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.

Changes in species richness and spatial distribution of mosquitoes (Diptera: Culicidae) inferred from museum specimen records and a recent inventory: a case study from Belgium suggests recent expanded distribution of arbovirus and malaria vectors.

Mosquito (Diptera: Culicidae) distribution data from a recent inventory of native and invading mosquito species in Belgium were compared with historical data from the period 1900-1960 that were retrieved from a revision of the Belgian Culicidae collection at the Royal Belgian Institute of Natural Sciences. Both data sets were used to investigate trends in mosquito species richness in several regions in Belgium. The relative change in distribution area of mosquito species was particularly important for species that use waste waters and used tires as larval habitats and species that recently shifted their larval habitat to artificial larval habitats. More importantly, several of these species are known as vectors of arboviruses and Plasmodium sp. and the apparent habitat shift of some of them brought these species in proximity to humans. Similar studies comparing current mosquito richness with former distribution data retrieved from voucher specimens from collections is therefore encouraged because they can generate important information concerning health risk assessment at both regional and national scale.

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.

Bionomics of the established exotic mosquito species Aedes koreicus in Belgium, Europe.

Adults of an exotic mosquito, Aedes (Finlaya) koreicus (Edwards) (Diptera: Culicidae) were identified by morphology and genotyping from one site in Belgium in 2008. In late summer of that year, the occurrence of adults and immature stages reconfirmed its presence. This is the first record of this species outside its native range and in particular in Europe. Two subsites of the original location were prospected from April until October 2009 with different traps to evaluate the extent of its presence and establishment in the area and to understand the dynamics of the species' population. Next to Ae. koreicus, 15 other mosquito species were collected. Adult individuals of Ae. koreicus were found from May to September and larvae were still found early October. Larvae were mainly retrieved from artificial containers both in 2008 as in 2009. Containers with eggs and/or larvae were found up to 4 km away from the initial location, indicating the species is spreading locally. Though the introduction route is unknown, it may have occurred via international trade as a large industrial center was located nearby. A comparison of different climatic variables between locations in Belgium with Ae. koreicus and putative source locations in South Korea, revealed similarities between winter temperatures and the number of freezing days and nights in four consecutive years (2004-2008), while humidity and precipitation values differed strongly. The introduction of a new potential disease vector into Europe seems to be a result of proper entrance points, created by intense worldwide trade and suitable environmental conditions.

Automatic segmentation of in-vivo intra-coronary optical coherence tomography images to assess stent strut apposition and coverage.

The implantation of intracoronary stents is currently the standard approach for the treatment of coronary atherosclerotic disease. The widespread adoption of this technology has boosted an intensive research activity in this domain, with continuous improvements in the design of these devices, aiming at reducing problems of restenosis (re-narrowing of the stented segment) and thrombosis (sudden occlusion due to thrombus formation). Recently, a new, light-based intracoronary imaging modality, optical coherence tomography (OCT), was developed and introduced into clinical practice. Due to its very high axial resolution (10-15 µm), it allows for in vivo evaluation of both stent strut apposition and neointima coverage (a marker of healing of the treated segment). As such, it provides valuable information on proper stent deployment, on the behaviour of different stent types in-vivo and on the effect of new types of stents (e.g. drug-eluting stents) on vessel wall healing. However, the major drawback of the current OCT methodology is that analysis of these images requires a tremendous amount of-currently manual-post-processing. In this manuscript, an algorithm is presented that allows for fully automated analysis of stent strut apposition and coverage in coronary arteries. The vessel lumen and stent struts are automatically detected and segmented through analysis of the intensity profiles of the A-lines. From these data, apposition and coverage can then be measured automatically. The algorithm was validated using manual assessments by two experienced operators as a reference. High Pearson's correlation coefficients were found (R = 0.96-0.97) between the automated and manual measurements while Bland-Altman analysis showed no significant bias with good limits of agreement. As such, it was shown that the presented algorithm provides a robust and fast tool to automatically estimate apposition and coverage of stent struts in in-vivo OCT pullbacks. This will be important for the integration of this technology in clinical routine and for the analysis of datasets of larger clinical trials.

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.

Personal protection by long-lasting insecticidal hammocks against the bites of forest malaria vectors.

OBJECTIVE: In Southeast Asia, malaria vectors bite outside the houses before bedtime, and forest dwellers rarely use insecticide-treated nets (ITNs). Thus, we tested the protection of long-lasting insecticidal hammocks (LLIH) using Olyset technology against exophagic vectors in two forest villages of Cambodia. METHODS: In each village, we conducted two entomological surveys (middle and end of the rainy season), each lasting 10 consecutive nights. These comprised human landing collections during the whole night by people sitting outside in the hammocks. Five households were selected per village, and for each household, two fixed positions were allocated: one for the control and one for the treated hammock. RESULTS: In total, 6449 mosquitoes were collected from control hammocks compared to 4481 in treated hammocks. Personal protection conferred by the hammocks was 46% (CI 95%: 35-55%) against the bites of Anopheles minimus. A significant reduction of An. dirus bites (46% CI 95%: 25-62%) was only observed at the end of the rainy season. An. maculatus and culicines bites were only reduced in one of the two study sites. CONCLUSION: Even if this LLIH is not inducing full protection against the bites of malaria vectors, it could prove effective in protecting forest workers and villagers before sleeping time. LLIH can be an additional and valuable tool in eliminating artemisinin-resistant malaria in the region.

Maturation of a Trypanosoma brucei infection to the infectious metacyclic stage is enhanced in nutritionally stressed tsetse flies.

We report on the effect of tsetse fly starvation on the maturation of an established Trypanosoma brucei brucei midgut infection, i.e., the development of procyclic infection into the infectious metacyclic parasites in the tsetse fly salivary glands. Glossina morsitans morsitans flies were nutritionally stressed 10 d after the uptake of a T. b. brucei-infected bloodmeal by depriving these flies from feeding for seven consecutive days, whereas the control fly group (nonstarved group) continued to be fed three times a week. After this period, both fly groups were again fed three times per week on uninfected rabbit. Thirty days after the infected bloodmeal, all surviving flies were dissected and examined for the presence of an immature midgut and a mature salivary gland trypanosome infections. Results showed a significantly increased proportion of flies with salivary gland infection in the nutritionally stressed fly group suggesting an enhanced maturation of the trypanosome infection. These data suggest that environmental factors that cause nutritional stress in a tsetse population do not only make tsetse flies significantly more susceptible to establish a midgut infection as was shown previously but also boost the maturation of these midgut infections.

Introduction and establishment of the exotic mosquito species Aedes japonicus japonicus (Diptera: Culicidae) in Belgium.

The establishment of the potential vector species Aedes (Finlaya) japonicusjaponicus (Theobald) (Diptera: Culicidae) in southern Belgium is reported. The species was most likely introduced through the international trade in used tires. It was first collected in 2002 on the premises of a second-hand tire company and was sampled using different sampling methods in the two consecutive years (2003-2004). It was only in 2007 and 2008, during a national mosquito survey (MODIRISK), that its presence as adults and larvae at the above-mentioned site and at another tire company in the area was confirmed based on morphological and molecular identification. This discovery is the first record for Belgium of an exotic mosquito species that established successfully and raises the question on the need for monitoring and control. Considering the accompanying species found during the surveys, we also report here the first observation of Culex (Maillotia) hortensis hortensis (Ficalbi) in Belgium.

Nutritional stress affects the tsetse fly's immune gene expression.

Tsetse-transmitted trypanosomiasis poses a serious threat to human and animal health in sub-Saharan Africa. The majority of tsetse flies (Glossina spp.) in a natural population will not develop a mature infection of either Trypanosoma congolense or Trypanosoma brucei sp. because of refractoriness, a phenomenon that is affected by different factors, including the tsetse fly's immune defence. Starvation of tsetse flies significantly increases their susceptibility to the establishment of a trypanosome infection. This paper reports the effects of nutritional stress (starvation) on (a) uninduced baseline levels of gene expression of the antimicrobial peptides attacin, defensin and cecropin in the tsetse fly, and (b) levels of expression induced in response to bacterial (Escherichia coli) or trypanosomal challenge. In newly emerged, unfed tsetse flies, starvation significantly lowers baseline levels of antimicrobial peptide gene expression, especially for attacin and cecropin. In response to trypanosome challenge, only non-starved older flies showed a significant increase in antimicrobial peptide gene expression within 5 days of ingestion of a trypanosome-containing bloodmeal, especially with T. brucei bloodstream forms. These data suggest that a decreased expression of immune genes in newly hatched flies or a lack of immune responsiveness to trypanosomes in older flies, both occurring as a result of fly starvation, may be among the factors contributing to the increased susceptibility of nutritionally stressed tsetse flies to trypanosome infection.

The efficacy of indoor CDC light traps for collecting the sandfly Phlebotomus argentipes, vector of Leishmania donovani.

The efficacy of light traps for collecting sandflies (Diptera: Psychodidae) varies both inter-specifically and intra-specifically (by gender and physiological status) as a result of significant differences in phototropic and other behavioural characteristics. The efficacy of miniature CDC light traps for collecting Phlebotomus argentipes Annandale & Brunetti, a vector of Leishmania donovani Laveran & Mesnil (Kinetoplastida: Trypanosomatidae), was assessed in the Indian state of Bihar. Sandflies were collected during the night from 16 houses in each of three villages over 3 months (four times at fortnightly intervals) using CDC light traps indoors, and by aspirator collection (carried out by one person for 30 min/house) from the walls of the same houses the following morning. Incidence rate ratios (IRRs) between CDC light trap collections and aspirator collections were obtained through a negative binomial regression with household as random effect. CDC light traps were especially effective in catching males (IRR 3.08, 95% confidence interval [CI] 2.12-4.46) and unfed females (IRR 3.50, 95% CI 2.37-5.16) of P. argentipes, and to a lesser extent gravids (IRR 1.77, 95% CI 1.07-2.93). However, only a relatively small proportion of all blood-fed P. argentipes were collected by light trap (IRR 0.45, 95% CI 0.28-0.73). Despite its limitations in collecting blood-fed female sandflies, the CDC light trap appears to trap a sufficient proportion of the indoor population of sandflies for sampling purposes, and as this light trap is also more convenient and more easily standardized than the aspirator method, we conclude that it is the most efficient method for monitoring P. argentipes populations in the Indian subcontinent.

Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review.

There is high diversity of Anopheles mosquitoes in Southeast Asia and the main vectors of malaria belong to complexes or groups of species that are difficult or impossible to distinguish due to overlapping morphological characteristics. Recent advances in molecular systematics have provided simple and reliable methods for unambiguous species identification. This review summarizes the latest information on the seven taxonomic groups that include principal malaria vectors in Southeast Asia, i.e. the Minimus, Fluviatilis, Culicifacies, Dirus, Leucosphyrus, and Sundaicus Complexes, and the Maculatus Group. Main issues still to be resolved are highlighted. The growing knowledge on malaria vectors in Southeast Asia has implications for vector control programs, the success of which is highly dependant on precise information about the biology and behavior of the vector species. Acquisition of this information, and consequently the application of appropriate, sustainable control measures, depends on our ability to accurately identify the specific vectors.

The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins.

The tsetse fly (Glossina spp.) is an obligate blood-sucking insect that transmits different human-pathogenic and livestock threatening trypanosome species in Africa. To obtain more insight in the tsetse salivary function, some general aspects of the tsetse fly saliva and its composition were studied. Direct pH and protein content measurements revealed a moderately alkaline (pH approximately 8.0) salivary environment with approximately 4.3 microg soluble proteins per gland and a constant representation of the major saliva proteins throughout the blood-feeding cycle. Although major salivary genes are constitutively expressed, upregulation of salivary protein synthesis within 48 h after the blood meal ensures complete protein replenishment from day 3 onwards. Screening of a non-normalised Glossina morsitans morsitans lambdagt11 salivary gland expression library with serum from a saliva-immunized rabbit identified three full-length cDNAs encoding for novel salivary proteins with yet unknown functions: a 8.3 kDa glycine/glutamate-rich protein (G. morsitans morsitans salivary gland protein Gmmsgp1), a 12.0 kDa proline-rich protein (Gmmsgp2), and a 97.4 kDa protein composed of a metallophosphoesterase/5'nucleotidase region with a glutamate/aspartate/asparagines-rich region (Gmmsgp3).

Preliminary field testing of a long-lasting insecticide-treated hammock against Anopheles gambiae and Mansonia spp. (Diptera: Culicidae) in West Africa.

The efficacy of an experimental long-lasting insecticide-treated hammock (LLIH) with a long-lasting treated net used as a blanket and made of the same fabric (polyethylene) was tested in a concrete block experimental hut, against the malaria vector Anopheles gambiae s.l. and the arbovirus vectors and nuisance mosquitoes Mansonia africana (Theobald) and Mansonia uniformis (Theobald). The LLIH was treated with the pyrethroid insecticide permethrin. It was evaluated concurrently with ignited mosquito coils over 20 successive weeks. In total, 2,227 mosquitoes (130 An. gambiae and 2,097 Mansonia spp.) corresponding to 27.8 mosquitoes per trap-night were collected in the untreated hut (control). The repellent effect of both coils and LLIH significantly reduced the number of mosquitoes entering the huts (35- 60%). There was no significant difference between LLIH and mosquito coils in blood-feeding inhibition (93-97%) or in mortality (88-98%). The LLIH is more cost-effective and user-friendly than mosquito coils, which need to be replaced nightly to protect people sleeping indoors from mosquito bites. The effects of LLIH on exophagic vectors also need to be investigated because most people that sleep in hammocks are outdoors.

Accuracy of the health information system on malaria surveillance in Vietnam.

The health information system (HIS) is a key component of control programs and its accuracy is necessary for the assessment of disease risks, the formulation of priorities and the evaluation of the cost-effectiveness of different interventions. In order to assess the quality of the HIS in estimating malaria morbidity in Vietnam, we compared data obtained by a 2-year active (ACD) and passive case detection (PCD) study with those routinely collected at the local commune health centres (CHC) at three sites having different malaria epidemiology. The majority of malaria cases (80-95%) detected by ACD were missed by the HIS. Similarly, most malaria cases (50-90%) detected by PCD were also missed by the HIS, and this was proportional to the number of active private practitioners. Reasons for this low sensitivity are low CHC attendance, high attendance at private health facilities, widespread self-medication and attendance at central health facilities. In conclusion, although malaria has sharply decreased in Vietnam over the past 10 years, the current HIS greatly underestimates the malaria burden. Involvement of the private sector and the establishment of sentinel sites might improve the quality of data and the relevance of HIS in malaria control.

Review of the Minimus Complex of Anopheles, main malaria vector in Southeast Asia: from taxonomic issues to vector control strategies.

BACKGROUND: The Minimus Complex of Anopheles subgenus Cellia is composed of two sibling species, A and C, on the Southeast Asian mainland, and a third allopatric species E that occurs in the Ryukyu Archipelago (Japan), a malaria-free region. Anopheles minimus s.l. is considered to be one of the main malaria vector in the hilly forested regions of Southeast Asia. Despite a large number of studies over its range of distribution, it is difficult to have a global view of the ecological and bionomical characteristics of the individual species as different identification methods were used, generally without specific identification of the sibling species. OBJECTIVES: (1) To review the main malaria studies on An. minimus s.l.; (2) to discuss recently published data on the biology and ecology of each sibling species; and (3) to identify gaps in our understanding of the Minimus Complex. REVIEW RESULTS: Major biological and ecological trends are addressed, such as the high plasticity of trophic behaviour and the sympatry of species A and C over the Southeast Asian mainland. Despite the availability of rapid molecular identification methods, we still lack important information concerning the biological characteristics of each sibling species. These gaps must be filled in the future because An. minimus species A and C may exhibit different abilities to transmit malaria. CONCLUSION: We expect that entomological surveys will employ molecular methods to clearly identify these two species, and thus elucidate the biological characteristics of each species. As a consequence, current vector control strategies will be improved by targeting the most efficient vectors.

Anopheles (Cellia) epiroticus (Diptera: Culicidae), a new malaria vector species in the Southeast Asian Sundaicus Complex.

Anopheles sundaicus species A of the Southeast Asian A. sundaicus complex is formally named Anopheles epiroticus Linton & Harbach based on DNA sequence differentiation of the whole nuclear ITS2 region and a portion of both the cytochrome b and cytochrome c oxidase I mitochondrial genes. Detailed comparative morphological studies of the adult, larval and pupal stages did not reveal any differential or diagnostic differences that reliably distinguish A. epiroticus from A. sundaicus s.s. Information is provided on the bionomics and systematics of the new species.