Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics.
Please upload your review as an attachment if it exceeds 20, characters. The data collection tools though not exhaustive lacking in the survey of school environment for breeding sites were appropriate and the data generated was adequate to address the set objective. The initial objective of explaining the burden of dengue and other arboviruses using the vector data in the diverse schools was clouded by the overemphasis on diversity indices. Additionally, interpretation of the diversity indices to explain the specific disease burden was not clearly brought how will these indices inform decision towards reducing disease burden?
However, abundance of specific species to explain transmission of the target diseases, DEN, JE and malaria has been articulated. In addition, the data analysis presented in the results made no reference to the said school characteristics and weather there was any correlation to the vector data abundance or diversity. Based on the above important major observation, the authors need to work further on the manuscript to improve on the listed issues and gaps before it can be considered further.
The information has been overgeneralized. There was focus on biodiversity as a conclusion. It is suggests that if the authors are not usual English writers, they may need to identify someone to edit the manuscript for grammar.
Were they identified in the field or in the lab? How were they transported? The methodology is very scanty with information. The authors evaluated the mosquito diversity using light traps and BG traps Shannon and Simpson indexes.
In the abstract, 61 species were described in schools, including Aedes, Anopheles and Culex genus. The manuscript proposal is interesting and promising to Cambodia authorities, especially because the authors mention that there are few data available on mosquito diversity in the country. However, the present manuscript needs to be thoroughly rewritten and more details should be given regarding experimental design, data analysis, results e.
The authors should be more didactic in the analysis section: what were the independent variables? What was the model? Was it a GLM? What probability distribution was used? Why did they use two models to analyze the same data? How the authors managed to overcome pseudoreplication problems that are common with this kind of study?
This section is confusing and is not clear. The authors should rethink their analysis strategy and probably update their results. The results are also very confusing as the authors use absolute abundance, relative abundance, the diversity indexes for the schools and seasons interchangeably and do not give depth to their analysis.
They should focus on their results separating by sections. The results could be more explored, and they are not suitable for PloS One such as they are presented. I suggest the authors to explore a spatial approach in their data so that they have more interesting material. The discussion and conclusion sections are very interesting but should be updated after a new batch of analysis are done.
Also, there are lots English mistakes and typos that need revision. PLOS authors have the option to publish the peer review history of their article what does this mean? If published, this will include your full peer review and any attached files. Do you want your identity to be public for this peer review?
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If this link does not appear, there are no attachment files to be viewed. To use PACE, you must first register as a user. Registration is free. If you encounter any issues or have any questions when using PACE, please email us at gro. Please note that Supporting Information files do not need this step. ONE-D : High diversity of mosquito vectors in Cambodian primary schools and consequences for arbovirus transmission. The figure 1 is a homemade map with no copyright. This map was never copyrighted until now and was never publish.
We did not use any satellite image or copyrighted background. We did ourselves this vectorial map by using a public administrative contour map. And we recorded school location by using a GPS. For the overall general comments, Reviewer 1 emphases our introduction and Reviewer 2 the discussion and conclusion, recommending to focus on Public Health areas.
It seems that the biodiversity indices that we wanted to introduce through this article, bring too much perturbation to this article. We removed these common indices of biodiversity, but still talking about the unexpected biodiversity of mosquito species we found inside class rooms. Overall we add 3 tables in the text, we removed 4 figures and replaced them by 3 other. We deleted the results parts and the figures and maps related to the diversity indices that are not related to the different diseases and induced more confusion.
But, we keep a result and discussion part on the diversity, even if we decrease the importance of this part, in order to illustrate also the huge diversity of mosquito species in the schools. I am totally agree that there is no relationship between the diversity of all mosquito species and the different diseases.
We are truly sorry with this major problem. AS usual, we add the tables as Excel files. We feel terribly sorry for the reviewers, and also a little disappointed against the editors work. These analysis were not made yet. The characterization of the landscape is very difficult and takes time. As you recommended, we reoriented the manuscript in regards of the observations you highlighted.
Particularly, we focused more on the vector species, and we developed the conclusion on the possible ways of research. In the abstract we add few sentences. In the conclusion as at the end of the abstracts, we focused now mainly on the importance of implementing new methods for vector controls.
The article was read again by a French native speaker that lived in U. Richard Paul, who is a English native speaker. We choose to let these information in the Discussion part. But we had a sentence regarding the incidence of Dengue in children in Cambodia. The only recent study is on the causes of acute meningoencephalitis in children population with But there is no global data to add in the introduction. The school characteristics are group in Table 1. You did not have this Table 1 in the previous version of the manuscript.
I am sorry for that. Now, you can have access to these data. They were tested but none of them was influencing the data. There is another huge work to do for each species related to each factors, including temperature and spatial effect, but this is another huge analysis work that we need to begin.
In the field and in the laboratory, mosquitoes were identified under stereomicroscope and microscope using morphological mosquito identification keys from Southeast Asia countries Rattanarithikul et al. The diversity as far as I know did not influence the risks of dengue, Japanese encephalitis and so on. As I wrote before, regarding the comments of the reviewers, we removed the parts on the biodiversity indices to focus on the vector species. And again, I really want to apologize for the absence of the table within the manuscript.
As for the reviewer 1, I really want to apologize for the absence of the 2 previous tables. Deeply sorry for that inconvenient. Following recommendations, some details were added in Material and Methods sections for experimental design, data analysis and also in the results part, as detailed before and below. The section statistical analysis was totally rewritten. You can no find lines None were significant except the month and school effects.
A deeper characterization of month and school effects and their comparisons were realized by using a Kruskal-Wallis test. Please respond to the reviewers' comments.
Additional experiments are not needed, but adjustment of the modelling can be warranted. We would appreciate receiving your revised manuscript by May 01 PM.
Reviewer 3: Authors have collected mosquitoes in school and have observed high diversity. Some of mosquitoes collected are known to be vectors. However, authors don't report any evidence of presence of parasite or virus in mosquitoes collected but have largely discussed the transmission risk in schools. Since it is possible not too difficult to detect parasites e.
Moreover, the description of the important presence of vectors in schools is an important step prior to vector control and for testing the implementation of new control methods adapted to schools. The authors really want to thank the technicians that did the sampling in the field, Suor Kimhuor, Chhum Moeun, Chhuoy Kalyan and the different field authorities including all the Directors and Teachers of the 24 primary schools.
The authors also would like to thank Sylvaine Jego for statistical advices, Vincent Herbreteau for the help for mapping, and Richard Paul for correcting the manuscript. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Only few data exist in Cambodia on mosquito diversity and their potential role as vectors. Introduction Mosquito-borne pathogens such as arboviruses and malaria parasites are transmitted through a high diversity of species belonging to three genera: Aedes , Anopheles , and Culex.
Materials and methods Study area Table 1 shows the characteristics of the schools selected for the study and their GPS coordinates. Download: PPT. Fig 1. Map representing the spatial distribution of the 24 schools sampled in Kampong Cham and Tboung Khmum provinces, Cambodia. Table 1. Statistical analysis The association of the different parameters characterizing the schools and the period of collection, i. Results Relative abundance and diversity of mosquito species The list of the genera and mosquito taxa collected during the four collection sessions in the 24 schools is presented in Table 2.
Table 2. Mosquito vector species and pathogen transmission risk Of all the 61 mosquito species collected, at least 17 species are considered as potential vectors of pathogens Table 2 : Ae.
Fig 2. Relative abundance of Dengue virus vectors caught in 24 schools during 4 different months. Fig 3. Spatial and temporal distribution of the relative abundance of dengue virus vectors in the 24 schools in Kampong Cham and Tboung Khmum provinces, Cambodia. Discussion Diversity of mosquito species There was a large overall diversity of mosquito species caught across all schools and even reached 61 different mosquito species in one school.
Diversity and relative abundance of mosquito potential vector species Children spend their time at school during the day and consequently these specific places are most likely to be considered as hot spots for transmission of vector borne-diseases.
Conclusion The important biodiversity of mosquitoes discovered in schools in Cambodia is relevant and can represent a research axis, especially on the association between the different surrounding ecotypes forest, culture, rice fields and urbanity and the presence of specific mosquito species or vectors. Supporting information. S1 Data. List of mosquito species caught in the different schools. S1 File. Acknowledgments The authors really want to thank the technicians that did the sampling in the field, Suor Kimhuor, Chhum Moeun, Chhuoy Kalyan and the different field authorities including all the Directors and Teachers of the 24 primary schools.
References 1. The effect of malaria control on Plasmodium falciparum in Africa between and World Health Organization. World malaria report World Health Organization; The challenge of emerging and re-emerging infectious diseases. Trop Med Int Health. Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, — View Article Google Scholar 6. National dengue surveillance in Cambodia — epidemiological and virological trends and the impact of vector control.
Bull World Health Organ. Arima Y, Matsui T. Epidemiologic update of dengue in the Western Pacific Region, Western Pac Surveill Response J.
Epidemiology and burden of disease from Japanese encephalitis in Cambodia: results from two years of sentinel surveillance.
Adults have been collected in rainforests in baited traps type of bait unspecified [ 17 ]. Larvae can be collected in flood pools in rainforests [ 17 ]. Female unknown [ 17 ]. Distribution: Cambodia, Malaysia, Singapore and Thailand [ 64 ]. Immature stages can be collected in mangrove areas [ 17 ]. Immature stages and adult male unknown [ 17 ]. Distribution: Cambodia and Thailand [ 32 , 64 ]. Distribution: Cambodia, Indonesia and Malaysia [ 32 ]. Larvae unknown [ 17 ].
Immature stages can be collected in various types of temporary ground pools and in axils of the Colocasia plants. Adults are diurnal and can readily bite humans [ 17 ]. Distribution: Cambodia, India, Myanmar and Thailand [ 64 ]. Adults collected in crab holes, while larvae were found in footprints in ricefields near mangrove swamps in salt marshes and in brackish waters [ 17 ].
The genus Culex consists of species divided among 26 subgenera [ 25 ]. In Cambodia, five subgenera are represented: Culex 15 species , Culiciomyia five species , Eumelanomyia 11 species , Lophoceraomyia 18 species and Oculeomyia three species.
Culex larvae occur primarily in semi- or permanent bodies of groundwater and can be found in artificial containers. Culex females feed mainly during the night. Some species are reported to be of medical importance and can be vectors of numerous arboviruses and filariosis [ 25 ]. Species of medical importance, confirmed vector of Japanese encephalitis virus [ 6 ].
Larvae can be found in a variety of temporary and semi-permanent groundwater habitats such as pools, puddles and small streams, but also occasionally found in artificial containers such as barrels and water tanks. Females prefer to bite large domestic animals [ 7 ]. Species of medical importance, confirmed vector for Japanese encephalitis virus [ 6 ]. Larvae can be found in water bodies containing a high degree of organic pollution and close to human habitations.
Females highly anthropophilic and biting at night [ 9 , 81 ]. This species is a vector of avian malaria, confirmed vector for Japanese encephalitis virus [ 6 ], West Nile virus, St Louis encephalitis virus, Rift valley virus, Edge Hill virus, Eubenangee virus, Getah virus, Kokobera virus, Koongol virus, Kowanyama virus, Kunjin virus, Mapputta virus, Stratford virus, Trubanaman virus, Wongal virus, Chikungunya virus [ 3 ].
It is also a primary vector of W. Larvae are found in brackish, salt and fresh groundwater habitats and some artificial containers in coastal areas. Females feed primarily on birds and pigs, but can bite humans [ 24 ]. Possible vector of Japanese encephalitis virus.
Larvae are found in many temporary, semi-permanent and permanent groundwater habitats that are sunlit and contain vegetation. Habitats can be ground pools, streams, swamps, and low-salinity tidal marshes [ 7 ]. Females are primarily cattle- and pig-biters, but in their absence, they will feed on humans [ 7 ].
Larvae are typically found in ground pool habitats that include puddles, ditches, ponds, animal and wheel tracks, and rice fields containing emergent and aquatic vegetation.
Females feed primarily on pigs and birds, but in their absence will readily bite humans [ 81 ]. Important vector of Japanese encephalitis virus [ 81 ]. Species collected throughout Cambodia. Male and pupae unknown. Distribution: Cambodia and Thailand [ 66 ]. Female and larval stages unknown. Distribution: species only known from Cambodia [ 31 ]. Distribution: Cambodia and Thailand [ 31 , 66 ]. Pupae unknown. Female unknown. Distribution: Cambodia and Malaysia [ 82 ].
Distribution: Cambodia, Malaysia and Thailand [ 66 ]. Distribution: Cambodia, Indonesia and Malaysia [ 81 ]. Immature stages restricted to groundwater habitats containing Spirogyra algae. Females are primarily bird feeders in Thailand. In New Guinea, a form readily bites humans [ 7 , 24 ]. The genus Culiseta includes 39 species divided among 7 subgenera Allotheobaldia, Austrotheobaldia, Climacura, Culicella, Culiseta, Neotheobaldia, and Theomyia [ 25 , 85 ].
The Culiseta are large mosquitoes and cold-adapted species which occur only in warmer climates during the colder times of the year or at higher elevations where temperatures are low. Only the subgenus Culimacura is present in Cambodia with one species. Immature stages of Culiseta Climacura species are usually found in permanent bodies of water, mainly swamps, but may also inhabit semi-permanent pools.
Females primarily feed on birds but can occasionally feed on humans. Distribution: Cambodia and Malaysia [ 18 ]. The genus Lutzia includes 9 species divided among 3 subgenera: Insulalutzia , Lutzia , and Metalutzia [ 25 ].
Only the latter is represented in Cambodia by 3 species. Adults are large, and females feed on livestock, and rarely on humans. Very little information is known about the biology of the adults.
Larvae are predaceous and occur in a wide variety of groundwater habitats, from tree holes to artificial cavities. Generally, they appreciate water with high organic content [ 25 ].
Upon hatching, the larvae will immediately consume nearby, similar-sized, larvae. Adult female seems to feed preferentially on avian hosts [ 7 ]. The larvae can be found in various kinds of temporary and semipermanent groundwater habitats and occur frequently in artificial containers as well as rock pools, stream margins and tree cavities [ 7 ]. The larvae prefer water containing high organic content.
They are predaceous and can attack other arthropods nearby. The host preference for the adult female is not known, but humans have been reported to be an occasional host [ 7 ]. Collected in Kampong Cham and Battambang provinces. The genus Ficalbia is represented by 8 species present in the Afrotropical, Palearctic and Oriental regions [ 25 ]. Only one species is present in Cambodia.
Very little is known about the biology of this genus. Their larvae can be found in swamps, marshes, ponds, pools and river margins with abundant vegetation. Nothing is known about the biting habits of females [ 67 ]. Collected in Cambodia in primary forests, in shaded areas. The genus Mimomyia includes 45 species shared among 3 subgenera: Etorleptiomyia, Ingramia, and Mimomyia [ 25 ].
The 3 subgenera are present in Cambodia hosting 2, 2 and 3 species, respectively. Mimomyia are generally small mosquitoes. The larvae of most species occur in swamps and marshes with dense vegetation. The larval siphon of several species is modified for piercing aquatic plants to obtain oxygen.
Larvae of subgenus Ingramia are found in the leaf axils of plants. The adults of several species have been reported to bite humans, but none are serious pests. Most species appear to be active at night. Immature stages unknown. Distribution: known only from Cambodia [ 25 ]. Collected in Cambodia only from Battambang province using BG traps.
The genus Hodgesia consists of 11 species [ 25 ]. These mosquitoes are minute in size, and the immature stages can be found in swamps and marshes, in water containing very dense vegetation, often associated with larvae of Mimomyia spp.
The feeding habits of most species are unknown, but some species have been reported to bite humans [ 25 ]. The genus Coquillettidia consists of 58 species, generally large, and yellowish [ 25 ].
The genus is subdivided into 3 subgenera: Austromansonia , Rhynchotaenia, and Coquillettidia [ 25 ]. Only the latter is present in Cambodia and comprises 3 species. Their larvae attach themselves to aquatic plants to obtain oxygen. Adult females are known to bite humans and domestic animals [ 59 ]. The genus Mansonia includes 25 species subdivided into 2 subgenera [ 25 ]. The subgenus Mansonia comprises 15 species distributed in the New World, while Mansonioides occurs in the Old World and consists of 10 species.
Only the latter is present in Cambodia with 5 species. The larvae of Mansonia occur in permanent waters in association with aquatic plants and attach themselves to their floating roots in order to obtain oxygen. Females of several species are nocturnal, and are known to transmit several arboviruses [ 25 ].
Known to transmit Chikungunya [ ]. Immature stages can be found in shaded pools in swamp forest. Adults are crepuscular and females seem to prefer cattle but can feed on humans [ ]. Immature stages can be found in swamps and pools containing abundant vegetation [ 9 , 54 , 67 ]. The genus Orthopodomyia consist of 36 species distributed worldwide.
Very little is known about their biology. Larvae occur principally in tree holes, but some species are found in bamboo internodes, inside Bromeliaceae and Heliconia plants. The larvae can sometimes be found in artificial containers. Adults are nocturnal and are found mainly in forests. The feeding habits of females are largely unknown, but they are probably ornithophilic.
Two species in the Oriental Region are known to approach and bite humans. This genus is not considered to be of medical importance [ 25 ]. The immature stages have been observed to breed in tree holes, bamboo stumps and artificial containers [ ]. In Cambodia, the larvae were collected in artificial containers. The immature stages have been observed in tree holes, bamboo stumps and artificial containers [ ].
Distribution: this species was only known from Thailand [ 65 ]. Species collected in Mondul Kiri and Rattanak Kiri provinces. The genus Malaya is a small group represented by only 12 species [ 25 ]. The immature stages can be found in plant cavities, such as leaf axils, tree holes or water-filled nests of arboreal ants.
The adults are diurnal and incapable of taking a blood meal. Their feeding habits are unique among mosquitoes: they feed on the regurgitation of ants: specifically, both sexes obtain a sugar meal from ants that have collected honeydew from scale insects Hemiptera: Coccoidea. To accomplish this, the mosquito accosts an ant and brings the tip of its proboscis into contract with the mouth of the ant until a drop of liquid is produced. The regurgitated liquid is rapidly sucked up and the ant goes away unharmed.
This genus is represented by two species in Cambodia. The genus Topomyia includes 65 species in 2 subgenera: Miyagiella and Topomyia. Only the latter is present in Cambodia represented with 4 species. Topomyia are considered forest mosquitoes. Very little is known about the biology of the adults, but as females do not feed on humans they are not considered to be of medical importance.
The larvae can be found in leaf axils, Nepenthes pitcher plants, bamboo internodes and stumps and sometimes tree holes [ 25 ]. Distribution: Cambodia and Thailand [ 34 , 65 ]. Described from Cambodia [ 34 ], recorded from Philippines Palawan islands. According to Miyagi et al. The larvae preys upon small crustacean and chironomid larvae.
Larvae can be found in leaf axils of plants [ 45 , 88 ]. The genus Tripteroides hosts species divided into five subgenera: Polylepidomyia , Rachionotomyia , Rachisoura , Tricholeptomyia and Tripteroides.
Only 2 Rachionotomyia and Tripteroides are present in Cambodia. The larvae inhabit small collections of water in tree holes, bamboo, coconut shells and husks, fallen leaves, pitcher plants or even snail shells. They feed on arthropods or their remains. Adults are diurnal. A few species feed on humans, but nothing is known about the feeding habits of most species [ 25 ]. Distribution: India, Sri Lanka and Thailand [ 65 ]. Collected in Cambodia from larvae reared from Nepenthes water, collected in the primary forest of Rattanak Kiri and Mondul Kiri provinces.
The larvae can be found in bamboo, tree holes, pitcher plants [ 96 ]. Among the Oriental species, only Tr. Distribution: Cambodia, Malaysia and Thailand [ 65 ]. Collected in Cambodia from clear water in tree holes, in the primary forest of Mondul Kiri province. Distribution: Indonesia, Malaysia and Thailand [ 65 ].
Species found in bamboo forests [ 96 ]. The genus Toxorhynchites comprises 89 species worldwide, divided into 4 subgenera: Afrorhynchus, Ankylorhynchus, Lynchiella and Toxorhynchites [ 25 ].
The Cambodian species belong exclusively to the subgenus Toxorhynchites. The larvae are predators, feeding mainly on other mosquito larvae. They can be found in plant cavities, mainly tree holes and bamboo stumps [ 95 ]. Adults are covered with iridescent scales and are typically large and colorful mosquitoes. Both males and females are diurnal and feed exclusively on nectar and other sugary substances.
They do not display blood-sucking behavior and are not considered to be of medical importance, but their larvae can be used for pest control [ 14 , 87 ]. Distribution: India, Malaysia and Thailand [ 99 ]. Specimens obtained from larvae collected in tree holes in Mondul Kiri province.
This genus includes species worldwide divided into 2 subgenera: Pseudoficalbia and Uranotaenia. Both are present in Cambodia, hosting 13 and 14 species, respectively [ 25 ]. The feeding preferences of most species are currently unknown but field observation tends to indicate that amphibians, reptiles, birds and mammals serve as hosts. Females can bite humans but do not seem to be involved in pathogen transmission. Immature stages can be found in a wide range of habitats: most species inhabit groundwaters, including swamps, marshes, stream margins and temporary pools with vegetation, but many also use crab holes, tree holes, bamboo, plant parts on the ground, leaf axils, flower bracts, pitcher plants or artificial containers [ 25 ].
Distribution: Cambodia and Thailand [ 55 ]. Immature stages seem to be associated with crab holes, and occur essentially in small freshwater crab holes at the margin of shallow running mountain or foothill streams, or at the edge of springs or seepages and generally under the cover of secondary or primary forests.
Adults can be observed resting on rocks over streams, up to an elevation of m [ 55 ]. Distribution: Thailand [ 67 ]. Collected in Rattanak Kiri province only. According to Peyton [ 55 ], this species is the most common of the genus in Southeast Asia, immature stages tend to colonize a very wide range of aquatic habitats. Distribution: Thailand and Cambodia [ 55 ]. Immature stages can be found in swamps, seepage pool or bog, or stream pool [ 67 ].
Most of the collection of immature stages was done in crab holes and marsh swamps areas [ 55 ]. Distribution: Cambodia, Thailand and Vietnam [ 55 ]. Species restricted to forested hills and mountainous areas. Immature stages can be collected in crab holes on the banks of shallow fresh running streams and elephant footprints in bogs where crab holes were present.
Adult often rest on vegetation or rocks along stream margins [ 55 ]. Prefer secondary forests where bamboo habitats are abundant. Immature stages show a preference for a variety of bamboo habitats, located on or near the ground, bamboo internodes, with small or moderate entrance holes, bamboo stumps, cut bamboo, or tree stumps.
Most collections were done at an elevation below m [ 55 ]. Collected in Cambodia from primary forest in Mondul Kiri province, the immature stages were collected from transparent water colored with tannin in bamboo tree holes.
Distribution: China, India, Malaysia and Thailand [ 67 ]. Collected in Mondul Kiri and Kampong Thom provinces. Immature stages collected from tree stumps tree holes and artificial containers.
Mainly found in forests and anthropic environments at elevations up to m [ 55 ]. Immature stages usually found in bamboo stumps, tree holes and sometimes artificial containers. Immature stages are usually associated with larvae of Aedes sp. Adults do not bite humans but seems to feed on toads [ 55 ]. Numbers below the bars indicate the total number of mosquitoes. Zero in the graph indicates no infected mosquitoes.
In the CBNTs, the prevalence of Plasmodium -positive mosquitoes was significantly higher during the dry season 5. The infectious mosquitoes collected in the HBNTs belonged to 8 different mosquito species molecular identification; Table 2.
Interestingly, an additional 12 Anopheles species were also found infected by human malaria parasites among the mosquitoes collected in the CBNTs A. We assessed the inherent mosquito host preference by calculating the anthropophily index AI for the different species, defined as the number of Anopheles sp. Anopheles dirus —A total of An. Human Plasmodium sp. The anthropophily index was calculated in the forest sites only and was Infectious mosquitoes were significantly more anthropophilic Anopheles dirus females were more anthropophilic during the dry season There was no difference in the anthropophily index of A.
Anopheles kochi —A total of A. Anopheles kochi females were significantly more anthropophilic during the daytime Indeed, A. We genotyped previously characterized mutations in ace-1, kdr, and rdl genes, GS 45 , LF 45 and AS 52 , respectively. The few missing genotypes were due to PCR amplification failure. There were four species of total that carried resistant mutations. Two A. Interestingly, a single species, A.
To gain a better understanding of this apparent enrichment of known insecticide resistance mutations in A. When resistant allele frequencies were compared between the A.
Of the total A. For the LF mutation, the distribution of alleles by ecological site was not uniform Fig. All heterozygotes were verified by amplifying and resequencing the necessary fragment and visualizing the resultant sequence trace files.
Insecticide resistance mutations are very frequent in Anopheles peditaeniatus. All A. Each row of three boxes represents genotype data from a single A. Out of A. A single individual was homozygous for the rdl resistant mutation and heterozygous for the ace-1 mutation.
Five individuals carried a resistance mutation at only one of the 3 loci tested, 3 were ace-1 mutation homozygotes, 1 was heterozygous for rdl resistance and 1 was homozygous for rdl resistance with no sequence available for ace-1 resistance.
Geographic distribution of insecticide resistance mutations in A. Allele frequencies for ace-1 GS top , kdr LF middle and rdl AS bottom are shown as a function of sampling location. A similar pattern is observed for rdl , but here the total number of susceptible alleles does not provide the power to detect significant differences.
Only those 8 sites with at least 5 A. Resistant res and susceptible sus alleles are colored according to the legends, with resistant alleles in the darker shade. We also examined the frequency of the kdr LF, ace-1 GS and rdl AS insecticide resistance mutations in rarer mosquito species that were detected in the random subset of samples molecularly typed for species.
In these rare species, rates of resistance alleles were high for rdl AS with resistance being present in 8 of the 14 species and in the unknown Anopheline.
In contrast no mosquitoes carried the LF mutation in kdr and only A. A 2 km buffer zone around each mosquito collection site was used to determine the percentage of the different land cover classes present and showed high variability between sites Fig. Patches of evergreen forest, forest and plantation forest were observed in all sites. Croplands were observed in all buffer zones, from 0. While more A.
The most conserved forest sites Forests 3 and 4 were also associated with lower densities and lower diversities of mosquitoes as also observed in Plantations 2 and 3 Fig. Sample sizes are given in parentheses. Although Forests 3 and 4 had high proportions of natural forests, their high PD and ED values indicate more fragmented landscapes than Forests 1 and 2. Patch richness only varies between 4 and 5 types of land cover classes therefore an increase value of SHDI and SIDI corresponded to an increase evenness between patch types, with the highest patch evenness observed in Forests 2, 3 and 4 as well as Plantations 2 and 3.
Villages and forest near the villages exhibited uneven patch distribution associated with fragmented landscapes. Interestingly, sleeping overnight in the field sites was reported in 3. A recent study in Cambodia found similarly high mosquito diversity although the 27 species were collected over three different provinces Three species were unique to the latter collection while 5 other species were unique to our collection, further highlighting the high Anopheles mosquito diversity in Cambodia.
Anopheles dirus mosquitoes were collected even in highly degraded and fragmented forest sites, suggesting that they can adapt to human-altered habitats. Interestingly, mosquito densities were similar in the dry and the rainy seasons in the forest sites whereas densities strongly dropped in the dry season in the other sampled sites. Thus, patches of forests might represent refuges for the mosquito populations during the dry season until they disperse again to the field and villages in the rainy season, as has been suggested previously for A.
Consequently, these forest refuges might not only maintain Anopheles populations, but also serve as a transmission reservoir during the dry season since human activity is also high in the forest sites during this period.
This is in line with the local epidemiology where although malaria cases exhibit a seasonal pattern in Cambodia, symptomatic cases are still presenting at the health centers during the dry season. The efficiency of cow baited traps, either collecting resting mosquitoes from the interior of a surrounding tent or directly on the surrounding vegetation, in collecting large number of mosquitoes has been shown previously 28 , Historically the CBNT sampling technique has been dismissed as unrepresentative of malaria parasite vectors.
Here, we investigated the innate host-preference of mosquitoes using exactly the same trapping techniques for both human and cow. We showed that the collected species of mosquitoes were all generalist or zoophilic in their preferences.
The random subset of molecularly identified species showed that among the 19 species collected in both type of traps, A. These 5 species enriched in one type of trap represented 47 and 25 samples for the CBNTs and the HBNTs respectively, out of the infected samples which were typed Table 2.
Furthermore, the overall malaria parasite prevalence in HBNTs 4. Moreover, as CBNTs are a more efficient means of capturing mosquitoes, a greater numbers of species of mosquitoes are able to be studied and the risk to human volunteers is eliminated. Overall, these results are of importance for two reasons.
First, screening for malaria parasites only in mosquitoes collected on humans is likely to miss species involved in malaria parasite transmission unless very large collection efforts are carried out. Second, it suggests that many of these mosquito populations are largely maintained by feeding on animals, and that, different from African malaria vectors, the notion of highly preferential human feeding may be inaccurate in the SEA region.
Knowing that opportunistic feeding is very common could inform the design of new vector control tools to control malaria infection levels. Labeling of a particular mosquito species as zoophilic or anthropophilic does not represent the reality of more opportunistic feeding preferences. We found a total of 20 different Anopheles species infected with malaria parasites Table 2.
Laurent et al 26 found 5 different species infected with P. These two studies are in strong contrast with another collection in Cambodia which found malaria parasites in only three mosquito species: A. However it should be noted that only human landing catches were used in Durnez et al. Laurent et al 26 were captured in cow baited tents and In addition, high malaria parasite prevalence was observed in the local human population 8.
These persisting pockets of high malaria risk as well as our selection of forest sites based on recent P. Anopheles dirus and Anopheles minimus are usually considered the main malaria vectors in the Greater Mekong Subregion 21 , Whereas A. Interestingly, the work from Durnez et al 19 carried out in in three Cambodian provinces found Several hypotheses could explain these differences in species composition.
First, collections using cow baited traps in our study and the study by St. Laurent and colleagues likely increased species diversity and numbers, consequently decreasing individual species relative abundance.
Secondly, variability between sampling sites is likely to be an important factor influencing the relative abundance of mosquito species collected. Thirdly, the increased human influence upon Cambodian landscapes including the studied area 55 , 56 , 57 is likely to impact mosquito ecology over time and space resulting in large changes in Anopheles communities.
Finally, malaria vector control measures might have shifted the Anopheles species involved in malaria parasite transmission as observed in Africa 58 , It is generally accepted that the vast majority of mosquito feeding happens in the period between dusk and dawn.
Malaria vectors are traditionally collected between 6 pm and 6 am since these 12 h usually encompass their peak biting times. Unlike their African counterparts, SEA Anopheles are known to be mainly early-biters and extended collections have sometimes been carried out 14 , 17 , 18 , 19 , with two studies sampling mosquitoes over 24 h 20 , Although these studies did collect small numbers of Anopheles during the day, the overall sample sizes were considerably lower than those we report here 20 , The non-negligible proportion of Anopheles mosquito biting during the daytime, when people are active and not protected by a bed net, highlights the importance of residual transmission in this area.
Similar estimates would be needed to adequately quantify the importance of residual transmission in SEA, still, with local vectors being mainly early and outdoor -biters, SEA estimates are likely to be at least equal if not largely higher than those reported for Africa.
Malaria infection rate in the HBNTs was not significantly correlated with the different collection sites, which is likely due to the comparatively low numbers of mosquitoes sampled from villages, forest near the villages and plantations as compared to the forest sites as well as the absence of infectious mosquitoes collected in these sites during the dry season Fig. There were 6 times more infectious bites per day in the forests compared to the villages during the rainy season Table 1 further supporting the higher malaria risk in forests in at least northern Cambodia.
Few people reported sleeping overnight when working in their field or plantation sites 3. Coupled with the fact that a non-negligible proportion of vectors bite during the day time, these observations call for new malaria vector control tools adapted to forest work activities. These control tools would ideally be easy to use and carry, and provide protection during both day and night hours. The random subset of mosquitoes genotyped for known insecticide resistance mutations ace-1 GS, rdl S, and kdr LF indicated that individuals carried mutations associated with response to all the different classes of insecticides.
Indeed, among the mosquitoes genotyped only 4 out of the 12 species represented were found carrying any of the three genotyped insecticide resistance mutations: A. Interestingly, two A. Since A. However, with fragmentation and degradation of forests, human activities are increasingly in closer contact with sites harbouring forest mosquitoes leading to potential insecticide resistance emergence even in forest-associated species.
Interestingly, insecticide resistance genotypes were overrepresented in A. This mosquito species is mainly zoophilic, outdoor biting and breeding in ponds and rice fields 29 , 62 , 63 , 64 , 65 , Therefore exposure to agricultural pesticides could explain the observed pattern of insecticide resistance mutations.
Even though A. Thus, A. Overall, this study illustrates the importance of diverse collection methods, sites and seasons to better characterize Anopheles mosquito ecology in SEA. In addition, the increased patchiness and landscape changes following economy-driven human activities is likely to result in large changes in Anopheles communities over time and space.
The frequency of opportunistic feeding and day-biting behavior highlights key mechanisms driving residual human malaria transmission in Cambodia. Further investigations on the importance of day-biting in various Anopheles species and ecological sites would be interesting to better quantify its frequency. Finally, the low frequency of known insecticide resistance mutations with the exception of a single vector species suggest that new integrated vector control methods could be added as a component in the malaria vector control toolbox, which would help accelerate malaria elimination in the Greater Mekong sub region.
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