Geographical distribution of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and genetic diversity of invading population of Ae. albopictus in the Republic of the Congo

Background: The arbovirus vector, Aedes albopictus, originating from Asia, has recently invaded African countries, including the Republic of the Congo, where it was associated with a chikungunya outbreak. Up until now, little was known about its distribution in relation to the native Aedes aegypti and how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution of Ae. albopictus and Ae. aegypti in the Republic of the Congo and explored the genetic diversity of the invading species, Ae. albopictus. Methods: Immature stages of Aedes were collected in nine locations in the Republic of the Congo in 2017 following a north-south transect and reared to adult stage. Adults were morphologically identified, counted and grouped according to species and location. Genetic diversity of Ae. albopictus was assessed by analyzing the cytochrome oxidase I ( COI) gene. Results: Ae. albopictus and Ae. aegypti were found together across the country in all the locations investigated. The invasive species is predominant over the native species in all locations except Brazzaville, suggesting that Ae. albopictus is displacing Ae. aegypti across Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire, Ae. albopictus was more prevalent than Ae. aegypti in the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity of Ae. albopictus with only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed that Ae. albopictus from Congo originated from other tropical Asian countries such as China, likely as a result of increasing trade links. Conclusion: These findings are important for the implementation of vector control strategies and can serve as a foundation for further research on these vectors in the country.


Introduction
Arthropod-borne viral diseases such as dengue, zika and chikungunya have emerged or re-emerged in several countries of the world during the past decades [1][2][3][4] .
These viruses are transmitted to vertebrates, including humans, by the bites of infected mosquitoes that share the same ecological niche as the host organism. Indeed, two distinct ecological cycles, enzootic and urban epidemic cycles, have been well documented 5,6 . The enzootic cycle occurs in the sylvan environment, involving non-human primates and wild mosquitoes, while urban epidemic cycle occurs in urban environments, implicating human beings and urban mosquitoes such as Aedes aegypti Linneaus 1762 and Ae. albopictus (Skuse) 1894. Other potential modes of Zika virus transmission to humans have been evoked notably via sexual intercourse or via blood donor 5 . Both epidemic vectors, Ae. aegypti and Ae. albopictus, are found in sub-Saharan Africa, where Ae. aegypti is native. Two subspecies of Ae aegypti, Ae. aegypti formosus and Ae. aegypti aegypti, were formally identified by Mattingly in 1957 7 . Ae. aegypti formosus, is a dark colored mosquito confined to African forests while Ae. aegypti aegypti is light-colored with white abdominal scales and is found in human-dominated habitats primarily outside Africa. Generally, Ae. aegypti collected in central Africa match Ae. aegypti formosus 8 .
While Ae. albopictus is a native of South East Asia, it has now invaded all the five continents during the past 30-40 years 9,10 . This rapid global spread has been caused mainly by sales and distribution of used tires across the world 11 coupled with the ecological plasticity of the species, enabling its adaptation to various environments 9 . Ae. albopictus was reported for the first time in Central Africa in early 2000 12 and is currently present in almost all central African countries 13 , where it tends to supplant the indigenous species Ae. aegypti in human-domesticated environment 14,15 . The predominance of Ae. albopictus over Ae. aegypti in sympatric areas has been shown to result from the higher mating competitiveness of Ae. albopictus over Ae. aegypti 16,17 . Previous studies in Central Africa showed that both Ae. aegypti and Ae. albopictus can be found together in the same location and often share the same larval habitats 14,15 . In this region, the immature stages of both species develop in stagnant water found mainly in peri-domestic containers such as used tires and discarded tanks. However, in the sympatric area, Ae. albopictus prefers containers surrounded by vegetation whereas Ae. aegypti prefers containers located in neighbourhoods with high building densities 15,18 .
Dengue, Zika and chikungunya were for a long-time considered to be rare in Central Africa, because only sporadic epidemics were reported in the rural environment, with isolation of the viruses in wild mosquitoes and humans 19,20 . In the past decades, several outbreaks have been reported in this part of the world, notably a concurrent dengue/chikungunya outbreak in Gabon in 2007, with more than 20, 000 cases of chikungunya 2 , and a large chikungunya outbreak in 2011 in the Republic of the Congo with more than 11, 000 cases 21 . This suggests an epidemiological modification of arboviral diseases in the region. During these outbreaks, Ae. albopictus was established as the major vector particularly in Gabon 22,23 , where Zika was detected in this species 24 . In Congo, both Ae. aegypti and Ae. albopictus were found to be positive for chikungunya virus 25 , implicating both species in virus transmission. This investigation was the first to confirm the presence of Ae. albopictus in the Republic of the Congo. Since then, no study has been undertaken to compare the geographical distribution and prevalence of Ae. aegypti and Ae. albopictus in the Republic of the Congo as well as the genetic diversity of the invading species. Indeed, previous studies in Central Africa based on polymorphisms to the cytochrome oxidase subunit 1 (COI) gene indicated that Ae. albopictus populations in Cameroon are related to tropical rather than temperate or subtropical out-groups 26 . However, the Central African Republic population segregated into two lineages: the first encompassed specimens from tropical areas including all the haplotypes from Cameroon and the second lineage encompassed temperate and subtropical areas 15 , suggesting multiple sources of Ae. albopictus.
To improve entomological surveillance and the control of these arbovirus vectors in the Republic of the Congo, we present here the current nation-wide geographical distribution and prevalence of Ae. aegypti and Ae. albopictus in this country, and establish the genetic diversity of the invading population of Ae. albopictus using the COI gene.

Sampling sites
Mosquitoes were collected in May and November 2017 (Pointe Noire only) corresponding to the rainy season in nine locations in the Republic of the Congo across the north-south transect (Table 1 and Figure 1). The Republic of the Congo is located in Central Africa, straddling the equator. Two main types of vegetation are found. The forest in the north, covering 60% of the national territory, and the savannah, which occupies the remaining parts of the country. There are three types of climate. The equatorial climate is found in the north of the country, characterized by high humidity and rainfall greater than 1,700 mm per year, with an average temperature between 24°C and 26°C. The humid tropical climate in the southwest, where annual average precipitation varies from 1,200 mm to 1,700 mm, with an average monthly temperature between 21°C and 27°C. The subequatorial climate, experienced at the plateau and basin regions, has an average annual rainfall of about 1,600 mm. Because the spread of Aedes mosquitoes mainly relies on human activities, sampling was focused on human-domesticated

Mosquito collection, rearing and identification
In Brazzaville and Pointe Noire, the two most populated cities of the Republic of the Congo, the difference between downtown and suburban was examined during the investigation. In the other locations, however, samples were collected randomly throughout each city and pooled together. In each selected location, all containers with water were inspected and positive containers (with at least one Aedes larva or pupa) were recorded. Immature stages of Aedes were collected, transported to the insectaries, pooled according to the location and reared to adult stage for morphological identification. G0 adults were stored at -20°C for molecular and genetic analyses. The comparisons between the prevalence of Ae. aegypti and Ae. albopictus in each location, across the country were performed using multiple chi-square test.
Mitochondrial DNA analysis for Ae. albopictus Genomic DNA was extracted from 20 whole Ae. albopictus per location (nine locations) using the Livak protocol as previously described 27 . DNA extracts from each location were used as templates to amplify 700-bp fragment of COI gene. The sequences of primers used are: albCOIF 5'-TTTCAACAAA TCATAAAGATATTGG-3' and albCOIR 5'-TAAACTTCTGGA TGACCAAAAAATCA-3' 28 . Polymerase chain reaction (PCR) amplification was performed using a Gene Touch thermal cycler (Bulldog Bio, Portsmouth, USA), as described previously 28 . PCR products were detected by agarose gel electrophoresis in Tris-Acid-EDTA buffer (TAE). The gel was prepared with Midori green, staining dye, and visualized with the aid of UV light. PCR products from each location with very good amplification were purified using the Exo-SAP protocol and sent to the Centre for Genomic Research (Liverpool, UK) for sequencing.

Results
Containers inspected and prevalence of Ae. aegypti and Ae. albopictus A total of 640 containers with water were investigated across the Republic of the Congo (Table 2). Among them, 42.9% were positive for immature stages of Aedes. Containers were classified into three main groups: domestic (flower pot and water storage tanks), peridomestic (used tires, discarded tanks and car wrecks) and natural (axil of plants). Used tires were the most prevalent habitat and most productive containers in all the locations, ranging from 18.1% in Pointe Noire to 100% in Lefini ( Table 2). The presence of Aedes in other containers was very limited.   In total, 6,684 specimens of immature stages of Aedes were identified, comprising 72.24% of Ae. albopictus, 27.70% of Ae. aegypti and 0.06% (four specimens collected in Brazzaville suburb) of Aedes simpsoni. Ae. aegypti and Ae. albopictus were found together in all the locations investigated ( Figure 1 and Table 3). However, Ae. albopictus was predominant in all the locations except in Brazzaville. When samples from the two major cities, Brazzaville and Pointe Noire, were divided according to the environment (downtown versus suburb), Ae. albopictus was found more prevalent in the suburbs (95.62% and 75.39% in Brazzaville and Pointe Noire, respectively) than Ae. aegypti, whereas the reverse was true for the downtown areas (Table 3).
Mitochondrial DNA analysis of Ae. albopictus In total, 127 specimens of Ae. albopictus from nine locations across the Republic of the Congo were analysed using the COI gene. Sequence analysis, based on 638 nucleotides, revealed a low polymorphism, with only two mutational sites defining three haplotypes namely H1, H2 and H3 ( Figure 2B). Consequently, this resulted in low haplotype diversity (Hd=0.24) and nucleotide diversity (π=0.00005) indexes (Table 4). The most frequent haplotype, H1 (86.6%), was detected in all the locations. Supplementary Table 2 shows the haplotype distribution per location. The haplotypes H2 (10.2%) and H3 (3.2%) were found in three (Brazzaville, Ouesso and Oyo) and two (Brazzaville and Lefini) locations, respectively (Table 4 and Figure 2). The dominant haplotype matches perfectly with the COI gene sequence deposited in GenBank that originated from China (KU738429.1). A higher genetic diversity was reported in Brazzaville where all the three haplotypes were reported. The haplotype network showed that each haplotype was separated from the others by one mutational step ( Figure 2). Overall, Tajima's D (D=-0.294) and Fu's Fs (Fs=-0.024) statistics were negative, but not statistically significant. Phylogenetic tree generated and analysed based on 445 nucleotides previously published in GenBank showed that the Republic of the Congo's haplotypes were closely related to the sequences from China, Singapore, Papua New Guinea and Christmas Island (Figure 3). In both major cities where samples were analysed according   to peri-urban and downtown environment, results revealed the predominance of Ae. aegypti in downtown areas but less in peri-urban areas. Similar findings to these were reported previously in Central Africa 15,18,40 . These observations are consistent with former studies indicating the segregation of habitats in sympatric areas according to urban environmental gradients as the main factor responsible for the coexistence of Ae. aegypti and Ae. albopictus 42,43 . Used tyres were the most common container found positive for Aedes in all the locations. This is in accordance with previous studies in Central Africa showing that used tires are the main productive for both Ae. aegypti and Ae. albopictus 13,15 . However, the current study targeted mainly garages and tire shops to increase the chances of discovering immature Aedes.

Acknowledgments
We thank the people living around all the sampling sites for their cooperation during the field investigations.

Supplementary material
Supplementary

Kamgang
. conducted this study with the overall aim of assessing the geographical distribution of et al and and the genetic diversity of in the Republic of Congo.

Aedes aegypti
Aedes albopictus Ae. albopictus Specifically, the authors sampled immature mosquitos among their usual breeding sites in 9 sites Aedes across several eco-regions, reared immature to adult stage for species morphological identification, and analyze the diversity of using CO1 gene method.

Ae. albopictus
This study merits a careful and particular attention. While it is not novel to describe the Aedes mosquito species in Africa, this study is the one focusing on the invading Asian tiger which has been Ae. albopictus newly introduced into the African continent by the means of transportation and trade. The authors identified three Aedes species ( , and ) that may have an Aedes aegypti Aedes albopictus Aedes simpsoni important impact on public health as they may carry and transmit pathogens. The study shows some clear results, which include the variation of among mosquito species abundance of and Aedes aegypti Aedes and genetic diversity of species as a function of location. The outcomes were albopictus Ae. albopictus represented as texts, figures and tables. The outcomes of this work could be explored for better understanding of the distribution of and species that can influence the Aedes aegypti Aedes albopictus transmission of arboviruses to humans and may contribute the control arboviral diseases in the Republic of Congo.
Despite its strength, there are a number of weaknesses and gaps to the study that need to be addressed. The study is inherently limited to represent the species and specimens of non-mosquitoes and Aedes some features of the containers. The authors did not characterize the larval habits by looking at some ecological features of the containers. Some important characteristics such as the exposure of containers to sunlight, the presence or absence of microbial food inputs, air temperature, rainfall, temperature of water, turbidity of water, color of containers and some biological interactions (predation, competition, sympatry…) involving predators, competitors and other associations among mosquito larvae were not reported. The report of these factors may probably help to better understand the difference in ecological distribution and patterns between and species. The authors talked about Aedes aegypti Aedes albopictus the displacement of due to the presence of in the abstract without clearly Aedes aegypti Aedes albopictus indicating the strength of the displacement and presenting data showing this important ecological phenomenon. The objective of this study is not clear to me. The hypothesis of this study was not provided. The authors should provide the objective and hypothesis of their works.
The writing throughout the manuscript is good to me. However, the writing needs to be improved in some The writing throughout the manuscript is good to me. However, the writing needs to be improved in some places. In addition, as the authors did not sample the ecological characteristics of sampling areas and containers, they discussed their findings on the basis of the findings of other studies -this seems to consider them as speculations -the authors should focus on the own data, discuss them on the basis of their own data sampled and drawn inferences after comparing the data with previous studies.
Ultimately, above all, more importantly are the paper's weaknesses that should be addressed. I have provided more detailed and specific comments below.

2) Specific comments Background
Line 3 (It….associated): Please replace "was" with "has been" Line 4-5 (how ….. diseases): It seems that this paper did not focus on the epidemiology of arboviral diseases, but the ecology of Aedes species. Please, it suggested that the authors delete or provide clearer explanation.

Abstract Background
Line 2 (in 2017): It suggested that the authors clarify the period (month) of the study. The authors should write the date at the start of the sentence or at the end of the sentence, but not at the middle. Please, correct.

Results:
The authors stated that has been newly introduction to Congo and that has Ae. albopictus Ae. albopictus displaced . I am concerned -please, what was the distribution of Ae aegypti before the Ae. aegypti introduction of ? Please, how do the authors know that is displacing Ae.

Ae. albopictus
Ae. Albopictus aegypti since I did not see data on such an ecological issue in this manuscript. Please, correct. Line 4-5 (suggesting …….across Congo): The portion of the sentence seems to be a discussion -may I suggest that the authors displace it into the Conclusion. Line 9-10 (supporting …….the Congo): The portion of the sentence seems to be a discussion -may I suggest that the authors displace it into the Conclusion. Line 11-12 (likely …….trade links): The portion of the sentence seems to be a discussion -may I suggest that the authors displace it into the Conclusion.

Introduction
Please replace "zika" with "Zika" in the whole document (please use a capital letter "Z" Line (This rapid …….caused mainly…): Please, replace "was" with "has been" Line (This suggests …… in the region): This sentence is not clear to me -may the authors clarify? May I suggest that the authors clearly provide the objective and hypothesis of this study?

Methods
Line (The humid tropical climate…..in the southwest): It seems that words are missing here -maybe "is found"? Line (In Brazzaville…..downtown and suburban…). How did the authors make the difference between "downtown" and "suburban"? What were the criteria? Line (with at least one Aedes larvae or pupae) -please, may the authors replace "larvae" with "larva" (singular form!) and "pupae" and "pupa" (singular form)? Line (….stage for morphological identification.): please, may the authors specify the determination key used for morphological identification and the reference? used for morphological identification and the reference? Lines (The comparisons between …………chi-square test): Is this a statistical analysis of the data? I am not sure that this corresponds to the title of this paragraph -usually, the statistical analysis of data is separated from the lab works -may the authors write an independent paragraph clearly describing the statistical analysis procedures? It will be interesting if the authors perform geospatial analysis of the data to clearly show the distribution of and across sampling areas.

Ae. albopictus
Ae. aegypti Lines (in each selected location ALL containers, ….were recorded) -I am not sure that it is possible to sample ALL containers in a given site -usually, only readily visible and accessible containers can be sampled -please, it is suggested that the authors reword this sentence? It appears that the authors did not sample the characteristics of containers and habitats, but only the mosquito immatures -If so, how will they explain the distribution of species? It should be important to collect information on the biotic and abiotic characteristics/variables of the sampling sites and breeding containers. May the authors explain or justify the importance of using method to assess the origin of CO1 Ae. instead of other technique? What are the advantages of using method? albopictus CO1

Results
As I said previously, while the authors stated that ALL containers were sampled in such large areas, only 640 containers were sampled? In my experience, it is not possible to sample all containers in such large areas.
Were the breeding sites and land cover not characterized? The characteristics of the breeding containers and land cover are likely to affect species distributions. It is suggested that the authors described the characteristics of breeding sites and land covers surrounding the containers. Aedes It is suggested that the authors add a paragraph focusing on the distribution of both main Aedes species, and , among the containers samples? May the authors also specify the Ae. aegypti Ae. albopictus proportions of containers that both species co-habited? Ae. albopictus suggested that the authors add other mosquito species if they collected.

Discussion
Line (This study has assessed….): please, it is suggested deleting "has". Line (Analyses showed…..is….Brazzaville): please, may the authors replace "is" with "was" (past simple / preterit) -as the authors are reporting their own data? Line (The co-occurrence of …….environmental factors……..both species) -please, may the Ae. aegypti authors explain which environmental factors that influence the co-occurrence of both Aedes species? I did not see a clear link/association/correlation between such environmental factors and and Ae. aegypti distribution. It should be good if the authors sampled and reported the environmental Ae. albopictus variables (vegetation, land cover, temperature, humidity, rainfall, season, sunlight radiation, shade, nature of water of the containers, type of breeding sites, foods, predators, competitors,) in each specific location and look at the relationships between these variable with the presence/or abundance of the species. May the authors provide such relationship between environmental parameters and the occurrence of Aedes species? Line (The ecological plasticity….): The data did not clearly report the ecological plasticity/elasticity in Ae.
which means that the species colonize different habitats (for instance, vegetated/open albopictus environment), containers (natural, peridomestic, domestic…), land covers (rural, urban, forests, environment), containers (natural, peridomestic, domestic…), land covers (rural, urban, forests, savannah…)…. Please, may I suggest that the authors report such findings in the Results season to support their discussion? Line (The prevalence……….peri-urban areas) -Here the authors seem to link the difference in the distribution of Aedes species between downtown and peri-urban environment to eggs tolerance/desiccation to temperature variations. But, as they did not record the temperature among both environments, it is difficult to understand such an inference… Why not other environmental parameters such as human density, human activities/behaviors, shade/sunlight, the nature of breeding sites or containers? Line (This is in accordance with ……….the main productive for both Ae. aegypti and Ae. albopictus): It seems that some words are missing in the sentence -it appears to be "…main productive -breeding sitesfor.." Line (However, the …………..garages and tire shops…) -may I suggest that the authors in the Methods that the larval collection was performed mainly in the garages and tire shops? It seems that the authors were mixing UK/British spelling and US spelling! As the overall manuscript is written using UK English, they should write "tyre" /UK English) instead of "tire" (US English). Please, correct in the whole document. Line (…. was found to be infected….) -please, it suggested deleting coma ",". Ae. albopictus,

Conclusion
Line (…SEVERAL emergence or re-emergence of arboviruses….) -may I suggest "….. emergence or re-emergence of SEVERAL arboviruses…" Line (Assessing the susceptibility……….future outbreaks) -yes, but I do see the link between the current study and insecticide susceptibility issue. Please, it is suggested to delete this sentence.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The study is important and essential in the Public Health domain, especially in the Republic of Congo where data on vectors are missing, and where doing field study is not that easy. Even knowing it is difficult to do field studies, from a scientific point of view, there is no replicate in this experiment. The authors went into the field one time in May 2017 and one time in November 2017. It should not be count as two independent samples of the same sites, but it should be considered as one sample during one season, and one sample during the other season. It is better to have a representation of the geographic distribution in the both season to observe if there is a variation than to have some chi-square tests that are not really useful here. It didn't deserve the study. As I mentioned earlier, I truly chi-square tests that are not really useful here. It didn't deserve the study. As I mentioned earlier, I truly think that the data are important here, including the percentage of positive container for each species, but is should be presented per season also, with no replicate.
For representing the data illustrated in the Figure 2, it should be better to represent it by chart by city. Here we didn't see very well what is the percentage of H1, H2 and H3 in Brazzaville for example. It is the same problem for the proportion of H1 and H2 in Ouesso, H1 and H2 in Oyo and H1and H3 in Lefini. By the way, it is very interesting to observe that these 3 haplotypes are nearby Asia and SouthEast Asia haplotypes.
In total, the study is well realized, and the conclusions are directly dependent of the results. But I suggest 2 only minor changes regarding the presentation of the results, especially for the sampling in May and November. I also suggest to replace the Figure 2 by a map with the % of each haplotype (circle or histograms) on each city.

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Thank you so much for your comments. Mosquitoes were sampled in May in 8 locations (Brazzaville, Lefini, Ngo, Owando, Makoua, Ouesso, Oyo and Gamboma, and in November in Pointe Noire only) corresponding to rany season in each location. Precision was make in the manuscript. The number of haplotypes detected per city is presented in supplement Table S2.
It is true that the number of pupae and larvae has not been counted, but the number of mosquitoes emerging from each type of larval habitat has been counted.
No competing interests were disclosed. Competing Interests: