Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.

X-ray sterilization of the An. arabiensis genetic sexing strain 'ANO IPCL1' at pupal and adult stages.

With a view to area wide integrated pest management programs with a sterile insect technique (SIT) component against the malaria vector Anopheles arabiensis, the effects of X-ray exposure of males of the genetic sexing strain (GSS) of An. arabiensis, "ANO IPCL1" have been tested. The suitability of X-ray was assessed in comparison to gamma-ray for mosquito sterilization in operational settings where the acquisition of gamma ray irradiators may be problematic. Pupae and adult males were sterilized using X-ray irradiation at 5 increasing doses and effects were observed in terms of fecundity and fertility. Irradiated pupae and adults were further observed for effects on longevity. The adult stage appeared to be more sensitive to radiation exposure than the pupal stage, with a residual fertility of 1% in adults irradiated at 75Gy and 0% at 105Gy, compared to 8%at 75Gy and 4% at 105Gy in adults irradiated at the pupal stage. All treatment groups except those irradiated at 60Gy were statistically different from the control groups in terms of adult longevity post treatment, however overall survival was not highly compromised in the 75Gy treatment group which showed 96% of induced sterility. To ensure the fine balance between the induced sterility and the fitness of the insect produced is achieved, irradiation of adults at this dose may be a good compromise for the SIT as applied for this species, especially in specific cases such as sterilization after female elimination by spiking blood meals. Investigation into the combined effects of blood spiking and irradiation at the adult stage and additional competitiveness studies both in laboratory and field cages will provide more insight into the radiation dose and mosquito stage to be used in the frame of future SIT programs targeting this important malaria vector.

Efficiency of three diets for larval development in mass rearing Aedes albopictus (Diptera: Culicidae).

A fundamental step in establishing a mass production system is the development of a larval diet that promotes high adult performance at a reasonable cost. To identify a suitable larval diet for Aedes albopictus (Skuse), three diets were compared: a standard laboratory diet used at the Centro Agricoltura Ambiente, Italy (CAA) and two diets developed specifically for mosquito mass rearing at the FAO/IAEA Laboratory, Austria. The two IAEA diets, without affecting survival to the pupal stage, resulted in a shorter time to pupation and to emergence when compared with the CAA diet. At 24 h from pupation onset, 50 and 90% of the male pupae produced on the CAA and IAEA diets, respectively, had formed and could be collected. The diet received during the larval stage affected the longevity of adult males with access to water only, with best results observed when using the CAA larval diet. However, similar longevity among diet treatments was observed when males were supplied with sucrose solution. No differences were observed in the effects of larval diet on adult male size or female fecundity and fertility. Considering these results, along with the relative costs of the three diets, the IAEA 2 diet is found to be the preferred choice for mass rearing of Aedes albopictus, particularly if a sugar meal can be given to adult males before release, to ensure their teneral reserves are sufficient for survival, dispersal, and mating in the field.

Anopheles arabiensis sperm production after genetic manipulation, dieldrin treatment, and irradiation.

The use of the sterile insect technique relies on the release of sterilized mass-reared male insects which, before field releases, endure several unnatural treatments. In the case of Anopheles arabiensis (Patton) sterile insect technique program in Sudan, the genetic background of the original strain was first changed to create a genetic sexing strain that is based on a dieldrin-resistant mutation. Secondly, the eggs of the genetic sexing strain require treatment with dieldrin to allow complete elimination of female L1 larvae to enable the release of males only. Finally, male mosquitoes receive an irradiation dose of 70 Gy as pupae for sterilization. The effects of these treatments on sperm production were tested separately and in combination. Irradiation alone significantly decreased the initial sperm number and prevented new sperm production. However, the dieldrin treatment, aimed at eliminating females, appears to have an unexpected radioprotectant effect.

Different blood and sugar feeding regimes affect the productivity of Anopheles arabiensis colonies (Diptera: Culicidae).

The success of the sterile insect technique for the management of mosquito populations depends on the release of large numbers of competitive sterile male insects. Sustainable mosquito production can only be obtained when proper mass-rearing equipment and adequate methods are available, including those to feed blood to the female mosquitoes. The blood feeding apparatus Hemotek consists of a small aluminum plate to which a collagen membrane is fixed and filled with blood kept warm by an electric heating element. A larger aluminum plate was developed to feed a larger number of female mosquitoes with blood that is kept at a constant temperature. The effect of different blood feeding regimes (feeding frequency and time the blood is kept in the Hemotek) and sugar deprivation before blood feeding on egg production of female Anopheles arabiensis Patton was tested. Egg production was higher when blood was offered to the mosquitoes every day as compared with every 2 or 4 d. Sugar deprivation for 7 h before blood feeding enhanced egg production by 50% compared with female mosquitoes that had continuous access to sugar. Neither male nor female survival was impaired. Finally, we showed that the same blood could be kept warm and used over several hours to feed mosquitoes in multiple cages without any impact on egg production or hatch rate. Being able to use the same blood over extended periods would save considerable time, handling, and funds.

Nationwide inventory of mosquito biodiversity (Diptera: Culicidae) in Belgium, Europe.

To advance our restricted knowledge on mosquito biodiversity and distribution in Belgium, a national inventory started in 2007 (MODIRISK) based on a random selection of 936 collection points in three main environmental types: urban, rural and natural areas. Additionally, 64 sites were selected because of the risk of importing a vector or pathogen in these sites. Each site was sampled once between May and October 2007 and once in 2008 using Mosquito Magnet Liberty Plus traps. Diversity in pre-defined habitat types was calculated using three indices. The association between species and environmental types was assessed using a correspondence analysis. Twenty-three mosquito species belonging to traditionally recognized genera were found, including 21 indigenous and two exotic species. Highest species diversity (Simpson 0.765) and species richness (20 species) was observed in natural areas, although urban sites scored also well (Simpson 0.476, 16 species). Four clusters could be distinguished based on the correspondence analysis. The first one is related to human modified landscapes (such as urban, rural and industrial sites). A second is composed of species not associated with a specific habitat type, including the now widely distributed Anopheles plumbeus. A third group includes species commonly found in restored natural or bird migration areas, and a fourth cluster is composed of forest species. Outcomes of this study demonstrate the effectiveness of the designed sampling scheme and support the choice of the trap type. Obtained results of this first country-wide inventory of the Culicidae in Belgium may serve as a basis for risk assessment of emerging mosquito-borne diseases.

An inexpensive and effective larval diet for Anopheles arabiensis (Diptera: Culicidae): eat like a horse, a bird, or a fish?

A successful sterile insect technique program depends upon mass production of good-quality sterile insects for release into a target area. Specifically, to control Anopheles arabiensis Patton (Diptera: Culicidae) in a pilot area in northern Sudan, 1 million An. arabiensis sterile males per day are needed. To reach this production objective, mosquito mass rearing is indispensable and larval diet, a key parameter for the production of healthy male mosquitoes, needs to be cost-effective. The Koi Floating Blend fish food, previously used at the Food and Agriculture Organization/International Atomic Energy Agency Insect Pest Control Laboratory for the routine rearing of the An. arabiensis colony, is no longer available. The aim of this study was to find a cheap and effective substitute for the discontinued diet. Several candidate powdered diets that are commercially available were tested to determine the best diet. By using mixture experiment principles and response surface methodology, the combination of two components, bovine liver powder and tuna meal, showed the best results in terms of larval survival, developmental, rate and adult size. The addition of a vitamin mixture further improved the diet. These positive production results coupled with the relatively low cost of our blend demonstrated the possibility of its use for mass rearing purpose.

Human-induced expanded distribution of Anopheles plumbeus, experimental vector of West Nile virus and a potential vector of human malaria in Belgium.

For the majority of native species, human-created habitats provide a hostile environment that prevents their colonization. However, if the conditions encountered in this novel environment are part of the fundamental niche of a particular species, these low competitive environments may allow strong population expansion of even rare and stenotopic species. If these species are potentially harmful to humans, such anthropogenic habitat alterations may impose strong risks for human health. Here, we report on a recent and severe outbreak of the viciously biting and day-active mosquito Anopheles plumbeus Stephens, 1828, that is caused by a habitat shift toward human-created habitats. Although historic data indicate that the species was previously reported to be rare in Belgium and confined to natural forest habitats, more recent data indicate a strong population expansion all over Belgium and severe nuisance at a local scale. We show that these outbreaks can be explained by a recent larval habitat shift of this species from tree-holes in forests to large manure collecting pits of abandoned and uncleaned pig stables. Further surveys of the colonization and detection of other potential larval breeding places of this mosquito in this artificial environment are of particular importance for human health because the species is known as a experimental vector of West Nile virus and a potential vector of human malaria.

Gamma radiation consequences on desert locust Schistocerca gregaria (Forsk.) digestive system.

Schistocera gregaria (Forsk.) (Orthoptera, Acrididae) remains a major insect pest in Africa, more particularly in the Sahelian zone. Present control methods are only partially efficient. In a previous study, we tested the potentiality of a sterile insect technique (SIT). Males of S. gregaria appeared to be much radiosensitive as already a dose of 3 Gy limited their survival. Gamma-radiations are known to damages the epithelial tissue of midgut, which affects the alimentation in insects. In this work, we show how digestive system of S. gregaria males is affected when submitted to a dose of 4 gamma rays. Nutrition is affected as males stop feeding soon after irradiation and progressively lose weight. Histological analyses on the midgut showed important epithelium damages. The regenerative cells by which the epithelial cells are replaced were damaged on the first days following irradiation. Consequently, regenerative cells are unable to divide and replace the normal loss of midgut cell. After nine days, the entire midgut epithelium was destroyed and only longitudinal muscles layer remained intact. This indicates that low radiation doses should be used if SIT will be applied.

The effect of age on sperm stock and egg laying in the parasitoid wasp, Dinarmus basalis.

Sperm quantity and quality during storage may be constraints acting on female fecundity and hence fitness. In Hymenoptera, the importance of sperm quality has rarely been considered, despite its central role in reproductive strategies and especially in sex ratio control. In these insects, fertilized eggs develop into females and unfertilized eggs into males. Experiments were conducted on the female wasp, Dinarmus basalis, in the laboratory with and without egg-laying resources (hosts). The first point was to test if sperm age influenced sperm storage by measuring sperm count and viability using a sperm viability test (SYBR14 : propidium iodide). The second point was the influence of prolonged storage in the female genital tract on the quantity, sex ratio and fitness of offspring produced. Results show that sperm viability in the spermatheca does not change significantly with maternal age, and that the sperm stock is not affected when females are deprived of hosts. Egg-laying is gradually restored after 21 days of host deprivation but remains at a low level after 115 days. The fitness of mated D. basalis females is therefore not constrained by sperm quantity or quality and seems to depend on host availability and female age.