Stegomyia mosquitoes in Mayotte, taxonomic study and description of Stegomyia pia n. sp.

Four mosquito species, including a new species of the genus Stegomyia, are reported from Mayotte in the western Indian Ocean. The most abundant species were Stegomyia aegypti and St. albopicta. Only one species of the St. simpsoni group was observed, St. bromeliae. The fourth species is Stegomyia pia Le Goff & Robert n. sp. of which the larva, pupa, male and female are here described. The larval stages of St. pia n. sp. are morphologically similar to St. aegypti but differ in the number of branches of the seta 1-X; the adult is morphologically distinct for a number of characters, for instance the scutal ornamentation. Stegomyia pia n. sp. is uncommon but not rare, and largely distributed across Mayotte. Its larval habitats are natural and diverse including rock pools, tree holes, and cut and severed bamboos. The biology of adults remains unknown, especially female biting behaviour. Both morphological characters and nucleotide sequences of the ITS2 and COI genes indicate that this species is best placed in the genus Stegomyia. Dichotomous keys to the four species of Mayotte Stegomyia are presented for adults and fourth-instar larvae. The potential vector role of these mosquitoes is hypothesised. This paper underlines advances in knowledge of the biodiversity in the French overseas departments and territories.


Introduction
The study of the entomofauna in the Comoros archipelago commenced in earnest in 1947, under the direction of Jacques Millot working at the Muséum National d'Histoire Naturelle in Paris, who organised several scientific missions. The observations were published in a synoptic paper on the origin of the Comorian entomofauna composed of 1106 taxa grouped in 767 genera [33]. In 1973In -1974, another mission of the Muséum National d'Histoire Naturelle, headed by Loïc Matile, took place in the archipelago, focusing on a small number of insect orders (Coleoptera, Lepidoptera, Homoptera, Phasmatoptera and Diptera); these collections significantly increased knowledge of the fauna [29]. During approximately the same period, Jacques Brunhes, a mosquito specialist, studied Diptera of medical and veterinary interest. Since these comprehensive studies [4][5][6], little work has been devoted to the morphotaxonomy of haematophagous Diptera in the Comoros archipelago. The exceptions concern Culicidae and Psychodidae Phlebotominae. They include the mention of the presence of two species belonging to the Stegomyia simpsoni complex [19,20] and the description of a new species, Zavortinkius brunhesi Reinert, 1999, endemic in Mayotte [36]. More recently, in a synthesis of the genus Uranotaenia in the Malagasy biogeographic subregion, a new species was described, Uranotaenia (Pseudoficalbia) comorensis da Cunha Ramos & Brunhes, 2004, which is endemic in the Comoros archipelago [7]. A review of Phlebotomine sandflies was published [35], including the description of new species and subspecies.
Aedini is the largest tribe of mosquitoes in the world with 1252 species classified in 80 genera, including the genus Aedes Meigen, 1818. Depending on the taxonomic preferences of different authors, the taxon Stegomyia Theobald, 1901 [43] is considered as a subgenus of the Aedes genus [23] or a genus of the tribe Aedini [37]. The Mosquito Taxonomic Inventory [16] recognises 127 Stegomyia species and subspecies worldwide, and Huang [21] distinguishes 59 of them in the Afrotropical Region. From a medical point of view, the genus Stegomyia is extremely important because it groups a number of species that are vectors of human arboviruses such as dengue, chikungunya and yellow fever. Hence, different aspects of Aedini taxonomy, distribution and biology have been studied in some detail, at least with respect to most other insects.
In 2011, we had the opportunity to conduct some inventories, specifically to update information on the mosquitoes of Mayotte via a project aiming to evaluate the culicid biodiversity on several islands of the western Indian Ocean [25,39]. With these aspects in mind, the collection of immature stages was performed in natural environments, as well as in humandegraded, agricultural and urban areas, giving priority to the best preserved environments. The goal of the study was to obtain a better picture of mosquito diversity, especially those species that are potential vectors of pathogens. Here we present only the results concerning the genus Stegomyia. We collected material of the species St. aegypti (of the form formosa, with the exception of a single specimen of the form aegypti), the species St. albopicta and one species of the St. simpsoni complex, St. bromeliae. Surprisingly, we also obtained material of a Stegomyia species that is new for science.

Study area
Geographically, Mayotte Island, at the northern end of the Mozambique Channel, belongs to the Comoros Archipelago, which encompasses four principal islands: Grande Comore, Mohéli, Anjouan and Mayotte (in Shikomory language: Ngazidja, Mwali, Ndzouani and Maoré, respectively). These islands are all of volcanic origin. Mayotte is the oldest in the archipelago at about 8 million years. It is located 250 km W of Madagascar, 30 km SSW of Anjouan and 450 km E of the Republic of Mozambique coast. The emerged land of Mayotte reaches its maximal altitude at 660 m, including a large lagoon that covers a surface area of 1500 km 2 . The principal land features are Grande Terre, 363 km 2 (about 39 km long and 22 km wide), Petite Terre, 11 km 2 , and about 30 islets. More than 1000 ha of natural forest remain on mostly Grande Terre, with about 15 000 ha of degraded forests in the island complex. The original forest has all but vanished and covers only 3% of the surface area. All remaining humid and semi-humid forests are located in the summital zones of the principal peaks on Grande Terre. Some reforestation projects have been undertaken, mostly with deciduous trees in the drier and southern end of Grande Terre (e.g., Choungui-Sohoa littoral forest and Dapani clump), and on some islets (e.g., Ilot M'bouzi, 84 ha). A large part of Grande Terre is now degraded in ''padzas'', places scoured by erosion and exposed subsoils. Petite Terre does not have any remaining forest [11]. There is no rice cultivation, with or without irrigation. Mayotte, with its estimated 212 000 people in 2012, is densely populated (567 individuals/km 2 ) and in continuous demographic increase (23 364 inhabitants in 1958).

Inventory
Our mosquito inventory was mainly focused on collections of immature stages. Entomological prospecting was carried out by two of us (GLG & VR) with the help of ''Service de la Lutte antivectorielle -Agence Régionale de Santé -Océan Indien, Mayotte''. We concentrated on natural environments, presumably with high biodiversity, but urban and agricultural areas were also prospected. The larval habitats of natural origin were mainly bamboo and tree holes, fallen coconuts and rock pools; the artificial containers were mainly abandoned wastes, piece of plastic and tyres. The first field mission took place during the end of rainy season from 22 March to 5 April 2011, and a second mission at the beginning of the next rainy season from 28 November to 2 December 2011. In total, 420 aquatic habitats with larval and/or pupal stages were inventoried. During the fieldwork, female mosquitoes were collected when biting us, although no formal human landing catch was performed.

Systematics
Following the Aedini classification of Reinert and collaborators [37,38] and the online catalogues of the Walter Reed Biosystematic Unit [48], the taxon Stegomyia is here considered at genus level [15]. Anatomical nomenclature and chaetotaxy follow Harbach and Knight [17,18] and updated terms listed in the Anatomical Glossary of the Mosquito Taxonomic Inventory website [16]. The morphotypes of St. aegypti are those of McClelland [28].

Molecular study
Molecular study targeted three genes: (i) the ribosomal internal transcribed spacer 2 (ITS2) gene, (ii) the mitochondrial cytochrome c oxidase subunit I (COI) gene and (iii) the mitochondrial NADH dehydrogenase subunit 4 (NDH4) gene. Molecular techniques were detailed previously for COI [34], and ITS2 and NDH4 [25]. Briefly, the following steps were successively performed: DNA extraction, gene amplification, purification and sequencing of the amplified fragment, electropherogram analysis and alignment of nucleotidic sequences. According to the objective of this study, which is not a phylogenetical analysis, a Neighbor-Joining analysis was performed for ITS2 and COI using MEGA5 software [42], with the Kimura-2 parameter model and using uniform rates among sites; bootstrap values were obtained after 1000 replications. Analyses were performed on the sequences obtained in this study along with sequences from GeneBank, of which one from Stegomyia mascarensis was collected in 2007 at Rivière Noire, Mauritius [8] and two from the St. simpsoni group (i.e., Stegomyia subgenus Mukwaya [16]) collected in Uganda [32].

Results
Of the 420 aquatic habitats with mosquitoes, Stegomyia were found in 159 (38%). The following four Stegomyia species were collected.

Stegomyia aegypti
This species was the most abundant Stegomyia in our surveys -70 of the 420 (17%) surveyed sites with 46 (66%) natural habitats. The most frequent habitat was cut bamboo (n = 13). It is a container-breeding mosquito, found in a wide range of vessel types; consequently, larval stages are very common in zones inhabited by people with a large number of anthropogenic habitats.
All 36 adults found in rural areas are referable to St. aegypti formosa, with only black scales on abdominal tergum I (morph F, n = 25) or with this tergum brindled of grey scales (morph G, n = 11). Contrarily, one female collected biting a man in the town of Mamoudzou (23 March 2011) had a large median patch of grey scales on the first abdominal tergum, typical of St. aegypti aegypti and corresponding to the morph Hap. Therefore, the two forms, formosa and aegypti, are present in Mayotte.

Stegomyia albopicta
This species is the second most abundant Stegomyia in our surveys -57 of the 420 (14%) surveyed sites with 27 (47%) natural habitats. The most frequent habitat was cut bamboo (n = 9). Like St. aegypti, St. albopicta is a container-breeding mosquito and these two species were found in association in 24 habitats. Stegomyia albopicta, more so than St. aegypti, is able to adapt to unusual habitats such as crab holes and transient pools of water.

Stegomyia bromeliae
Stegomyia bromeliae was quite common in our surveys -42 of the 420 (10%) surveyed sites with 38 (90%) natural habitats. All males of the St. simpsoni group show leg ornamentation (n = 21) and genitalia (n = 10) typical of St. bromeliae. All females (n = 19) had the ungues of the fore-and midlegs equal and toothed, a character shared by St. bromeliae and St. lilii. All of these females have leg ornamentation typical of St. bromeliae with one exception. This latter has fore-and midtarsomeres I and II with ornamentation typical of St. lilii, but the scutum and hindtarsomere V with ornamentation typical of St. bromeliae; and was collected as a larva in taro leaf axils with two other females of the St. simpsoni group typical of St. bromeliae, an indirect argument to consider the doubtful female as St. bromeliae rather than St. lilii.
Larval habitats were mainly leaf axils, particularly Typhonodorum, and less commonly banana, pineapple and taro. Other types of sites included dead leaves on the ground, bamboo pots and bamboo stumps, and tree holes in both urban and rural areas. Genus: Stegomyia Theobald, 1901 Authorship: Note that the authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of International Code of Zoological Nomenclature [22]. Etymology: The term ''pia'' means ''fresh'' or ''new'' in the Shimaore language, the main language spoken in Mayotte. In addition, Pia is the first name of a stepdaughter of V. Robert.
GenBank Accession numbers for the ITS2, COI and NDH4 sequences of two males of Stegomyia pia (T5 and T6: MY 648 bamboo, Convalescence, Mayotte, 2 December 2011) are included in Table 3.
This species was the least abundant Stegomyia in our surveys -27 of the 420 (6%) surveyed sites.

Information on type series
Type locality: near the Governor House, at the locality known as ''Convalescence'' in the ''Réserve forestière Majimbini'', located at 3. The pupal exuviae of the five adults permitted the description of the pupa.
Type deposition: the holotype, allotype, two paratypes, four pupal exuviae and one larva used in the descriptions of the male, female, pupa and larva are deposited in the ''collection d'Arthropodes d'intérêt médical (Arim)'', Laboratory of Vector Taxonomy, IRD, 911 avenue Agropolis, Montpellier, France. One complete male paratype (1#), one male pupal exuviae and one larva will be deposited in the Muséum National d'Histoire Naturelle (MNHN), Paris, France. Scutum. Integument pale brown, mainly covered with small black scales. Scutal scales all narrow. Falciform acrostichal scales delineated by a complete median thin line that reaches prescutellar area; these scales yellowish except on anterior promontory that has silvery scales. Around median scutal fossa 2 large crescentic spots of iridescent silvery scales, with anterior side of spots slightly stretched and posterior sides widened slightly. Posterior to each large spot, a line of yellowish scales from posterior dorsocentral to lateral prescutellar areas and a small spot of yellowish supraalar scales. Prescutellar area without scales on its margin.
Scutellum. Large iridescent silvery scales on all three lobes, except posterior part of midlobe covered with small black scales.
Pleura. Integument yellowish. Several spots of silvery scales cover antepronotum, paratergite and proepisternum, form one small spot on proepimeron and subspiracular areas, and form two small spots on the meskatepisternum (one in upper and one in lower area) and the mesepimeron (one in upper and one in anterior area). No lower mesepimeral setae. No scales on postspiracular area.
Wing. All veins and wing fringe with black scales; female sometimes with a few silvery scales at base of costa. Midleg. Coxa, femur and tibia as foreleg. Tarsomeres 1 and 2 as foreleg with exception of a slightly more developed basal white spot, on basal 1/8 and 1/5, respectively. Tarsomeres 3-5 and midungues as foreleg.
Hindleg. Coxa as foreleg. Femur with light milky scales on proximal half and entire anterior surface; black scales, sometimes iridescent, on distal half of posterior surface; knee with a spot of silvery scales, with protruding scales on terminal part, spot slightly more developed than on fore-and midlegs. Tibia as foreleg. Tarsomeres 1-3 mainly black. Tarsomere 1 with basal ring of light yellowish scales on proximal. Tarsomeres 2-4 with basal ring of light milky scales on proximal 1/5, 1/5-1/4 and 2/3, respectively. Tarsomere 5 entirely with light milky scales. Hindungues equal and simple in female and male.

Abdomen
Terga. All terga mainly covered with black scales, with following exceptions. Basal bands of yellowish scales on terga III-VII. Large spots of silvery scales anterolaterally on terga I-VII in female and terga I-VIII in male; with triangular form on terga I-VI, with decumbent scales on basal part and protruding scales on terminal part; these spots less developed and more central on tergum VII, permitting an easier observation on dorsal face. Visible part of tergum VIII of female covered with black scales.
Sterna. Sterna mainly covered with light whitish scales without silver reflection. Sternum I not observed. Sterna II-IV covered with light whitish scales. Sterna V and VI with apical bands of black scales on about distal 1/4. Strenum VII mainly covered in black scales, with three round spots of light whitish scales, one basal and central and two apico-lateral. Visible part of sternum VIII in female with black scales.
Male genitalia ( Figure 1D) Gonocoxite wide, coniform, two times longer than wide; few thin setae on dorsomesal surface; numerous, long and strong setae and large, ridged and spatulate scales on lateral and ventral surfaces. Claspette a round tubercle, with thin setae on external part and more pronounced setae on central part, these latter structures form a right angle in their terminal half. Gonostylus short, rather thin, evenly curved; gonostylar claw with obtuse termination. Apical margin of tergum IX clearly concave in middle, with three or four thin spinulate setae on lobes.
Pupa Integument light, except trumpet. Median part of terga II to V with 2-4 small round light brown patches. Setae of cephalothorax and abdomen pale, usually thin and short, except seta 1-I, 9-VII,VIII.
Paddle. Inner and outer margins finely spiculate on distal half; midrib distinct whole length of paddle; seta 1-Pa single, long, brownish. Larva ( Figure 3 and Table 1)

Head
Antenna. Smooth, short; no longer than third length of head. Seta 1-A single, straight, inserted between 3/5 and 2/3 from base.

Diagnosis with regard to larval stage in other Stegomyia present in Mayotte
A quick examination of larval specimens allows separation of Stegomyia in two groups, the first group with a large, prominent and strongly sclerotised tooth on the plate that supports the meso-and metathoracic pleural setal groups (St. pia and St. aegypti), and the second group with these teeth poorly or normally developed (St. albopicta and St. bromeliae). The distinction between St. pia versus St. albopicta and St. bromeliae at larval stage, in addition to the aspect mentioned above, is simple using the comb scales and the distribution of the denticles on the pecten spines. On the other hand, the distinction between St. pia versus St. aegypti is much more difficult. Three morphological characters are relevant, none which are diagnostic. The general aspect of the larval setae is a means to separate these species, with setae largely developed for St. pia and notably smaller and less dense in St. aegypti. A character easy to observe, although not diagnostic, is the number of branches of the saddle seta 1-X: usually more than two-branched (range 1-7) in St. pia and usually two-branched (range 1-3) in St. aegypti (Figure 4). Another character is the number of branches of seta 4-P (usually with 2-3 branches [range [1][2][3] in St. pia and usually single [range [1][2][3] in St. aegypti). Using these three characters, the identification of St. pia larvae was possible, and indeed, confirmed by molecular techniques for 86% of specimens (Table 2).
Neighbor-Joining trees were drawn using ITS2 and COI sequences of five species of Stegomyia distributed in the western Indian Ocean ( Figure 5). The specific status of these species was strongly supported.
Justification for placing St. pia within the genus Stegomyia Following Huang [21], Stegomyia is characterised by the following combination of characters: adults (both sexes). Vertex with all broad, flat decumbent scales, erect forked scales not numerous, restricted to occiput; maxillary palpus of male not particularly short, more than 0.5 length of proboscis, five-segmented, palpomeres 4 and 5 subequal, slender and with only a few short setae, total length of apical two palpomeres not very short, at least 0.4 length of the remaining palpomeres; in females about 0.14-0.32 length of proboscis, with three or sometimes four palpomeres, palpomere 4 minute when present;  T  I  II  III  0 - maxillary palpus with white scales; acrostichal setae absent; prespiracular setae absent; postspiracurar setae present; postprocoxal membrane without scales; scutum with all, or mainly with narrows scales; scutellum with broad scales on all lobes; mesopostnotum bare; wing with narrow plume scales; hindtarsus with basal white band on at least one tarsomere. Male genitalia. Aedeagus strongly toothed; claspette well developed, with numerous setae; gonostylar claw present. Female genitalia. Insula longer than broad, with minute setae and 2-10 larger setae on apical 0.25-0.50; cerci short and broad; three spermathecal capsules, one larger than the other two. LARVAE. Seta 4-C well developed, branched, closer to 6-C than 5-C, inserted cephalad and mesad of 6-C; 4-C and 6-C inserted cephalad of antennal base; 6-C inserted cephalad of 5-C and 7-C; seta 12-I not developed; seta 2-VIII distant from 1-VIII; comb scales in a single row; ventral brush (seta 4-X) with four to five pairs of setae on grid; without precratal setae. In addition of this combination of characters, all the supplementary characters listed in the Mosquito Taxonomic Inventory website [16] that distinguish the genus Stegomyia from other genera of subfamily Culicinae were verified (except those related to female genitalia, not observed in the present study). Therefore, we confirm the placement of St. pia within the genus Stegomyia. Following Huang [21], the diagnosis for the Aegypti Group is based on the following combination of characters: Maxillary palpus with white scales; scutum with dorsocentral setae; scutum with a distinct patch of broader crescent-shaped white scales on the fossa; subspiracular area with broad white scales; postspiracular area without scales; paratergite with broad white scales; scutellum with broad white scales on all lobes; white knee-spot present on all femora (except in mascarensis); all tibiae anteriorly dark, without any white band; hindtarsus with a basal white band on tarsomeres 1-4; and hindtarsomere 5 all white. In addition of this combination of characters, all the supplementary characters listed in the Mosquito Taxonomic Inventory website [16] that distinguish the subgenus Stegomyia from other subgenera of genus Stegomyia were verified, except two characters. Indeed, St. pia exhibits larger foreungues of males without tooth and larval seta 1-VIII slightly longer than 2-VIII. However these characters are considered of minor importance. Therefore, we confirm the placement of St. pia within the subgenus Stegomyia.

Distribution, bionomics and potential vector role
On the basis of current information, St. pia is considered endemic to Mayotte. It is an uncommon species although not rare.
It was collected across the elevational range from 20 to 450 m ( Figure 6). It is distributed in the northeast (Bouyouni) and west (Lac Karihani) coastal plains, as well as in forested areas along the paths leading to upper elevational zones (Monts M'tsapéré, Combani, Bénara). Our records indicate that it is broadly distributed across Grande Terre, from the foothills of Mont M'tsamboro in the northwest to the surroundings of Mont Choungui in the south. It was also collected on a small-uninhabited islet (Ilot M'bouzi) where introduced lemurs and rats are numerous. We do not have any records of it on Petite Terre, but its presence there is possible.
The distribution of St. pia encompasses the remaining forests on Grande Terre in reserve zones, which are the last remaining natural habitats, slightly or non-degraded by human activities. Although not strictly dependant on natural forested environments, this species needs woody zones. In other words, its geographical range excludes urban areas, villages and cultivated zones. However, its verified presence in habitats close to inhabited buildings is confirmed, as observed in March 2011 in Bouyouni within 30 m of houses and in the ''centre'' of Bougouni, a traditional village in the commune of Kahani.    Table 3 for GenBank Accession numbers. The larval habitats are of natural origin: bamboo and tree holes, fallen coconuts and rock pools (Table 4). More specifically, the vast majority of these sites are associated with vegetation, particularly bamboos and trees, which constituted about 75% of the documented locations. In the case of bamboos, the sites varied from being green and standing or dry and on the ground. In most cases, cut bamboos occurred in the peripheral area of the clump and in the shade; on a few occasions, St. pia was also collected in bamboos severed in longitudinal sections, used as field fences and exposed to the sun.
Tree holes were mainly of small diameter, about 10 cm, at various heights, ranging from the lower portion of the tree (10-20 cm high) up to at least 2 m on the trunk. Tree species were not identified, except in one case for a hole at 1.8 m high, in an adventitious root of a ''badamier'' (Terminalia catappa L.). These places ordinarily contain 20-100 mL of water (max 500 mL), heavily laden with organic materials, and often a dark coffee colour.
Rock pools are important habitats, during the dry season, when ovipositioning in different types of vegetation disappears Figure 6. Schematic map of Mayotte with indication of the 11 sites where Stegomyia pia n. sp. has been recorded. Red disc, adults reared from pupae with confirmation by DNA sequencing; red circle, adults reared from pupae; yellow square, larvae with confirmation by DNA sequencing; blue triangle, larvae. because of the lack of water. At such sites, there is little water, and the openings to the sources are notably small, 2 or 3 cm in diameter, which presumably reduces evaporation. These types of small cavities are common in the volcanic rock, particularly in the bed of small mountain torrents and downstream from waterfalls where there is shade and a constant source of rain and river water. The presence of plant matter is common. The only exception to known sites for this species in natural situations is one found in abandoned plastic waste hidden in dense vegetation on the track leading to Mont Combani. This habitat can be considered as ''natural'' with regard to the availability of rain water, and the co-occurrence at the same site of the larvae of Ur. (Psc) comorensis, a culicid species typical of forest. Larval development is slow, at least with regard to other Stegomyia encountered. Adults reared from fourth-instar larvae and pupae were obtained after 3-4 days in the insectary in only five sites among 19 (26%). With St. aegypti, for instance, this proportion was much higher (39/70 = 56%).
The bionomics of adults are unknown, including the trophic and resting preference of females and their longevity.

Associated culicid fauna
In 67% of cases (18/27), St. pia shared its larval habitats with other mosquito species that ordinarily occupy habitats of small size (stenotrophe mosquitoes) in woody zones on Mayotte. In decreasing order, the following have been found in association with St. pia (number of occasions in parentheses): St. aegypti (7), Eretmapodites subsimplicipes Edwards, 1914 (6), species of the Zavortinkius monetus group (Edwards, 1935) (5) Key to the fourth-instar larvae of the Stegomyia of Mayotte

Discussion
Stegomyia aegypti is a polymorphic species. Mattingly [30] described two subspecies based on overall body colour and the extent of white scaling on the first abdominal tergum. According to Mattingly, (i) the darker and presumably ancestral St. aegypti formosa is confined to Africa, where it prefers natural, as opposed to artificial, larval habitats and is predominantly zoophilic in its biting behaviour, and (ii) the lighter St. aegypti aegypti is an invasive taxon distributed throughout the tropics outside Africa, where it is strongly anthropophilic and endophilic. The queenslandensis form is the palest form of the subspecies aegypti (see also [28]). More recently these two subspecies were observed nearly in sympatry in some places along the coast of East Africa, including some localities of Kenya ( [41] and other references herein), where aegypti inhabits in villages and formosa in surrounding forests, with little gene flow between the two subspecies [3]. The Neighbor-Joining trees ( Figure 5) indicate the close proximity of St. aegypti and St. mascarensis, in agreement with their placement in the subgenus Stegomyia (the Aegypti Group of Huang [21]).
The first record of St. aegypti in Mayotte was in 1942 (as Aedes fasciatus) [24] and regularly observed thereafter [1,2,4,13,27]. Brunhes [4] reported the presence of several forms from formosa to queenslandensis in the Comoros Archipelago and, indeed, in the Collection Arim This species is suspected to have been the major vector of a supposed Dengue epidemic that raged across the island, especially in Dzaoudzi, in 1943 [27].
Stegomyia albopicta, the ''Asian tiger mosquito'', is native to Southeast Asia. Initially described from the Indian Peninsula since the early 20th century this species has been observed in Madagascar [10], the Seychelles [31,47], Mauritius (de Grandpré et de Charmoy in [14]) and La Réunion [10]. For the last 30 years, this species has progressively adapted to human environments and has spread from its native area. Like St. aegypti, it is now distributed on every continent, Antarctica excluded. The first record of St. albopicta in Mayotte was in November 2001 for immature stages found in old waterfilled tyres at Mamoudzou, the biggest city, prefecture and harbour [12]. That this species was ''recently introduced'' in Mayotte is interestingly confirmed by its close proximity in the Neighbor-Joining trees ( Figure 5) with specimens from Greece, where the species was also recently introduced [40], and the distance to specimens from China which lies within the native area of the species. In 2005-2006, a Chikungunya epidemic hit Mayotte, affecting 37% of the human population [26]. The presence of St. albopicta on the island probably played an important role in the emergence of this infectious disease. The vector implicated in the transmission of the disease has not been precisely identified, although it was most probably St. aegypti and St. albopicta [1]. An outbreak of Dengue 3 occurred on Mayotte in March 2010 [26], and, once again St. aegypti and St. albopicta were the suspected vectors.
Based on our findings in urban areas St. aegypti populations are being replaced by St. albopicta, which confirms the observations of other researchers [1,2,8]. The increasing urbanisation of the island seems to considerably favour the presence of the invasive St. albopicta.
The subgenus Mukwaya (previously the Simpsoni Group [16]) includes 10 species in the Afrotropical region [21], with three of these forming the St. simpsoni complex: St. simpsoni (Theobald, 1905) [44], St. lilii (Theobald, 1910) [45] and St. bromeliae (Theobald, 1911) [46]. These three species were formerly placed in synonymy [9], but all have been restored to specific status [19,21]. According to Huang [19,20], St. simpsoni is only distributed in the Republic of South Africa and Swaziland, St. lilii in West Africa, Uganda, Sudan and Ethiopia, and St. bromeliae with a larger distribution forming a triangle between Guinea, Ethiopia and South Africa. Stegomyia simpsoni has not been documented in the Comoros Archipelago [20]. Stegomyia lilii was noted in the ''Comores Is.'' ( Table 2 in [19]) and on Mayotte [1]. Stegomyia bromeliae was noted in the ''Comores Is.'' ( Table 2 in [19]) and on Grande Comore (Figure 1 in [20]). Females of St. simpsoni are easily distinguished by having the fore-and midlegs with simple ungues, whereas St. bromeliae and St. lilii have the fore-and midlegs with toothed ungues and different leg ornamentation [19,20]. Males of these three species differ by genitalia characters [21]. The first record of the subgenus Mukwaya on Mayotte was in 1955 by Brygoo and Escolivet at Coconi village [13], with regular records thereafter [4]. Today, individuals of this subgenus are common on the island. In line with published literature [19,20], our study confirms that St. simpsoni is not present on Mayotte. Conversely, our data suggests that St. bromeliae is the only species of the St. simpsoni complex present on Mayotte; this view is today shared by Dr. Yiau-Min Huang (pers. com.). The considerable morphological similarity between St. bomeliae and St. lilii may explain possible confusion between these two species [32]. Interestingly, the nucleotide sequences of St. bromeliae in Mayotte are closer to the anthropophilic population of the subgenus Mukwaya in Uganda than to the non-anthropophilic ones (see [32]), suggesting that the anthropophilic mosquitoes in Uganda belong to St. bromeliae. Curiously, species of the subgenus Mukwaya, specifically St. bromeliae, are not known on Madagascar, some 250 km from Mayotte.
Stegomyia bromeliae is an important vector of yellow fever virus in East Africa [20].
Despite the relative abundance of St. pia on Mayotte and its distinctive characters, this species has never been identified until now. This is puzzling and only tentative explanations can be proposed. First, the larval stages are morphologically similar to St. aegypti, which might explain some of the confusion. Second, we did not observe any males or females in our field collections. All adults were obtained in the insectary from larvae or more often from pupae collected in the field. When the first adult emerged, it was immediately clear that it belonged to a Stegomyia species that was hitherto unknown on Mayotte and the Comoros Archipelago. With these points in mind, we checked specimens in the Arim collection, obtained in the Comoros Archipelago and identified as St. aegypti. We found among about 30 St. aegypti one female of St. pia in a good state of preservation, collected by Dr. Alexis Grjebine on 19 February 1956 at larval stage in Coconi village on Mayotte. This is clear evidence that this species belongs to the Mayotte entomofauna but has been misidentified as St. aegypti. This location (12°50 0 024 00 S, 45°08 0 132 00 E) was added as an 11th site in the distribution map of the species (Figure 6).
The recognition of St. pia as a species new to science is clearly supported by a number of morphological characters in both males and females, and to a lesser extent in larvae. This recognition is reinforced by molecular data for all the gene sequences examined.
Due to the larval habitats utilised by St. pia, this species is of little consequence for the evaluation of stegomyian indices (aiming to estimate the density of potential vectors, an important criterion for risk estimation in public health) in anthropic and peri-domestic sites. However, immature stages of St. pia need to be taken into account on Mayotte when looking at relatively natural environments. Of interest is our observation of larval habitats in close proximity to villages in rural areas. The ability of this species to occupy woody zones in the periphery of towns needs further investigation.
Stegomyia pia is considered endemic on Mayotte. Further studies in the sister islands of The Union of the Comoros would be useful to determine if this species occurs elsewhere in this island group.
The bionomics of the adults are unknown, including the blood-feeding preference and longevity of females. However the potential vector role of St. pia may be hypothesised taking in mind the paramount importance of a number of Stegomyia species as vectors of many pathogens of medical and veterinary importance, including the West Nile, Yellow fever, St. Louis encephalitis, Dengue and Chikungunya viruses, as well as avian malaria such as Plasmodium relictum and several filarial nematodes such as Dirofilaria immitis.