fed-individuals preferred animals than sucrose source. Ae. aegypti has a high degree of domestication and probably they find feeds from human sources. The olfactometer showed high degree of reproducibility in the results.

Singing on the wing as a mechanism for species recognition in the malarial mosquito Anopheles gambiae

C. Pennetier, B. Warren, R. Dabiré, I. Russell, G. Gibson

Natural Resources Institute, Univ of Sussex, IRSS (Burkina Faso)

The complexity of malaria epidemiology and control in sub-Sahara Africa is due, in part, to remarkable genetic structure of Anopheles gambiae, which facilitates rapid ecological speciation, enabling colonisation of an ever widening range of human-influenced habitats. Although reproductive isolation is essential for speciation, virtually nothing is known about how it occurs in sympatric populations of incipient species. We show that in a particular population of the ‘M’ and ‘S’ molecular forms in Burkina Faso a novel mechanism of sexual recognition (male-female frequency-matching), also confers the capability of mate recognition, an essential precursor to assortative mating. We found that frequency-matching occurs more consistently in same-form pairs than in mixed-form pairs (P > 0.001), and that the key to frequency-matching is ‘difference tones’ produced in the nonlinear vibrations of the antenna by the combined flight-tones of a pair of mosquitoes, and detected by the auditory Johnston’s organ. Through altering their wing-beat frequencies to minimise these difference tones, mosquitoes can match flight-tone harmonic-frequencies above their auditory range. This is the first description of close-range mating interactions between males and females of incipient An. gambiae species.

Studies on the ecology and behaviour of Anopheles gambiae s-l-from the village of Okyreko, Ghana, with a special emphasis on males and newly

emerged females

J.D. Charlwood, E.V.E. Tómas, P. Salgueiro, A, Egyir-Yawson We will report on the results of a three week field trip to the village of Okyreko, Ghana.

The village lies in an irrigation scheme and both M & S form An. gambiae are common there. Unfed female mosquitoes were collected in light and tent-traps and samples dissected to

determine gonotrophic condition and mated status. An. gambiae s.l. were common in the village. The numbers in the tent-trap increased from 130 to 985 and in the light-trap from 105 to 967, 1315 and 795 of which were dissected. Parous rates were always extremely low (0.31 and 0.33 for tent and light-trap respectively if the total is used or, the more respectable 0.73 for both traps when first feeding insects are excluded from the analysis). Such a result implies that the insects go through a pre-gravid phase following emergence. Although swarms of males, each circa 200+ males, were common over prominent objects within the environs of the village and although recently mated females were common in both tent and light-traps, mating was rarely seen. Swarms were not observed in the rice fields, the predominant breeding site. The emerging population of females following an initial decline (following the maturation of the rice in a very productive site) increased. Despite the probable location of the principal source of mosquitoes (fields in which the rice is young) changing relative to the traps during the study, the proportion of newly emerged females being virgin or with mating plugs in the traps remained more or less constant During the study the proportion of parous females collected the night following oviposition appeared to increase. Either dissector bias or a change in relative distance from the oviposition site may be responsible for this. With the exception of one night both tent and light-trap collected similar numbers and, with the exception of nulliparous females with ovaries at Christophers’ Stage II, similar proportions of different aged females. On this night more young first-feeding females were collected inside and many fewer than expected outside. The night in question coincided with the start of the rains when 144mm of rain were recorded 7km from the village. The possible effect of enhanced feeding indoors by young mosquitoes coincident with enhanced availability of potential oviposition sites will be discussed as will the utility of the Furvela tent-trap for mosquito surveys of this sort and the expected interpretation value of specific form identifications.

Analyse of swarming and mating characteristics of the M and S

molecular forms of Anopheles gambiae in areas of sympatry, Burkina Faso (West Africa)

K.R. DABIRE1, S. SAWADODGO1, A. DIABATE1, H. TOE1, GOUAGNA L.C.2, F.

SIMARD3, G. GIBSON4 1Institut de Recherche en Sciences de la Santé (IRSS)/Centre Muraz, BP 390, Bobo-

Dioulasso, Burkina Faso 2LIN/UR016/IRD, B.P. 64501, 34394 Montpellier Cedex 5, France

3IRD/Institut de Recherche en Sciences de la Santé, BP 545 Bobo-Dioulasso, Burkina Faso 4Natural Resources Institute /University of Greenwich, Central Avenue Chatham Maritime

Kent, ME44TB, UK The swarming and mating system in natural populations of Anopheles gambiae M and S

molecular forms were investigated across longitudinal surveys from July 2006 to October 2009 in Soumousso and Vallée du Kou (VK7), two areas of western Burkina Faso where these forms are sympatric. In Soumousso, a wooded savannah, overall 205 swarms and 109 pairs caught in copula were sampled whereas in VK7 more than 250 swarms with 491 couples were sampled and effectively PCR analysed. In both sites no spatial segregation has been observed between the swarming places of M and S forms which shared the same type of visuals markers. Even though some mixed swarms were annually collected in varying frequencies from one site to other, the major swarming behaviour was that the two forms swarm separately in more than 90% of cases. Analysing the mating feature within the swarms, although collected within mixed swarms the frequencies of pure form pairings was higher and the mixed ones did not reach up 4%. The insemination rate estimated within natural swarming populations of M form averaged 68% without any mixed insemination corroborating the existence of low hybrids rate in the field. The relation between environmental factors and swarming behaviour showed that some parameters such temperature and relative humidity were not associated to the swarming formation. But the time point of swarms’ apparition seemed to be fairly conserved in An. gambiae s.s. natural populations and did not vary significantly between the two forms. However the swarms of the S form (Soumousso) appeared more precociously than those of the M form (VK7). They followed the seasonal fluctuation of photoperiodicity suggesting that the apparition of swarms was tightly regulated by circadian rhythms and/or biological horologe. The mechanisms regulating the recognition between inter or intra forms partners during the swarming and mating process are discussed.

Key-words: Anopheles gambiae s.s., mating, mixed-swarm, molecular S and M forms, Burkina

Faso.

Feasibility of control of emergent vector diseases by the Sterile Insect

Technique in La Reunion Island. Sexual competitiveness of sterile males and wild males in the two mosquito species, Aedes albopictus and Anopheles arabiensis

Clelia OLIVA

IRD / CRVOI / FAO-IAEA / University of Reunion Island, France This PhD work is in support of the mosquito Sterile Insect Technique (SIT) project recently

started on Reunion Island by the Institut pour la Recherche et le Dévelopment and the Centre de Recherche et de Veille sur les Maladies Emergentes dans l'Océan Indien (IRD-CRVOI), with the technical assistance of the FAO/IAEA. This project is designed to study the feasibility of implementating the SIT on Reunion Island targeting the mosquito species Aedes albopictus, vector of Chikungunya and Dengue, and Anopheles arabiensis vector of the malaria parasite Plasmodium ssp.

Present knowledge of the effect of irradiation on the sexual development, the competitiveness and the survival of males’ mosquito is limited. The goal of this PhD is to develop the most appropriate procedure for a mass production of sterile males with emphasis on limiting the negative effects of irradiation. The study has three main objectives. (i) the first objective deals with developing an optimal technique for irradiating An. arabiensis and Ae. Albopictus. All the crucial points of the sterilisation steps will be characterized (sterilizing dose, irradiation protocol, radioprotective treatments effect and recovery of fertility) and the parameters favouring/limiting the competitiveness of males will be determined. (ii) for the second objective, the mechanism of action of the irradiation will be studied in an attempt to find possible indicators of induced somatic damages, which are thought to be responsible of a loss of competitiveness in irradiated males. The usual competitiveness tests are time-consuming and it is desirable to find an alternative method that could reflect precisely the impact of an irradiation and the protective effect brought by a radioprotectant treatment. In this view, groups of bad and good competitors will be compared for some physiological parameters and metabolic indicators (protein or enzyme analysis, C/N ratio, fatty acids analysis, etc.). (iii) for the last objective, some life history traits of irradiated and wild males An. arabiensis and Ae. albopictus will be studied in semi-field conditions. Prior to a release of sterile males on Reunion Island, we will have to assess their ability to compete with wild males in semi-field conditions and to assess which parameters have to be improved. This will encompass studies on longevity, flight capacity, sexual activity period, frequency of mating, capacity of inseminating females and competitiveness.

Radioprotection studies have been conducted and suggest that competitiveness of fully sterile males can be improved by adding chemical substances (bee venom, chitosan or Nordihydroguaiaretic acid) to the larval diet. Longevity studies showed that high doses of irradiation induced a slight decrease in lifespan; however there was no significant difference for the first 10 days between fully sterilized and non irradiated males. Males from of our laboratory strain An. arabiensis Dongola were able to mate less than 24 hrs after emergence, suggesting that the rotation of terminalia would occur earlier than in wild males. These first results will be presented during the RCM.

Analysis of genes expressed in the Anopheles gambiae testis

Jaroslaw Krzywinski

Liverpool School of Tropical

Processes associated with testis and spermatozoa development are virtually unexplored in mosquitoes. Yet, because of their high complexity and numerous molecular interactions involved, they may offer many targets for novel anti-vector interventions. To gain insights into these developmental pathways we sought to identify genes transcribed exclusively in testes of the Anopheles gambiae pupae. Using a cDNA subtraction strategy, we isolated five genes with male-specific or highly male-biased expression. Spatial and temporal expression patterns suggest that each of these genes is involved in the mid-late stages of spermatogenesis. Their sequences are rapidly evolving; however, two genes possess clear homologs in a wide range of taxa and one of these probably acts in a sperm motility control mechanism conserved in many organisms. The other three genes have no match to sequences from non-mosquito taxa, thus can be regarded as orphans. RNA in situ hybridization demonstrated that one of the orphans is transcribed in spermatids, which suggests its involvement in sperm maturation. The role of two other orphans cannot be deduced from properties of their sequences or their expression profiles. Discovery of testis-expressed orphans in mosquitoes opens exciting new prospects for the development of innovative control methods. The orphan encoded proteins may represent unique targets of selective anti-mosquito sterilizing agents that will not affect non-target organisms. An ongoing research focuses on functional analysis of the orphans to provide information regarding their suitability as targets.

The ecology of sugar feeding in males Anopheles gambiae from Burkina Faso.

SIMARD F, MAIGA H, DABIRE KR, DIABATE A, GOUAGNA LC

IRD / UR016 (France), IRSS/DRO (Burkina Faso) Advances in our understanding of male mosquito ecology are needed to maximize the

likelihood of success of vector control interventions based on population suppression (e.g., Sterile Insect Technique) or replacement (e.g., through the use of genetically modified mosquitoes) that require large scale releases of mass reared male mosquitoes. The focus of IAEA Research contract 14777 “Assessing the ecology of interactions between nutritional resources and adult males of African malaria vectors” is on male foraging and feeding behavior. Resources acquisition and use are major determinants of individual fitness in the wild, and a better knowledge of the behavioral and physiological components of food intake and resource utilization by male African malaria vectors should provide relevant insights towards improvement of lab-reared mosquito fitness and competitive ability upon release under natural conditions. Sugar is the primary source of energy for emerging mosquitoes of both sexes, and sugar is the only resource upon which males feed during their whole life. Our recent results in Burkina Faso suggested that males An. gambiae are selective when a large variety of flowers are available and that different blossoms might differentially attract distinct species/populations of mosquitoes. Based on these findings, we devised a series of experimental tests to explore the ultimate consequences of these feeding preferences on mosquito fitness. We will provide an update of the results obtained so far and on ongoing research activities that are being conducted at IRSS, Burkina Faso towards the evaluation of the fitness gain obtained by males exposed to different sugar feeding regimens.

Keywords: Anopheles gambiae, male, plant sugar, foraging, feeding behavior, fitness.

Behaviour of Aedes albopictus, population from La Reunion Island

Sébastien Boyer, Didier Fontenille, Guy Lempérière Institut de Recherche pour le Développement (IRD) Research Unit 16 "Biology and Control of Vectors"

Aedes albopictus is present worldwide, from Asia to Europe, the Indian Ocean region, South

Africa and North America. This mosquito is the major vector of dengue and also the major vector of Chikungunya virus outbreak which occurred in 2005, 2006 and 2009. It is also the vector of other diseases. Its rapid spread in Europe is a major concern. It has been recorded in Italy 10 years ago, and after invading the whole of Italy, Ae. albopictus is now in most of the northern mediterranean countries from Italy to Spain. It has been observed in the French Alps where it has established a colony in 2009. It is able to invade most of Europe despite vector control measures. The use of insecticides has no effect on its establishment in Europe. A research project assessing the feasibility of a sterile insect technique as an alternative vector control measure is under progress at La Reunion Island. This presentation includes the first results on the biology of this invading species, and focuses on the fecundity of males and their ability to disperse.

Their mating ability has been tested with several batches of females under laboratory conditions. Tests were conducted in large and small cages with 2, 10 and 20 females. Females were used during all the experiment or removed every 24h. All females were desiccated in order to observe their spermathecal capsules and assess whether they were mated or not. In regard to the different methods used during this experiment, results showed that Ae. albopictus male was able to inseminate an average of 9-10 females with a maximum of 16, with a constant mating rate during the first 7 days. A decrease of their mating ability was then observed until day 10. After this date, males did not mate females.

In the SIT program, before any hypothetic release, flight behaviour, dispersal of released mosquitoes and population of wild mosquitoes must be estimated. The second part of the study focused on the dispersal of males and females Aedes albopictus in a semi-urban area which is the most common case on the island. The size of the wild population of Ae. albopictus was also estimated during the different seasons. Mice-baited BG-sentinel traps were used to catch mosquitoes in mark-release-recapture experiments carried out every 3 months (March, June, September and December 2009). An overall 5% of released males were recaptured (15% for the females). The dispersal behaviour and the distribution of Aedes albopictus showed high disparities. Wild population was estimated between 60 000 and 25 000 on the same released site (40 000m²), depending on the season. Females seemed to disperse more than males but when males were released, they covered more distance. The relationship with their physical environment is also discussed.

In the same time, a complementary work on the chemically mediated behaviour of both males and females of Aedes albopictus and Anopheles arabiensis is currently carried out together with colleagues of the Department of Chemistry, University of la Réunion in order to identify volatiles emitted by the insects.

Keywords. Aedes albopictus; Chikungunya; TIS; Fecundity; Dispersion

Biological and reproduction studies of Mosquitoes

in relation to SIT program in Syria

Hayat Makee Atomic Energy Commission, Damascus, Syria

A survey was conducted in two regions: coastal areas and Gape regions during 2008 2009 to

collect mosquito larvae and adults. A number of female mosquitoes and larvae were captured and examined. A complex of mosquito species were detected including Culex and Anopheles species. One of the vectors of malaria, Anopheles sacharovi, was collected from several sites.

Mosquitos larvae were reared in trays containing ± 2 liter of tap water (water depth 2 cm). Larvae were fed a diet of fish-food. Emerged adults were maintained in the mating cage at room temperature 28°C and the light regime was L12:D12 with a one hour simulated dusk and dawn period. Adult cages will be supplied with 10% sucrose solution.

After 3 days of adults' emergence the females were fed on blood to lay eggs. Fresh blood (from goats and human) was collected with a syringe and put in a sterile 15 ml Falcon tube containing 1ml of CPD (anticoagulant) for each 10 ml of blood, and was then mixed.

The blood was added to the metallic plate feeder which then covered by parafilm as an artificial membrane. The plate was put on the upper side of mating cage and warm water-jacket (37oC) placed over the plate feeder. By this way the mosquitoes had an access to the membrane surface. The mosquitoes were allowed to feed for ~30 min.

Management of Mosquitoes using Sterile Insect Technique (SIT)

Shakil A. Khan, Humayra Akter, Mahfuza Momen1 and M. Saidul Islam Insect Biotechnology Division, Institute of Food and Radiation Biology

Bangladesh Atomic Energy Commission, Dhaka, Bangladesh 1 Department of Zoology, University of Dhaka, Bangladesh

Rearing protocols for mass production of the mosquito Aedes aegypti was optimized. Larval diets,

larval density and adult food supplement were assessed for developmental period, pupal recovery, adult body size and male longevity. Three larval diets viz., biscuit, fish feed and poultry feed were used to mass rear the larvae of Ae. aegypti. Among the diets tested fish feed was found to be the most suitable ones for larval rearing of Ae. aegypti. Developmental period from larva to adult was shorter when reared on fish feed diet. Pupal recovery were found to be similar in fish and poultry feed diet. However, pupal recovery from biscuit reared colony was low. Quality of the adult mosquitoes was assessed on their wing size of the both sexes. Diets had significant influences on wing length of both the sexes. It appeared that the wing of Ae. aegypti developed from the larvae reared on fish feed was larger in size compared to those from poultry feed and biscuit reared mosquitoes. Larval density had significant influence on the wing size. A gradual decrease of wing size was observed with increasing larval density/tray. Fecundity and hatching were recorded. Pupal separation on the basis of their size was attempted. Preliminary results on the radiosensitivity on pupae and adults of Ae. aegypti are also discussed.

Mosquito Control - Lessons From Fruit Fly Sit

Boaz Yuval Department of Entomology, Hebrew University of Jerusalem

Transgenic mosquitoes, showing varying levels of refractoriness to infection, are a reality, and

the release of such creatures in the field, whether Aedes in dengue afflicted regions or Anopheles in malarial ones, is only a matter of time. The success of this approach hinges on myriad factors- biological, legal, social, political and logistical. Ultimately, the genetically modified mosquitoes will have to be mass reared, transported to and released in the field, where they will face the ultimate test- spreading the refractory gene by mating with wild conspecifics. The Sterile Insect Technique faces similar conceptual and logistical problems. Decades ago, efforts to control mosquitoes by SIT were largely unsuccessful. However, the technique is currently employed against several species of tephritid fruit flies (Diptera: Tephritidae) in many regions of the world, with overall gratifying results.

I suggest that this success stems to a large extent from the pervading culture that has evolved in the tephritid research community. This culture fosters synergism between various disciplines, collaborating in a coordinated fashion in a manner that allows the application of advances in basic research to applied and operational goals. As a result, tephritid SIT is an industrial process with quality control and quality assurance parameters that are related not only to the design of the product, but to its performance in the field. Accordingly, operational procedures are regularly updated and improved by means of international collaborations and structured workshops in which behavioral ecologists, molecular biologists, economists and operational managers are intimately involved. In particular, basic research focused on field ecology and behavior provides insights on ways to improve the SIT. For example, studies on sexual behavior and nutritional ecology have suggested ways to improve the sexual competitiveness of mass reared sterile males by effecting relatively minor changes in the rearing and pre-release environments. Bibliometric data suggest that the research leading to transgenic mosquitoes has focused on design, and neglected performance. A behavioral-ecology perspective on mosquito mating systems will anticipate problems related to the process whereby mosquito SIT is developed, and will enable future programs based on this approach to define appropriate quality control and assurance parameters.

Molecular Biology and Functional Biochemistry of Olfaction

in Anopheles gambiae and Aedes aegypti.

LJ. Zwiebel, J. Pitts Department of Biological Sciences, Vanderbilt University - Nashville, Tennessee U.S.A The ability to sense and discriminate a large collection of chemical and other sensory cues is

central for several behaviors of insects that act as vectors for the pathogens that are responsible for many important human diseases. In particular, olfaction plays a major role in host seeking and selection behaviors of blood feeding female anopheline mosquitoes. A long-term objective of our research is centered on an examination of the molecular genetics of the chemosensory system in anopheline and other mosquitoes and its role in determining anthropophilic host preference in malaria vector mosquitoes. We have identified and continue to characterize the complete repertoire of 79 odorant receptor (OR) genes An. gambiae as well the complete set of 117 functional OR genes from the Dengue and Yellow Fever virus vector mosquito Aedes aegypti. Together with our collaborators, we have completed a detailed analysis of the olfactory physiology, molecular neurobiology and odorant-binding characteristics of ORs on the adult maxillary palp and proboscis as well as behavioral and molecular elements of the larval stage olfactory system of An. gambiae and Ae. aegypti to begin to define the functional “odor space” for these vector mosquitoes. Progress will also be presented on ongoing efforts to design novel anti-malarial programs that target chemosensory pathways and the behaviors they control in vector mosquitoes.

This work has received generous support by grants from the Foundation for the National

Institutes of Health through the Grand Challenges in Global Health Initiative as well as the U.S. National Institutes of Health.

Male and female volatiles affecting mosquito swarming behaviour

Anna-Karin Borg-Karlson

Royal Institute of Technology, Sweden

In our research for male and female volatiles affecting mosquito swarming behaviour, larva and adult Anopheles arabiensis, Dongola strain, were fed on U- 13C6 labeled glucose. Chemical analyses with SPME-GC-MS were made of the emissions from live and recently freeze killed mosquitoes from the two treatments. Different volatiles were labelled when fed on 13C-glucose as larvae compared to the adult 13C-glucose fed. Some of these compounds have recently been identified from gut microbes in Anopheles gambiae.

Wolbachia-mediated Sterile Insect Technique yields strategy for South Pacific Filariasis Vector Elimination

Hervé BOSSIN1, Limb HAPAIRAI1, Bethany PEEL2, Stephen DOBSON2

1 Institut Louis Malardé, Papeete, Tahiti, French Polynesia, 2 University of Kentucky, Lexington, Kentucky, USA

Despite its natural isolation, French Polynesia like other regions of the South Pacific is the stage

of an ongoing battle against mosquito–borne diseases such as lymphatic filariasis (LF) and dengue. The presence of highly competent mosquito species (e.g. Ae. polynesiensis and Ae. aegypti) along with favourable tropical conditions, a growing population and an increase in international trade all contribute to the maintenance or re-emergence of infectious diseases in the region.

Lymphatic filariasis, a painful and disfiguring parasitic disease, is the primary cause of disability in the Pacific region. In an attempt to curb LF transmission, mass drug administration (MDA) campaigns have been recommended and applied repetitively in French Polynesia since 1949. These campaigns have resulted in the spectacular reduction in LF prevalence (Esterre et al., 2001; Ichimori, 2001) and the decline of the disease’s most severe forms (hydrocoele and elephantiasis). In 1999, the WHO-led PacElf programme was launched with the goal to eliminate LF from the South Pacific by 2010. Involving 22 island countries and territories of the South Pacific, this programme relies on the annual distribution of a combination of drugs diethylcarbamazine (DEC or Notezine®) and albendazole (ALB or Zentel®) for a period of at least five years.

While the strategy has met with success in certain areas of the Pacific, the goal of filariasis elimination is clearly not attained in French Polynesia despite eight rounds of MDA treatment. Recent epidemiological studies sustain the idea that MDA alone will not be enough to end the LF transmission cycle (Burkot et al., 2006), at least in some Pacific countries. Supplemental control strategies are thus much needed to ensure the success of the global LF elimination campaign.

Obligate vector mosquitoes provide additional targets that can complement existing anti-filariasis approaches. Due to its ubiquitous nature, however, conventional methods have failed to successfully control Aedes polynesiensis, the primary LF vector throughout most of the South Pacific.

Such paucity in the arsenal of tools available to control Ae. polynesiensis has raised interest in the use of evolutionary genetics to fight vector-borne diseases. Several feasibility studies for the field assessment of genetic control strategies are under way at Institut Louis Malardé investigating the elimination or the replacement of naturally isolated populations of Ae. polynesiensis.

The testing of symbiont-based control strategies relies on sustained, inundative releases of incompatible males in the field. Such releases have just begun in French Polynesia to assess the efficacy of the Wolbachia-mediated mosquito suppression strategy. These releases target a geographically isolated population of Aedes polynesiensis mosquitoes on a privately owned 3,6 ha. islet off of Raiatea.

The improvement of field evaluation of released mosquitoes entails developing methods for assessing male competitiveness, dispersal and monitoring of released and targeted mosquitoes. The research objectives investigated to this date have focused on sexual behaviour, bionomics and dispersal and longevity post-release. Male mating competitiveness is one key parameter influencing the strategy success in the field. Here we are reporting on behavioural features (male mating competitiveness) characterizing the incompatible males released. Incompatible (CP) male competitiveness was assessed in a field cage trial. Cages of virgin wild type Ae. polynesiensis females are combined with differing ratios of incompatible:wild type males. The results show a decreasing number of hatching eggs, correlated with increases in the ratio of incompatible males. The results are consistent with the hypothesis that increased frequencies of incompatible males induce sterility in wild type females. This bioassay demonstrated equal CP male mating competitiveness with their wild conspecifics under semi-natural tropical conditions.

A description of the release and monitoring methods developed and progress of the ongoing release operations will also be presented.

References Burkot T.R., Durrheim D.N., et al. (2006). "The argument for integrating vector control with

multiple drug administration campaigns to ensure elimination of lymphatic filariasis." Filaria Journal 5(1): 10.

Esterre, P., C. Plichart, et al. (2001). "The impact of 34 years of massive DEC chemotherapy on Wuchereria bancrofti infection and transmission: the Maupiti cohort." Trop Med Int Health 6(3): 190-5.

Ichimori, K. (2001) Entomology of the filariasis control programme in Samoa, Aedes polynesiensis and Ae. samoanus. Medical Entomology and Zoology, 52, 11-21

Copulation vigor of Aedes aegypti males in the laboratory and the field

in Trinidad

Dave D. Chadee

University of West Indies

Transglutaminase-Mediated Semen Coagulation Controls Sperm Storage in the Malaria Mosquito

David W. Rogers1., Francesco Baldini2., Francesca Battaglia3.¤, Maria Panico3., Anne Dell3,

Howard R. Morris3, Flaminia Catteruccia1,2 1 Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom,

2 Universita degli Studi di Perugia, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Terni, Italy,

3 Division of Molecular Biosciences, Imperial College London, London, United Kingdom

Insect seminal fluid proteins are powerful modulators of many aspects of female physiology and behaviour including longevity, egg production, sperm storage, and remating. The crucial role of these proteins in reproduction makes them promising targets for developing tools aimed at reducing the population sizes of vectors of disease. In the malaria mosquito Anopheles gambiae, seminal secretions produced by the male accessory glands (MAGs) are transferred to females in the form of a coagulated mass called the mating plug. The potential of seminal fluid proteins as tools for mosquito control demands that we improve our limited understanding of the composition and function of the plug. Here, we show that the plug is a key determinant of An. gambiae reproductive success. We uncover the composition of the plug and demonstrate it is formed through the cross-linking of seminal proteins mediated by a MAG-specific transglutaminase (TGase), a mechanism remarkably similar to mammalian semen coagulation. Interfering with TGase expression in males inhibits plug formation and transfer, and prevents females from storing sperm with obvious consequences for fertility. Moreover, we show that the MAG-specific TGase is restricted to the anopheline lineage, where it functions to promote sperm storage rather than as a mechanical barrier to re-insemination. Taken together, these data represent a major advance in our understanding of the factors shaping Anopheles reproductive biology.

Studies on mating behavior of male Anopheles arabiensis Patton (Diptera: Culicidae) in Dongola, northern Sudan

Mo’awia M Hassan, Ph. D.,

Tropical Medicine Research Institute, National Centre for Research,

The failure of the conventional mosquito control methods leads to develop an alternative method such as the Sterile Insect Technique (SIT) to suppress populations of the malaria vector Anopheles arabiensis in Northern State, Sudan. This method entails the release of large numbers of irradiated males to compete against wild conspecific for mating with virgin females in the field. In this study, attempt was made to investigate the swarming behaviour of An. arabiensis. Two surveys were conducted during March and June 2009 in Dongola area, northern Sudan, where project of SIT will take place. Observations on swarming behaviour were made both in the field sites and a contained semi-field condition. In the field, three swarming stations were identified nearby Maraga (2 stations) and Kabotood (one station) villages (10-20 m from the villages). The same observations were recorded from a contained semi-field condition. The males An. arabiensis was observed to start swarming at 19:35 evening in the stations near to Maraga village and + 1 min at station near Kabtood village. The maximum number of swarming males was at 19:45 at all station. However, swarms occurred in a place where donkeys and cow stay. The swarms occurred at 2.5–3 m above the ground level 8-25 m from a breeding site. In the contained semi-field system male swarm was observed to start at 19:38 and end at 20:05. Coupling pairs were observed both in field an in a contained semi-system field and the field sites. Twenty males identified as An. arabiensis were collected from one swarm from the field. Three females were collected the contained semi-field system. None of the females were found to be inseminated. The findings obtained enhance the planning to select the future release point of SIT programme in Northern State-Sudan.

Use of stable isotopes for understanding male mosquito behavior.

Rebecca Hood-Nowotny Ph.D, MBA Department of Chemical Ecology and Ecosystem Research

Faculty of Life Sciences, University of Vienna Stable isotope labeling of mosquitoes is a marking technique which offers the opportunity to

trace the source, distribution and movement of vector populations across the landscape. It also allows estimates of vector population size and enables determination of which males, wild, sterile or genetically modified, are inseminating the native female population. All factors which are critically important in any vector control program, but are essential in implementing the sterile insect technique or genetic based eradication strategies. Many current malaria control projects reiterate that for optimal use of intervention and resources information on the density and movement of the mosquito population is required. Stable isotope tools can provide this essential information unambiguously.

Stable isotopes are safe, non-radioactive and omnipresent in the environment. Insect populations can be intrinsically labeled with compounds enriched with stable isotopes either in-situ in the field or in a mass rearing setting with no detrimental environmental effects. The labeling does not affect the biology or ecology of the insect in anyway and making it an ideal marker especially in small insects such as mosquitoes where other markers may have such a profound influence on their behavior and survival.

Stable isotope tools offer a cataclysmic leap in vector-ecology methodology but until now their applications have been constrained by the access to expensive, complex, laboratory-based isotope ratio mass spectrometers (IRMS) for high precision isotope measurement, which are generally only fully operational in developing countries. Robust field-deployable laser-based instruments for high-precision isotope ratio measurement have recently become commercially available, which require minimal technical training and infra-structural support.

These instruments present new opportunities and expand the scope and applicability of the isotopic methods in terms of vector control, these new opportunities and issues will be presented.

Appendix 4 RCM report male biology.doc

APPENDIX 4 - LIST OF PARTICIPANTS

BANGLADESH Mr. Islam Saidul Bangladesh Atomic Energy Commission (BAEC); Atomic Energy Research Establishment (AERE); Institute of Food and Radiation Biology Ganakbari, Savar P.O. Box 3787 Dhaka 1344 Phone: 880-2-7701172 Fax: 880-2-8130102 Email: ibd@aitlbd.net BURKINA FASO Mr. Roch K. Dabire Centre Muraz Organisation de coopération et de coordination pour la lutte contre les grandes endémies 01 B.P. 153 Bobo-Dioulasso Phone: 226-20-97-01-02 Fax: 226-20-97-04-57 Email: dabire_roch@hotmail.com Mr Frederic Simard Institut de recherche en sciences de la santé Délégation régionale de l’Ouest-Bobo-Dioulasso 399 Avenue de la Liberté B.P. 545 Phone : 226 2097 1269 Fax : 226 2097 0942 or 4868 Email : frederic.simard@ird.fr CUBA Mr. Rene Gato Armas Instituto de Medicina Tropical "Pedro Kourí" Mariano 13 Apartado 601 La Habana Phone:53-7-2020650 Fax: 53-7-2046051 Email: rene@ipk.sld.cu

DENMARK Mr. Jacques Charlwood DBL Institute for Health Research and Development 1 D Jaergersborg Alle 2920 Charlottenlund Phone: 0045-77327732 Fax: 0045-77327733 Email: jdcharlwood@gmail.com FRANCE Mr Guy Lemperiere Institut de Recherche pour le Dévelopment (IRD) Plateforme Technologique du Centre de Recherche et de Veille sur les maladies Emergentes dans l’Océan Indien (CRVOI) 2, rue Maxime Riviere 97 490 Sainte Clotilde Ile de la Réunion Phone : 00262 938821 Fax : 00262 21 66 71 Email : guy.lemperiere@ird.fr FRENCH POLYNESIA Mr. Herve C. Bossin Institut Louis Malarde (ILM); Laboratoire de recherche en entomologie médicale rue des Poilus Tahitiens B.P. 30 Papeete, Tahiti Phone: 689-53-31-56 Fax: 689-53-28-73 Email: hbossin-ilm@mail.pf GHANA Mr. Alexander Egyir-Yawson Ghana Atomic Energy Commission (GAEC); Biotechnology and Nuclear Agriculture Research Institute P.O. Box 80 Legon Accra Phone: 233-21-40303 Fax: 233-21-402286/400807 Email: egyiryawson@hotmail.com

Mobile: 233-277-839821

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ITALY Mr Romeo Bellini Centro Agricoltura Ambiente « G. Nicoli » Via Argini Nord 3351 40014 Crevalcore Phone : 0039 051 6802211 Email : rbellini@caa.it SUDAN Mr. Mo'awia Mukhtar Hassan Epidemiology Department, Tropical Medicine Research Institute, Khartoum, P. O. Box 1304, Phone: 249-11-779 246 Fax: 249-11-781 845 Email: moawia65@hotmail.com SWEDEN Ms. Anna-Karin Borg-Karlson Royal Institute of Technology (KTH); Department of Chemistry 100 44 Stockholm Phone: 46 8 790 8449 Email: akbk@kth.se SYRIA Ms. Hayat Makee Atomic Energy Commission of Syria (AECS) P.O. Box 6091 Damascus Phone: 46 8 790 8449 Email: atomic@aec.org.sy hmakee@aec.org.sy TRINIDAD AND TOBAGO Mr Dave Chadee University of the West Indies St. Augustine Phone: 1 8686622002 Fax: 1 868663 5241 Email: dave.chadee@sta.uwi.edu UNITED KINGDOM Mr. Jaroslaw Krzywinski Liverpool School of Tropical Medicine Pembroke Place Liverpool, L3 5QA Phone: (+44) 151 705 3155 Fax: (+44) 151 705 3369 Email: jarek@liv.ac.uk J.Krzywinski@liverpool.ac.uk

Ms. Gabriella Gibson University of Greenwich Chatham, Kent ME4 4TB Phone: 44-1634-883171 Fax: 44-1634-883379 Email: g.gibson@gre.ac.uk UNITED STATES OF AMERICA Mr. Jason Pitts Vanderbilt University P.O. Box 1596 Nashville, TN 37235 Phone: 1-615-343-1894 Fax: 1-615-936-0129 Email: j.pitts@vanderbilt.edu CONSULTANTS ISRAEL Mr Boaz Yuval Dept. of Entomology Hebrew University of Jerusalem P.O. Box 12 Rehovot 76100 Phone: 972 2 6586638 Fax: 972 2 5664740 Email: yuval@agri.huji.ac.il OBSERVERS FRANCE Mr. Louis Clement Gouagna IRD UR016 911, av. Agropolis, BP 64501 34394 Montpellier Cedex 5 Phone: +33(0)467416331 Fax: +33(0)467416330 Email: Louis-Clement.Gouagna@ird.fr UNITED KINGDOM Mr David Rogers MRC Career Development Fellow Division of Cell and Molecular Biology Imperial College London, SAF Building Imperial College Road, London SW7 2AZ Phone: +44 20 75895111 ext 55409 Email: d.rogers@imperial.ac.uk

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UNITED STATES OF AMERICA Mr Paul Howell Centers for Disease Control and Prevention 4770 Buford Highway NE MS F42, Atlanta, GA 30341 Phone: 770 488 3593 Email: bsr7@CDC.GOW AUSTRIA Ms. Rebecca Hood-Nowotny Department of Chemical Ecology and Ecosystem Research Faculty of Life Sciences University of Vienna Althanstrasse 14, 1090 Vienna, Austria Phone: +43 699 1 8622506 Email: rebecca.clare.hood-nowotny@univie.ac.at