First observation of Aedes albopictus in the Tshuapa province (Boende) of the Democratic Republic of the Congo

In May-June 2021, we detected Aedes albopictus adults near the central hospital in Boende, the capital city of the Tshuapa province of the Democratic Republic of the Congo (DRC). We identified the mosquitoes using morphological and molecular techniques (COI barcoding). This is the first report of this species in the DRC outside of Kinshasa and Kongo Central. Given the central location of Boende in the Congo Basin, our finding suggests that the vector might also have spread to other cities which are located upstream of the Congo River and its major tributaries. Because Aedes albopictus is an important vector for human arbovirus transmission, we highlight the need to investigate its distribution range and to update disease risk maps in Central Africa.


Introduction
Aedes albopictus (Skuse 1895) (Diptera: Culicidae) is considered to be the most invasive mosquito species worldwide 1 . The species originated in Southeast Asia and has successfully expanded to other parts of the world during the last decades 2 . This range expansion is primarily caused by human activities related to global commerce (e.g. trafficking of used tires) and the mosquito's adaptation to various environments in both tropical and temperate regions (e.g. ability to breed in different container forms) [3][4][5] . This rapid expansion has also been linked to multiple outbreaks of Aedes-borne disease, such as Chikungunya (CHIKV), Zika, yellow fever and Dengue [6][7][8] .
Although the first observation of Ae. albopictus in Africa was reported four decades ago (Nigeria), its presence in the Democratic Republic of Congo (DRC) was only described for the first time in 2018 9 . One year later, we linked a major CHIKV outbreak to its recent colonization in that area (the provinces Kinshasa and Kongo Central during 2019-2020) 10,11 . Indeed, Ae.
albopictus was the main Aedes species around human CHIKV cases in Kinshasa (>80% of the adults and larvae), Kasangulu (>99% of the adults) and Matadi (>95% of the adults and larvae).
The only other Aedes species detected during this outbreak was Aedes aegypti, a primary vector of arboviruses indigenous to Africa. Furthermore, we could link the CHIKV outbreak to an amino acid substitution in the viral envelope gene E1 (E1-A226V) which makes the virus more suitable for dissemination by Ae. albopictus 10 . These previous results indicate that Ae.
albopictus has now well-established populations in South-Western DRC, highlighting the potential for future outbreaks of Aedes-borne diseases and the urgent need for additional surveillance and control methods in the entire country 11,12 .
To prevent and control future outbreaks of Aedes-borne viruses in the Congo Basin, it will be crucial to monitor both the viruses and their vectors in the region 13 . While research in the DRC is often restricted to Kinshasa due to logistic reasons, the eco-epidemiological drivers of disease transmission could be significantly different in other regions (e.g. through differences in seasonal dynamics, insecticide resistance, landscape elements, human-social behaviours, immunological responses, etc…). Here, we provide data on the presence of Ae. albopictus in Boende to encourage scientists to investigate the ecology of Ae. albopictus upstream of the Congo River and warn policymakers for a potential increase of Aedes-borne viral outbreaks in the area.

Methods
In May 2021, we were bitten by mosquitoes while walking in the garden of our hotel in Boende (Tshuapa province, 0°16'40.4"S and 20°52'39''E). We identified these mosquitoes as Ae.
albopictus by their characteristic white stripe on the scutum. Six female Ae. albopictus were collected during human-landing and confirmed morphologically following Walter Reed's identification keys 14 . Later that day we went to the central hospital in Boende (0°16'38.5''S 20°52'59''E) and were again bitten by the same species. One month later (June 2021), Ae.
albopictus mosquitoes bit us again when we returned to the same two locations.
The morphological identifications were validated by DNA-barcoding, a technique based on the amplification of the partial mitochondrial cytochrome c oxidase subunit I (COI) gene 15 . Sanger sequencing of the 658-base pair barcode and phylogenetic analyses followed laboratory protocols and methodologies described in Wat'senga Tezzo et al 2021 16 . In short, PCR amplicons (EPPO 2016; LCO1490 and HCO2198 universal primers) and negative controls were checked on an agarose gel, purified and sequenced in both directions on an ABI 3230xl capillary DNA sequencer using BigDye Terminator v3.1 chemistry (ThermoFisher Scientific). Generated sequences were processed using Geneious ® R11 (Biomatters Ltd., Auckland, New Zealand) for quality control, pairing of bi-directional strands, and to extract one consensus sequence per specimen. The generated consensus sequences (NBoende = 5) were compared against the BOLD Identification System with Species Level Barcode Records.
To check the phylogenetic relation of the sequences, we compiled a list of Aedes species (belonging to the genus Stegomyia) that are considered to be of medical importance in the Afrotropical region by consulting the WRBU and the APHC databases 16 . Publically available COI sequences from these species were cleaned and aligned using ClustalW in Geneious® R11. Culex quinquefasciatus was added as outgroup to the alignment. A Neighbour-joining tree was constructed assuming the Kimura 2-paramers and branch support assessed by 500 bootstrap (BS) replicates using MEGA7 [17][18][19] . The generated sequences were deposited in GenBank with following accession numbers: MZ673312-MZ673316.

Results and Discussion
The observed Ae. Albopictus during the months of May and June 2021 in Boende suggest that established populations of the mosquito species occur close to the Tshuapa river in Boende. All Ae. albopictus COI sequences including the newly generated sequences group together in a highly supported cluster (100 BS, Figure 1), validating the morphological identifications.
Indeed, all similarity percentages reached 100% to Ae. Albopictus in the BOLD identification system, with the identification of one haplotype in Boende that also occurred in Kinshasa.
Although Boende is situated more than 750km from Kinshasa in the middle of the rain forest, the finding of Ae. albopictus in the city is not unexpected 13 . Boende is located on the left bank of the Tshuapa river (one of the tributaries of the Congo River) and boats arrive daily from downstream large cities such as Mbandaka and Kinshasa. These boats are usually very crowded and stacked with various goods, including water containers and used tires, making them ideal vehicles for Ae. albopictus larvae and eggs 20 . This assumption is further supported by the fact that the identified haplotype in Boende is shared with ones recorded in Kinshasa and Kongo Central (Figure 1) 16 , also being the most frequent in the latter study (Haplotypefrequency = 0.71).
Furthermore, the average precipitation (210.9 cm per year) and daily temperatures (ranging between 24°C and 30°C year-round) fall within the optimal boundaries for Ae. albopictus development 8,21 . Given that many cities in the DRC have similar climate conditions and transportation links to Kinshasa, we expect that established populations of Ae. albopictus also exist in other cities located near the Congo River or its tributaries throughout the Congo Basin.
If this is the case, we predict that the frequency and magnitude of Aedes-borne viral outbreaks will increase significantly the following years across Central DRC, similar to what we currently observe in South-Western DRC 10 .

Ethics statement
Ethical approval is not required for this type of study.