Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

The Crimean-Congo haemorrhagic fever tick vector Hyalomma marginatum in the south of France: Modelling its distribution and determination of factors influencing its establishment in a newly invaded area.

We developed a correlative model at high resolution for predicting the distribution of one of the main vectors of Crimean-Congo haemorrhagic fever virus (CCHFV), Hyalomma marginatum, in a recently colonised area, namely southern France. About 931 H. marginatum adult ticks were sampled on horses from 2016 to 2019 and 2021 in 14 southern French departments, which resulted in the first H. marginatum detection map on a large portion of the national territory. Such updated presence/absence data, as well as the mean number of H. marginatum per examined animal (mean parasitic load) as a proxy of tick abundance, were correlated to multiple parameters describing the climate and habitats characterising each collection site, as well as movements of horses as possible factors influencing tick exposure. In southern France, H. marginatum was likely detected in areas characterised by year-long warm temperatures and low precipitation, especially in summer and mostly concentrated in autumn, as well as moderate annual humidity, compared to other sampled areas. It confirms that even in newly invaded areas this tick remains exclusively Mediterranean and cannot expand outside this climatic range. Regarding the environment, a predominance of open natural habitats, such as sclerophyllous vegetated and sparsely vegetated areas, were also identified as a favourable factor, in opposition to urban or peri-urban and humid habitats, such as continuous urban areas and inland marshes, respectively, which were revealed to be unsuitable. Based on this model, we predicted the areas currently suitable for the establishment of the tick H. marginatum in the South of France, with relatively good accuracy using internal (AUC = 0.66) and external validation methods (AUC = 0.76 and 0.83). Concerning tick abundance, some correlative relationships were similar to the occurrence model, as well as the type of horse movements being highlighted as an important factor explaining mean parasitic load. However, the limitations of estimating and modelling H. marginatum abundance in a correlative model are discussed.

High dispersal capacity of Culicoides obsoletus (Diptera: Ceratopogonidae), vector of bluetongue and Schmallenberg viruses, revealed by landscape genetic analyses.

BACKGROUND: In the last two decades, recurrent epizootics of bluetongue virus and Schmallenberg virus have been reported in the western Palearctic region. These viruses affect domestic cattle, sheep, goats and wild ruminants and are transmitted by native hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Culicoides dispersal is known to be stratified, i.e. due to a combination of dispersal processes occurring actively at short distances and passively or semi-actively at long distances, allowing individuals to jump hundreds of kilometers. METHODS: Here, we aim to identify the environmental factors that promote or limit gene flow of Culicoides obsoletus, an abundant and widespread vector species in Europe, using an innovative framework integrating spatial, population genetics and statistical approaches. A total of 348 individuals were sampled in 46 sites in France and were genotyped using 13 newly designed microsatellite markers. RESULTS: We found low genetic differentiation and a weak population structure for C. obsoletus across the country. Using three complementary inter-individual genetic distances, we did not detect any significant isolation by distance, but did detect significant anisotropic isolation by distance on a north-south axis. We employed a multiple regression on distance matrices approach to investigate the correlation between genetic and environmental distances. Among all the environmental factors that were tested, only cattle density seems to have an impact on C. obsoletus gene flow. CONCLUSIONS: The high dispersal capacity of C. obsoletus over land found in the present study calls for a re-evaluation of the impact of Culicoides on virus dispersal, and highlights the urgent need to better integrate molecular, spatial and statistical information to guide vector-borne disease control.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Update of the species checklist of Culicoides Latreille, 1809 biting midges (Diptera: Ceratopogonidae) of Morocco.

BACKGROUND: Investigations of Culicoides fauna, including inventories, were carried out in Morocco at different periods after the country had faced major bluetongue and African horse sickness outbreaks. However, no comprehensive reference publication has provided a clear overview of the Culicoides species diversity. This study reviewed available data on Culicoides biting midge species in Morocco from 1968 to 2015 (published and grey literature in French and English) in order to revise the current checklist, in light of state of the art taxonomic and systematic knowledge, and confirmed the checklist with morphological and molecular identifications of specimens collected from the region of Rabat. METHODS: Literature related to Culicoides collections in Morocco was collated. Authors were contacted to obtain raw data and additional information for the collections. Fresh Culicoides material was collected and examined from two sites around Rabat, the capital of Morocco. Each collected individual was examined and morphologically identified, if possible, to the species level. In addition, molecular identification was performed to separate closely related species, to confirm difficult morphological identifications and to confirm new species records. RESULTS: A total of 6121 individuals of Culicoides spp. were collected and analyzed and at least 17 species were identified: C. cataneii/C. gejgelensis, C. circumscriptus, C. fagineus, C. festivipennis, C. imicola, C. jumineri, C. kingi, C. longipennis, C. montanus, C. newsteadi, C. obsoletus, C. paolae, C. parotti, C. puncticollis, C. sahariensis, C. scoticus and C. subfagineus. Seven species were confirmed using phylogenetic analyses. Two new species records for Morocco are reported: C. paolae and C. subfagineus. CONCLUSIONS: The Moroccan fauna of Culicoides now includes 54 valid species. Further work would certainly increase this total, as one of the clades we identified was not affiliated to any described and valid species.

DNA barcoding and molecular identification of field-collected Culicoides larvae in the Niayes area of Senegal.

BACKGROUND: Biting midge species of the genus Culicoides Latreille (Diptera: Ceratopogonidae) comprise more than 1300 species distributed worldwide. Several species of Culicoides are vectors of various viruses that can affect animals, like the African horse sickness virus (AHSV), known to be endemic in sub-Saharan Africa. The ecological and veterinary interest of Culicoides emphasizes the need for rapid and reliable identification of vector species. However, morphology-based identification has limitations and warrants integration of molecular data. DNA barcoding based on the mitochondrial gene cytochrome c oxidase subunit 1 (cox1) is used as a rapid and authentic tool for species identification in a wide variety of animal taxa across the globe. In this study, our objectives were as follows: (i) establish a reference DNA barcode for Afrotropical Culicoides species; (ii) assess the accuracy of cox1 in identifying Afrotropical Culicoides species; and (iii) test the applicability of DNA barcoding for species identification on a large number of samples of Culicoides larvae from the Niayes area of Senegal, West Africa. RESULTS: A database of 230 cox1 sequences belonging to 42 Afrotropical Culicoides species was found to be reliable for species-level assignments, which enabled us to identify cox1 sequences of Culicoides larvae from the Niayes area of Senegal. Of the 933 cox1 sequences of Culicoides larvae analyzed, 906 were correctly identified by their barcode sequences corresponding to eight species of Culicoides. A total of 1131 cox1 sequences of adult and larval Culicoides were analyzed, and a hierarchical increase in mean divergence was observed according to two taxonomic levels: within species (mean = 1.92%, SE = 0.00), and within genus (mean = 17.82%, SE = 0.00). CONCLUSIONS: Our study proves the efficiency of DNA barcoding for studying Culicoides larval diversity in field samples. Such a diagnostic tool offers great opportunities for investigating Culicoides immature stages ecology and biology, a prerequisite for the implementation of eco-epidemiological studies to better control AHSV in the Niayes region of Senegal, and more generally in sub-Saharan Africa.

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

Influence of temperature and relative humidity on survival and fecundity of three tsetse strains.

BACKGROUND: Tsetse flies occur in much of sub-Saharan Africa where they are vectors of trypanosomes that cause human and animal African trypanosomosis. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal and Ethiopia. Three Glossina palpalis gambiensis strains [originating from Burkina Faso (BKF), Senegal (SEN) and an introgressed strain (SENbkf)] were established and are now available for use in future AW-IPM programmes against trypanosomes in West Africa. For each strain, knowledge of the environmental survival thresholds is essential to determine which of these strains is best suited to a particular environment or ecosystem, and can therefore be used effectively in SIT programmes. METHODS: In this paper, we investigated the survival and fecundity of three G. p. gambiensis strains maintained under various conditions: 25 °C and 40, 50, 60, and 75 % relative humidity (rH), 30 °C and 60 % rH and 35 °C and 60 % rH. RESULTS: The survival of the three strains was dependent on temperature only, and it was unaffected by changing humidity within the tested range. The BKF strain survived temperatures above its optimum better than the SEN strain. The SENbkf showed intermediate resistance to high temperatures. A temperature of about 32 °C was the limit for survival for all strains. A rH ranging from 40 to 76 % had no effect on fecundity at 25-26 °C. CONCLUSIONS: We discuss the implications of these results on tsetse SIT-based control programmes.

Strong evidence for the presence of the tick Hyalomma marginatum Koch, 1844 in southern continental France.

Hyalomma ticks can transmit several human and animal pathogens in Eurasia and Africa. Interest in Hyalomma marginatum has increased since the recent (re)emergence of Crimean-Congo Hemorrhagic fever in the Palearctic region. Until now, continental France has been considered free of this tick species. Nevertheless, the existence of incomplete and occasionally incorrect records has maintained confusion about its status. Based on several tick sampling campaigns conducted on horses and birds from 2007 to 2016, we provided very strong evidence for the presence of reproducing populations of H. marginatum in parts of southern continental France. We also confirmed the introduction of immature developmental stages of H. marginatum, as well as H. rufipes, into France probably through trans-Mediterranean bird migrations.

Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events.

The role of the northward expansion of Culicoides imicola Kieffer in recent and unprecedented outbreaks of Culicoides-borne arboviruses in southern Europe has been a significant point of contention. We combined entomological surveys, movement simulations of air-borne particles, and population genetics to reconstruct the chain of events that led to a newly colonized French area nestled at the northern foot of the Pyrenees. Simulating the movement of air-borne particles evidenced frequent wind-transport events allowing, within at most 36 hours, the immigration of midges from north-eastern Spain and Balearic Islands, and, as rare events, their immigration from Corsica. Completing the puzzle, population genetic analyses discriminated Corsica as the origin of the new population and identified two successive colonization events within west-Mediterranean basin. Our findings are of considerable importance when trying to understand the invasion of new territories by expanding species.

Spatio-temporal genetic variation of the biting midge vector species Culicoides imicola (Ceratopogonidae) Kieffer in France.

BACKGROUND: Introduction of vector species into new areas represents a main driver for the emergence and worldwide spread of vector-borne diseases. This poses a substantial threat to livestock economies and public health. Culicoides imicola Kieffer, a major vector species of economically important animal viruses, is described with an apparent range expansion in Europe where it has been recorded in south-eastern continental France, its known northern distribution edge. This questioned on further C. imicola population extension and establishment into new territories. Studying the spatio-temporal genetic variation of expanding populations can provide valuable information for the design of reliable models of future spread. METHODS: Entomological surveys and population genetic approaches were used to assess the spatio-temporal population dynamics of C. imicola in France. Entomological surveys (2-3 consecutive years) were used to evaluate population abundances and local spread in continental France (28 sites in the Var department) and in Corsica (4 sites). We also genotyped at nine microsatellite loci insects from 3 locations in the Var department over 3 years (2008, 2010 and 2012) and from 6 locations in Corsica over 4 years (2002, 2008, 2010 and 2012). RESULTS: Entomological surveys confirmed the establishment of C. imicola populations in Var department, but indicated low abundances and no apparent expansion there within the studied period. Higher population abundances were recorded in Corsica. Our genetic data suggested the absence of spatio-temporal genetic changes within each region but a significant increase of the genetic differentiation between Corsican and Var populations through time. The lack of intra-region population structure may result from strong gene flow among populations. We discussed the observed temporal variation between Corsica and Var as being the result of genetic drift following introduction, and/or the genetic characteristics of populations at their range edge. CONCLUSIONS: Our results suggest that local range expansion of C. imicola in continental France may be slowed by the low population abundances and unsuitable climatic and environmental conditions.

New data regarding distribution of cattle ticks in the south-western Indian Ocean islands.

Recent studies have produced new insight into the origin and distribution of some cattle ticks in the south-western Indian Ocean islands. Rhipicephalus appendiculatus, introduced from Tanzania in 2002, is now well established on Grande Comore but has not yet reached the other islands of the archipelago (Mohéli, Anjouan and Mayotte). Only one of the two clades identified in Africa has settled so far. Amblyomma variegatum, which was not supposed to be able to persist in the Antananarivo region (1300 m) nor in other Malagasy regions of high altitude without regular introductions of ticks by infested cattle, is now endemic as a general rule up to 1600 m although other regions of lower altitude (1400 m) are still free of the tick. This species remains confined in a small area of the west coast on La Reunion Island. On the contrary, Hyalomma dromedarii could not settle on Madagascar where it was introduced in 2008 and Rhipicephalus evertsi evertsi is not yet present in Grande Comore despite regular introductions by infested cattle from Tanzania. A phylogeographic approach has been carried out at an intra-specific level for A. variegatum. This study has led to the identification of two main lineages, one covering all species distribution and one restricted to East Africa and the Indian Ocean area. These two lineages are in sympatry in Madagascar where a high genetic diversity has been described, whereas a lower genetic diversity is observed on other islands. These results seem to agree with the historical data concerning the introduction of the tick in the Indian Ocean area.

Clonal origin of emerging populations of Ehrlichia ruminantium in Burkina Faso.

Cowdriosis or heartwater is a major tick-borne disease on ruminants in Africa and the Caribbean. The causative agent is Ehrlichia ruminantium, an intracellular bacterium. Development of vaccines against heartwater has been hampered the limited efficiency of vaccine in the field, thought to be a consequence of the high genetic diversity of strains circulating in a same area. A sampling scheme was set to collect ticks over 2 years in a delimited area and well identified flock. Prevalence was low at about 3%. A set of 37 strains was considered for MLST analysis along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available, using a previously described scheme based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Two populations were identified both with limited genetic variability but with differing evolutionary patterns. Population 1 is in genomic stasis, in agreement with the paradigm for intracellular bacteria. The two reference strains, one from the Caribbean separated from West African strains three centuries ago and another one isolated in South Africa, belong to Population 1. Population 2 is on expansion following a recent clonal emergence from Population 1. The founder strain was identified as strain 395. Strain 623 displays a particularly high rate of mutations in groEL. Owing to the chaperone function of GroEL, this might indicate another clonal emergence under way. This work brings further insight in the genomic plasticity of E. ruminantium and its impact on vaccine strategy.

MLST scheme of Ehrlichia ruminantium: genomic stasis and recombination in strains from Burkina-Faso.

Heartwater, caused by the intracellular bacterium Ehrlichia ruminantium, is a major tick-borne disease of livestock in Africa also introduced in the Caribbean. The main problem encountered with the control of this disease is the lack of efficient vaccine in the field. This is thought to be related to the high genetic diversity of strains circulating in a same area. A set of eight circulating strains was isolated from a herd of cows in a small locality in Burkina-Faso and analyzed along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available. A MLST analysis was developed based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Phylogeny analysis was conducted both on concatenated MLST loci and on each individual locus. This showed differing phylogenies for each individual target gene. Most of the recorded polymorphism was borne by three strains: 331, 469 and 623. The neutrality hypothesis could not be rejected. Recombination and linkage disequilibrium were shown to have occurred. A core of seven strains displayed little polymorphism and signs of most likely ancient recombination events. The two reference strains, one from the Caribbean separated from west African strains three centuries ago and another one isolated in South Africa, were very closely related to the core strains whereas the three differing strains displayed recombination and most of the parcimony informative sites. These data suggest that some strains are in genomic stasis, as expected for intracellular parasites, while others emerge in the same area with DNA polymorphism. This work also shows that the MLST scheme developed can discriminate between these two kinds of strains.

Aedes aegypti in Senegal: genetic diversity and genetic structure of domestic and sylvatic populations.

Aedes aegypti is the main vector of dengue viruses. The epidemiology of dengue fever remains poorly understood in Senegal. A sylvatic transmission seems to predominate. However, despite the sylvatic circulation of the dengue virus and the presence of vectors in urban areas, only sporadic cases have been reported. Ae. aegypti is a polytypic species. In Senegal, a purely sylvatic form is found in the forest gallery areas and a domestic form is found in the villages in savannah and sahelian areas and in urban areas. Using allozymes, we analyzed the genetic diversity and the genetic structure of Ae. aegypti populations differing in their ecological characteristics. Populations from Senegal were significantly structured but with a low level of genetic differentiation. Ae. aegypti from the "domestic" populations show a decreased genetic diversity and a lower genetic differentiation compared with "sylvatic" populations. These findings suggest that environmental conditions, ecological factors, and human activities may impact the genetic structure of Ae. aegypti populations in Senegal.

A preliminary study of natural and experimental infection of the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) with Histomonas meleagridis (Protozoa: Sarcomastigophora).

Histomonas meleagridis is a protozoan parasite causing histomoniasis (histomonosis), a disease of gallinaceous fowl. In order to determine whether the lesser mealworm, Alphitobius diaperinus, is capable of harbouring H. meleagridis, the presence of the parasite was tested by polymerase chain reaction in lesser mealworms collected in the field. Parasite DNA was detected in two larvae from two farms undergoing an outbreak of histomoniasis. Insects were also artificially infected, killed after incubation and analysed by polymerase chain reaction for the presence of parasite DNA. After 4 days, two larvae (out of 20) remained positive. In another experimental infection to investigate the viability of histomonads in the larvae, living parasites were detected in five of 20 larvae 4 days after infection. These results indicate that although A. diaperinus can become infected with H. meleagridis it appears to have a low susceptibility to infection and would probably not be a major route of contamination between flocks.

Detection of Histomonas meleagridis in turkeys cecal droppings by PCR amplification of the small subunit ribosomal DNA sequence.

Histomonas meleagridis is a protozoan parasite that may cause histomoniasis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis and high mortality. Diagnosis of this disease is based on direct identification or on cultivation of the parasite. With the aim of developing more sensitive, rapid and useful tools for parasite detection, PCR that amplified a DNA target of 209 pb of the 18S rRNA gene was designed to detect the genome of H. meleagridis and to differentiate it from the genome of Tetratrichomonas gallinarum, another common protozoan parasite of fowl. The sensitivity of the test was evaluated using serial diluted samples of cultured H. meleagridis and showed positive amplification for concentrations comprised between 10 and 10(-1)parasites/ml of culture. The sensitivity for cecal droppings samples was assessed using spiked material and was comprised between 3 x 10(3) and 3 x 10(5)parasites/ml of stool. The reliability of the PCR for the detection of Histomonas infection was also evaluated by experimental infection of turkeys. Results of the PCR appeared to be in agreement with the development of the clinical signs and of the cecal lesions. The PCR developed in this study may be a useful tool in the detection and identification of H. meleagridis for rapid, routine screening as a supplement to direct identification or cultivation of the parasite.

Comparisons of amplified fragment length polymorphism (AFLP), microsatellite, and isoenzyme markers: population genetics of Aedes aegypti (Diptera: Culicidae) from Phnom Penh (Cambodia).

Aedes aegypti is the main vector of dengue viruses responsible for dengue hemorrhagic fever, which has become a major public health concern in tropical countries. Because vaccines are still under development, dengue prevention depends entirely on vector control. Knowledge of gene dispersal patterns is required to develop efficient vector control strategies. Here we report the use of amplified fragment length polymorphism (AFLP) to infer the genetic structure of Ae. aegypti populations at a local levels (Phnom Penh, Cambodia). The amount of variation and patterns of gene flow detected are compared with those obtained with two other more widely used markers, isoenzymes and microsatellites. The pattern of differentiation depicted by AFLP data were confirmed by comparison of the Fst values of the three markers. Even though Fst values estimated with AFLP markers are three- to fivefold higher than those estimated with isoenzymes or microsatellites, these different markers reveal the same population structure. This technique is useful for population genetic studies of Ae. aegypti and is especially advantageous when few individuals specimens are available because of the ability to AFLP to simultaneously amplify large numbers of polymorphic DNA fragments.

Influence of breeding sites features on genetic differentiation of Aedes aegypti populations analyzed on a local scale in Phnom Penh Municipality of Cambodia.

This study analyzed genetic differentiation of 20 Aedes aegypti populations collected along a street in Phnom Penh Municipality of Cambodia. Using allozyme and microsatellite variations, we demonstrated that populations were differentiated and the pattern of differentiation was dependent on the type of breeding sites. Moreover, insecticide treatments with temephos mostly affect the population functioning of discarded containers. Low gene flow detected could limit the natural diffusion of resistant populations that might instead take advantage of human displacements to spread.