Review: Improving our knowledge of male mosquito biology in relation to genetic control programmes.

The enormous burden placed on populations worldwide by mosquito-borne diseases, most notably malaria and dengue, is currently being tackled by the use of insecticides sprayed in residences or applied to bednets, and in the case of dengue vectors through reduction of larval breeding sites or larviciding with insecticides thereof. However, these methods are under threat from, amongst other issues, the development of insecticide resistance and the practical difficulty of maintaining long-term community-wide efforts. The sterile insect technique (SIT), whose success hinges on having a good understanding of the biology and behaviour of the male mosquito, is an additional weapon in the limited arsenal against mosquito vectors. The successful production and release of sterile males, which is the mechanism of population suppression by SIT, relies on the release of mass-reared sterile males able to confer sterility in the target population by mating with wild females. A five year Joint FAO/IAEA Coordinated Research Project brought together researchers from around the world to investigate the pre-mating conditions of male mosquitoes (physiology and behaviour, resource acquisition and allocation, and dispersal), the mosquito mating systems and the contribution of molecular or chemical approaches to the understanding of male mosquito mating behaviour. A summary of the existing knowledge and the main novel findings of this group is reviewed here, and further presented in the reviews and research articles that form this Acta Tropica special issue.

The roles of kairomones, synomones and pheromones in the chemically-mediated behaviour of male mosquitoes.

Despite decades of intensive study of the chemical ecology of female mosquitoes, relatively little is known about the chemical ecology of males. This short review summarizes the current state of knowledge of the chemicals that mediate male mosquito behaviour. Various trophic interactions including insect-plant, insect-host, and insect-insect responses are emphasized. The relevance of the chemical ecology of male mosquitoes in the context of vector control programmes is discussed.

Reproductive strategies of Aedes albopictus (Diptera: Culicidae) and implications for the sterile insect technique.

Male insects are expected to optimize their reproductive strategy according to the availability of sperm or other ejaculatory materials, and to the availability and reproductive status of females. Here, we investigated the reproductive strategy and sperm management of male and virgin female Aedes albopictus, a mosquito vector of chikungunya and dengue viruses. The dynamics of semen transfer to the female bursa inseminalis and spermathecae were observed. Double-mating experiments were conducted to study the effect of time lapsed or an oviposition event between two copulations on the likelihood of a female double-insemination and the use of sperm for egg fertilization; untreated fertile males and radio-sterilised males were used for this purpose. Multiple inseminations and therefore the possibility of sperm competition were limited to matings closely spaced in time. When two males consecutively mated the same female within a 40 min interval, in ca. 15% of the cases did both males sire progeny. When the intervals between the copulations were longer, all progeny over several gonotrophic cycles were offspring of the first male. The mating behavior of males was examined during a rapid sequence of copulations. Male Ae. albopictus were parceling sperm allocation over several matings; however they would also attempt to copulate with females irrespective of the available sperm supply or accessory gland secretion material. During each mating, they transferred large quantities of sperm that was not stored for egg fertilization, and they attempted to copulate with mated females with a low probability of transferring their genes to the next generation. The outcomes of this study provided in addition some essential insights with respect to the sterile insect technique (SIT) as a vector control method.

Effects of irradiation, presence of females, and sugar supply on the longevity of sterile males Aedes albopictus (Skuse) under semi-field conditions on Reunion Island.

BACKGROUND: The development of the sterile insect technique (SIT) for reducing populations of Aedes albopictus (Skuse), (the vector of Chikungunya and Dengue fever), was studied in Reunion Island. For some mosquito species the sterilization process and mating activity may alter male survival. Most previous studies were carried out in the laboratory and may inadequately reflect the field situation. We conducted a semi-field experiment to evaluate the impact of sugar supply and mating activity under natural climatic conditions on wild and sterile male Ae. albopictus longevity, using large cages set up in an open clearing between trees and shrubs in Reunion Island. RESULTS: Wild males had a mean longevity of 15.5 days in the absence of females and with an immediate sugar supply; longevity in sterile males was similar. The presence of females greatly reduced both wild and especially sterile male lifespan; however, an immediate sugar supply could counteract this effect and allow sterile males to live an average of 11.6 days. CONCLUSION: The outcomes indicate that sugar feeding could compensate for sterilization-induced damage, and that mating activity is not deleterious for well-fed males. This study stresses the critical importance of providing suitable sugar sources prior to release during SIT programmes.

The sterile insect technique for controlling populations of Aedes albopictus (Diptera: Culicidae) on Reunion Island: mating vigour of sterilized males.

Reunion Island suffers from high densities of the chikungunya and dengue vector Aedes albopictus. The sterile insect technique (SIT) offers a promising strategy for mosquito-borne diseases prevention and control. For such a strategy to be effective, sterile males need to be competitive enough to fulfil their intended function by reducing wild mosquito populations in natura. We studied the effect of irradiation on sexual maturation and mating success of males, and compared the sexual competitiveness of sterile versus wild males in the presence of wild females in semi-field conditions. For all untreated or sterile males, sexual maturation was completed within 13 to 20 h post-emergence and some males were able to inseminate females when 15 h old. In the absence of competition, untreated and sterile males were able to inseminate the same number of virgin females during 48 h, in small laboratory cages: an average of 93% of females was inseminated no matter the treatment, the age of males, and the sex ratio. Daily mating success of single sterile males followed the same pattern as for untreated ones, although they inseminated significantly fewer females after the ninth day. The competitiveness index of sterile males in semi-field conditions was only 0.14 when they were released at 1-day old, but improved to 0.53 when the release occurred after a 5-day period in laboratory conditions. In SIT simulation experiments, a 5:1 sterile to wild male ratio allowed a two-fold reduction of the wild population's fertility. This suggests that sterile males could be sufficiently competitive to mate with wild females within the framework of an SIT component as part of an AW-IPM programme for suppressing a wild population of Ae. albopictus in Reunion Island. It will be of interest to minimise the pre-release period in controlled conditions to ensure a good competitiveness without increasing mass rearing costs.

Evidence of multiple inseminations in the field in Aedes albopictus.

Studies on the biology and mating behaviour of male mosquitoes are of major importance in a frame of a Sterile Insect Technique which could be used against mosquito vector species. Most particularly, the assumption of possible multiple inseminations in mosquito species must be investigated in order to optimize alternative mosquito control methods (Sterile Insect Techniques with genetically modified mosquitoes, cytoplasmic incompatibility, radiation…). The occurrence of multiple insemination events was investigated after 2 field samplings of Aedes albopictus (Diptera: Culicidae) in La Reunion Island using microsatellite markers. Respectively, 14 and 13 females after the first and the second sampling laid eggs. Seven wild females out of the 27 laying females were found with a progeny involving more than one father. This result is important for the new alternative mosquito control methods and raises the importance of pre- and post-copulatory competition.

Abiotic and biotic factors associated with the presence of Anopheles arabiensis immatures and their abundance in naturally occurring and man-made aquatic habitats.

BACKGROUND: Anopheles arabiensis (Diptera: Culicidae) is a potential malaria vector commonly present at low altitudes in remote areas in Reunion Island. Little attention has been paid to the environmental conditions driving larval development and abundance patterns in potential habitats. Two field surveys were designed to determine whether factors that discriminate between aquatic habitats with and without An. arabiensis larvae also drive larval abundance, comparatively in man-made and naturally occurring habitats. METHODS: In an initial preliminary survey, a representative sample of aquatic habitats that would be amenable to an intensive long-term study were selected and divided into positive and negative sites based on the presence or absence of Anopheles arabiensis larvae. Subsequently, a second survey was prompted to gain a better understanding of biotic and abiotic drivers of larval abundance, comparatively in man-made and naturally occurring habitats in the two studied locations. In both surveys, weekly sampling was performed to record mosquito species composition and larval density within individual habitats, as well as in situ biological characteristics and physico-chemical properties. RESULTS: Whilst virtually any stagnant water body could be a potential breeding ground for An. arabiensis, habitats occupied by their immatures had different structural and biological characteristics when compared to those where larvae were absent. Larval occurrence seemed to be influenced by flow velocity, macrofauna diversity and predation pressure. Interestingly, the relative abundance of larvae in man-made habitats (average: 0.55 larvae per dip, 95%CI [0.3-0.7]) was significantly lower than that recorded in naturally occurring ones (0.74, 95%CI [0.5-0.8]). Such differences may be accounted for in part by varying pressures that could be linked to a specific habitat. CONCLUSIONS: If the larval ecology of An. arabiensis is in general very complex and factors affecting breeding site productivity sometimes not easy to highlight, our results, however, highlight lower populations of An. arabiensis immatures compared to those reported in comparable studies conducted in the African continent. Overall, this low larval abundance, resulting from both abiotic and biotic factors, suggests that vector control measures targeting larval habitats are likely to be successful in Reunion, but these could be better implemented by taking environmental variability into account.

Vegetation cover and long-term conservation of radioactive waste packages: the case study of the CSM waste disposal facility (Manche District, France).

The CSM is the first French waste disposal facility for radioactive waste. Waste material is buried several meters deep and protected by a multi-layer cover, and equipped with a drainage system. On the surface, the plant cover is a grassland vegetation type. A scientific assessment has been carried out by the Géophen laboratory, University of Caen, in order to better characterize the plant cover (ecological groups and associated soils) and to observe its medium and long term evolution. Field assessments made on 10 plots were complemented by laboratory analyses carried out over a period of 1 year. The results indicate scenarios and alternative solutions which could arise, in order to passively ensure the long-term safety of the waste disposal system. Several proposals for a blanket solution are currently being studied and discussed, under the auspices of international research institutions in order to determine the most appropriate materials for the storage conditions. One proposal is an increased thickness of these materials associated with a geotechnical barrier since it is well adapted to the forest plants which are likely to colonize the site. The current experiments that are carried out will allow to select the best option and could provide feedback for other waste disposal facility sites already being operated in France (CSFMA waste disposal facility, Aube district) or in other countries.

Spatial and temporal distribution patterns of Anopheles arabiensis breeding sites in La Reunion Island--multi-year trend analysis of historical records from 1996-2009.

BACKGROUND: An often confounding facet of the dynamics of malaria vectors is the aquatic larval habitat availability and suitable conditions under which they can thrive. Here, we investigated the impact of environmental factors on the temporal and spatial distribution of larval habitats of Anopheles arabiensis in different locations on La Reunion Island. METHODS: A retrospective examination was made from archival data which provided the complete enumeration of An. arabiensis breeding habitats in three distinct geographic zones--extending North-east, West and South of the island over 14 years, from January 1996 to December 2009. Data on the occurrence and the number of active larval habitats at each of a total of 4376 adjacent ellipsoid grid cells (216,506 square meters each) were used (1) to provide the geographic extent of breeding site availability from year to year and (2) to analyze associations with prevailing environmental factors, habitat types, and locations. RESULTS: Anopheles arabiensis utilized a spectrum of man-made and natural aquatic habitats, most of which were concentrated primarily in the rock pools located in ravines and river fringes, and also in the large littoral marshes and within the irrigated agricultural zones. The numbers of breeding site per sampling grid differed significantly in different parts of the island. In contrast to an originally more widespread distribution across the island in the 1950s, detailed geographic analyses of the data obtained in the period extending from 1996-2009 showed an intriguing clustered distribution of active breeding sites in three discontinuous geographic zones, in which aquatic habitats availability fluctuates with the season and year. Seasonality in the prevalence of anopheles breeding sites suggests significant responsiveness to climatic factors. CONCLUSIONS: The observed retreat of An. arabiensis distribution range to lower altitudinal zones (< 400 m) and the upward shift in the most remote littoral areas in the northeast and southwest regions suggest the possible influence of biogeographic factors, changes in land use and control operations. The results of this study would allow for a more rational implementation of control strategies across the island.

Laboratory selection for an accelerated mosquito sexual development rate.

BACKGROUND: Separating males and females at the early adult stage did not ensure the virginity of females of Anopheles arabiensis (Dongola laboratory strain), whereas two years earlier this method had been successful. In most mosquito species, newly emerged males and females are not able to mate successfully. For anopheline species, a period of 24 h post-emergence is generally required for the completion of sexual maturation, which in males includes a 180° rotation of the genitalia. In this study, the possibility of an unusually shortened sexual maturity period in the laboratory-reared colony was investigated. METHODS: The effect of two different sex-separation methods on the virginity of females was tested: females separated as pupae or less than 16 h post-emergence were mated with males subjected to various doses of radiation. T-tests were performed to compare the two sex-separation methods. The rate of genitalia rotation was compared for laboratory-reared and wild males collected as pupae in Dongola, Sudan, and analysed by Z-tests. Spermatheca dissections were performed on females mated with laboratory-reared males to determine their insemination status. RESULTS: When the sex-separation was performed when adults were less than 16 h post-emergence, expected sterility was never reached for females mated with radio-sterilized males. Expected sterility was accomplished only when sexes were separated at the pupal stage. Observation of genitalia rotation showed that some males from the laboratory strain Dongola were able to successfully mate only 11 h after emergence and 42% of the males had already completed rotation. A small proportion of the same age females were inseminated. Wild males showed a much slower genitalia rotation rate. At 17 h post-emergence, 96% of the laboratory-reared males had completed genitalia rotation whereas none of the wild males had. CONCLUSION: This colony has been cultured in the laboratory for over one hundred generations, and now has accelerated sexual maturation when compared with the wild strain. This outcome demonstrates the kinds of selection that can be expected during insect colonization and maintenance, particularly when generations are non-overlapping and similar-age males must compete for mates.

Ubiquitous water-soluble molecules in aquatic plant exudates determine specific insect attraction.

Plants produce semio-chemicals that directly influence insect attraction and/or repulsion. Generally, this attraction is closely associated with herbivory and has been studied mainly under atmospheric conditions. On the other hand, the relationship between aquatic plants and insects has been little studied. To determine whether the roots of aquatic macrophytes release attractive chemical mixtures into the water, we studied the behaviour of mosquito larvae using olfactory experiments with root exudates. After testing the attraction on Culex and Aedes mosquito larvae, we chose to work with Coquillettidia species, which have a complex behaviour in nature and need to be attached to plant roots in order to obtain oxygen. This relationship is non-destructive and can be described as commensal behaviour. Commonly found compounds seemed to be involved in insect attraction since root exudates from different plants were all attractive. Moreover, chemical analysis allowed us to identify a certain number of commonly found, highly water-soluble, low-molecular-weight compounds, several of which (glycerol, uracil, thymine, uridine, thymidine) were able to induce attraction when tested individually but at concentrations substantially higher than those found in nature. However, our principal findings demonstrated that these compounds appeared to act synergistically, since a mixture of these five compounds attracted larvae at natural concentrations (0.7 nM glycerol, <0.5 nM uracil, 0.6 nM thymine, 2.8 nM uridine, 86 nM thymidine), much lower than those found for each compound tested individually. These results provide strong evidence that a mixture of polyols (glycerol), pyrimidines (uracil, thymine), and nucleosides (uridine, thymidine) functions as an efficient attractive signal in nature for Coquillettidia larvae. We therefore show for the first time, that such commonly found compounds may play an important role in plant-insect relationships in aquatic eco-systems.

Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family.

Bacterial intracellular symbiosis (endosymbiosis) is well documented in the insect world where it is believed to play a crucial role in adaptation and evolution. However, although Coleopteran insects are of huge ecological and economical interest, endosymbiont molecular analysis is limited to the Dryophthoridae family. Here, we have analyzed the intracellular symbiotic bacteria in 2 Hylobius species belonging to the Molytinae subfamily (Curculionoidea superfamily) that exhibit different features from the Dryophthoridae insects in terms of their ecology and geographical spanning. Fluorescence in situ hybridization has shown that both Hylobius species harbor rod-shaped pleiomorphic symbiotic bacteria in the oocyte and in the bacteria-bearing organ (the bacteriome), with a shape and location similar to those of the Dryophthoridae bacteriome. Phylogenetic analysis of the 16S ribosomal DNA gene sequences, using the heterogeneous model of DNA evolution, has placed the Hylobius spp. endosymbionts (H-group) at the basal position of the ancestral R-clade of Dryophthoridae endosymbionts named Candidatus Nardonella but relatively distant from the S-clade of Sitophilus spp. endosymbionts. Endosymbionts from the H-group and the R-clade evolved more quickly compared with free-living enteric bacteria and endosymbionts from the S- and D-clades of Dryophthoridae. They are AT biased (58.3% A + T), and they exhibit AT-rich insertions at the same position as previously described in the Candidatus Nardonella 16S rDNA sequence. Moreover, the host phylogenetic tree based on the mitochondrial COI gene was shown to be highly congruent with the H-group and the R-clade, the divergence of which was estimated to be around 125 MYA. These new molecular data show that endosymbiosis is old in Curculionids, going back at least to the common ancestor of Molytinae and Dryophthoridae, and is evolutionary stable, except in 2 Dryophthoridae clades, providing additional and independent supplementary evidence for endosymbiont replacement in these taxa.

How human practices have affected vector-borne diseases in the past: a study of malaria transmission in Alpine valleys.

BACKGROUND: Malaria was endemic in the Rhône-Alpes area of eastern France in the 19th century and life expectancy was particularly shortened in Alpine valleys. This study was designed to determine how the disease affected people in the area and to identify the factors influencing malaria transmission. METHODS: Demographic data of the 19th century were collected from death registers of eight villages of the flood-plain of the river Isère. Correlations were performed between these demographic data and reconstructed meteorological data. Archive documents from medical practitioners gave information on symptoms of ill people. Engineer reports provided information on the hydraulic project developments in the Isère valley. RESULTS: Description of fevers was highly suggestive of endemic malaria transmission in the parishes neighbouring the river Isère. The current status of anopheline mosquitoes in the area supports this hypothesis. Mean temperature and precipitation were poorly correlated with demographic data, whereas the chronology of hydrological events correlated with fluctuations in death rates in the parishes. CONCLUSION: Nowadays, most of the river development projects involve the creation of wet areas, enabling controlled flooding events. Flood-flow risk and the re-emergence of vector-borne diseases would probably be influenced by the climate change. The message is not to forget that human disturbance of any functioning hydrosystem has often been linked to malaria transmission in the past.

Do herbicide treatments reduce the sensitivity of mosquito larvae to insecticides?

Invasive mosquitoes are economic and sanitary concerns especially in Europe and America. Most work has emphasized the role of resistance [Berrada, S., Fournier, D., Cuany, A., Nguyen, T.X., 1994. Identification of resistance mechanisms in a selected laboratory strain of Cacopsylla pyri (Homoptera: Psyllidae): altered acetylcholinesterases and detoxifying oxidases. Pesticide Biochemistry and Physiology 48, 41-47; Hemingway, J., Hawkes, N.J., McCarroll, L., Ranson, H., 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochemistry and Molecular Biology 34, 653-665] to insecticides. Compounds acting on larval sensitivity to insecticides are not well studied and their action remains poorly understood. Among several residual chemicals in ecosystems, particularly in wetlands, we identified a possible interaction of an herbicide on larval resistance to an insecticide. Our work contributes to the global control of mosquito populations by identifying possible pathways of resistance to insecticides of these vectors. Resistance or tolerance to insecticide treatments might contribute to successful invasion by mosquitoes. Here we report an ecotoxicological approach to test the hypothesis of an indirect effect of atrazine on mortality of an invasive vector. A brief contact (48h) between Aedes aegypti mosquito larvae and atrazine led to a modification of larval sensitivity to an insecticide: using atrazine as an inducer led to a decrease in the mortality of larvae treated with Bacillus thuringiensis var. israelensis (Bti).

Response of Aedes aegypti (Diptera: Culicidae) larvae to three xenobiotic exposures: larval tolerance and detoxifying enzyme activities.

The ability of mosquito larvae to tolerate toxic compounds (temephos, Bacillus thuringiensis var. israelensis, toxic vegetable leaf litter) was examined on a laboratory larval strain of Aedes aegypti L. Bioassays and detoxifying enzyme activity measurements were performed to compare tolerance/resistance capacities. The possibility of a functional plasticity of detoxifying equipment was investigated through experimental determination of the inductive effect of each xenobiotic within a given generation. In the same way, the selective effect of a toxic leaf litter was also investigated along successive generations. Results revealed that differential cytochrome P450 monooxygenase, esterase, and glutathione S-transferase activity levels correlated with the bioassay results. Both induction and selection increased larval tolerance to the xenobiotics used and increased the levels of larval detoxifying enzyme activities.