Culex camposi belongs to the Coronator complex together with Cx. coronator Dyar and Knab, 1906; Cx. ousqua Dyar, 1918; Cx. usquatissimus Dyar, 1922 and Cx. usquatus Dyar, 1922, this species has a wide distribution, being Cx. coronator the most widespread, from Argentina to the United States (Wilkerson et al., 2021), and found in a large range of breeding sites: stagnant or slow-moving water in ground pools and seeps, ditches, culverts, artificial containers, ground depressions, tire ruts, and even dredge sites, most commonly in open, sunlit aquatic habitats (Schluep et al., 2023). All the species of this complex are synanthropic in Colombia (Wilkerson et al., 2021) and as far as we know they share the same ecological characteristics and breeding (Bram, 1967), in this study, Cx. camposi larvae were collected in a ground puddle with no association with other mosquito larvae.
These five species can only be differentiated through the analysis of the genitalia of males. According to the description of Bram (1967), the feature that differentiate Cx. camposi is the presence of 10 subequal and gently curved setae that are evenly distributed in the subapical lobe of the gonocoxite; also, the presence of a small tubercle remote from the subapical lobe bearing one or two strong, gently curved spines, plus a small patch of setae at the apex of the gonocoxite reaching at least the midpoint of the gonostylus (Fig. 3A). However, Laurito et al. (Laurito et al., 2018) described the male genitalia of holotype Cx. camposi and mentions that “the proximal part of the subapical lobe bears five slender rod-like setae, two shorter than the other three. The distal part, a small prominence located well distomesad from the proximal part, bears a single strong seta”. These features in the sub apical lobe are different from those found in our specimens, which were as described by Bram (1967). This represents a morphological variation between populations of Cx. camposi, evidence that had already been reported in other species of this species complex (Demari et al., 2014; Laurito et al., 2018).
The difficulties raised by the attempted identification of the species of the Coronator complex using female specimens can lead, among other aspects, to inconclusive assessment of their vector status (Demari et al., 2015). Furthermore, the real distribution range of each one of these species remains uncertain. As far as it is known, only Cx. coronator was implicated as a potential vector of the West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), Venezuelan Equine Encephalitis Virus (VEEV), Murutucu virus (MURV) and Itaqui virus (ITQV), in Peru, Brazil, and the United States (Consoli & Oliveira, 1994; Alto et al., 2014; Demari et al., 2015; Turell et al., 2021).
In Peru, species of the Coronator complex were reported in some regions of the country. For example, Cx. coronator was reported in the eastern region (Loreto and Cusco), Cx. usquatissimus and Cx. usquatus the region in which the specimens were collected is unknown (Morales, 1971; Ayala et al., 2020), while Cx. camposi was reported by Bram (1967) only in the northeastern and central-eastern regions (Loreto and Ucayali), however, it is not considered in the mosquito list of Morales (Morales, 1971), and it was not recorded in any subsequent study in these same regions either, which generates expectation in its real distribution in Peru, our results confirm that Cx. camposi is present in the central region of the country.
Partial sequences of the CoxI gene of all five male specimens Cx. camposi, (LC750482-LC750486) were obtained. By using the BLASTn tool, these sequences showed 99.84%-100% identity with homologous sequences from Cx. usquatissimus, Cx. usquatus and Cx. coronator. When using the Boldsystems taxonomy tool, an identity of 99.36–100% with the homologues from Cx. usquatissimus, Cx. usquatus, Cx. coronator and Cx. maxi Dyar, 1928 was observed. Hence, neither BLASTn nor Boldsystems tools yielded the morphologically confirmed identification of Cx. camposi.
Regarding phylogenetic reconstruction analysis, the generated trees showed that our sequences of Cx. camposi grouped together with Genbank sequences corresponding to Cx. coronator, Cx. usquatus and Cx. camposi, in a strongly supported monophyletic clade where the only other Cx. camposi sequence, from Brasil, was equally placed (Fig. 4–5). In addition, the intraspecific variation analysis showed a value of 0.55 ± 0.18, while the interspecific variation varied between 0.50 ± 0.16 to 0.81 ± 0.19 with the Cx. usquatus and Cx coronator sequences (Table S1-1). These results are not totally surprising, as lack of segregation power of CoxI barcoding sequences for species groups or complexes have been reported in previous studies (Demari et al., 2015; Laurito et al., 2013, 2018; Montalvo et al., 2022). Demari et al. (2017) to differentiate the species Cx coronator and Cx usquatus, they used morphological data and nuclear and mitochondrial gene sequences (CoxI, NADH5, CAD, Hunchback), for his part, Vesgueiro et al. (2011) used sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA, however, their results did not resolve a clear separation between these two species. More studies are required, and the use of other genetic markers, such as the ACE2 gene, used to differentiate species of the Pipiens complex (Smith & Fonseca, 2004).
On the other hand, Cx. bonnei of the subgenus Carrollia Lutz, 1904 is so far considered a mosquito species without medical importance. The subgenus Carrollia is divided into two groups: the Bihaicolus Group composed of five species (Cx. bihaicolus Dyar & Núñez Tovar 1928; Cx. guerreroi Cova García, Sutil, & Pulido 1971; Cx. infoliatus Bonne-Wepster & Bonne 1920; Cx. metempsytus Dyar 1921 and Cx. rausseoi Cova García, Sutil O, & Pulido F. 1972) and the Iridescens Group composed of thirteen species, which in turn is divided into two subgroups: Iridescens Subgroup with eleven species (Cx. antunesi Lane & Whitman 1943; Cx. babahoyensis Levi-Castillo 1953; Cx. bonnei Dyar 1921; Cx. cerqueirai Valencia 1973; Cx. insigniforceps Clastrier & Claustre 1978; Cx. iridescens (Lutz 1905); Cx. kompi Valencia 1973; Cx. secundus Bonne-Wepster & Bonne 1920; Cx. soperi Antunes & Lane 1937; Cx. wannonii Cova García & Sutil O. 1976 and Cx. wilsoni Lane & Whitman 1943) and Urichii Subgroup with two species (Cx. anduzei Cerqueira & Lane 1944 and Cx. urichii (Coquillett 1906)) (Valencia, 1973; Wilkerson et al., 2021). In our study we followed on the characteristics of the mosquito male genitalia, in which the subapical lobe was observed, in agreement with that described by Valencia (1973), with three divisions: the distal division formed by a small bump; the accessory division in the form of a robust and long column that ends with four long and flattened seats, and the elongated and slightly curved proximal division with two to three simple distal setae and seta a and b with dilated and curved apex (Fig. 3B). In Peru, five species of Culex of the Carrollia subgenus were reported, including Cx. bonnei; Cx. urichii, Cx. infoliatus, Cx. iridescens and Cx. bihaicolus (Ayala et al., 2020, 2021). So far, Cx. bonnei has only been only reported in northern countries of South America (Wilkerson et al., 2021) reaching the 3rd parallel south in the region northeastern from Peru (Iquitos) (Lopes et al., 1985; Pecor et al., 2000). In this study we reported for the first time Cx. bonnei in the central region of Peru (Huanuco) to the 9th parallel south. This specie has been reported in many types of breeding: Broken or cut bambo, treeholes, fallen palm spathes, fallen cacao pod, fallen fruit, artificial containers metal and plastic (Valencia, 1973; Lopes et al., 1985; Patrick et al., 2002). In this study the larvae were collected in an artificial container of plastic, furthermore, we did not find it associated with other species.
Five sequences were obtained corresponding to the specimens identified as Cx. bonnei (3 males and 2 females) (LC750487 - LC750491), and these showed 90.53% − 99.84% identity with homologues from Cx. bonnei in BLASTn tool, and an identity of 99.35–99.84% with CoxI sequences from this same species when the Boldsystems tool was used, thus, differently from the previous specimens, confirming its taxonomic identity. In the phylogenetic analysis, these sequences were clearly clustered with the Cx. bonnei from Ecuador used as references, forming a monophyletic group strongly supported sister clade to the subgenus Culex (Fig. 4–5). Regarding the divergence analysis, the results showed an intraspecific variation of 0.13 ± 0.09, a lower result to that obtained by Demari et al. (2011) who reported a value of 0.6, while interspecific divergence value that varied between 4.83 ± 0.94 to 10.23 ± 1.14 with Cx. bihaicola, Cx. infoliatus, Cx. urichi and Cx secundus sequences (Table S1-1). On the other hand, it is observed that the grouping of the sequences does not correspond to the proposed informal groups. In other studies, only one or two species of this subgenera were included in their phylogenetic analysis, which limited the observation of this grouping (Demari et al., 2011; Linton et al., 2013; Viveros et al., 2022).
Studies regarding the analysis of mosquitoes from Peru are limited, outdated, and concentrated only in some regions of the country, which generates a gap in the knowledge of the distribution, ecology, and diversity of the species over the Peruvian national territory (Ayala et al., 2020). In the department of Huanuco, 15 genera including 36 species of Culicidae have been registered, and of these, six are Culex species. On the other hand, our study reports the occurrence of Cx. camposi, in addition to adding the record of Cx. bonnei to the central region of Peru (Huanuco). However, we suspect an underestimation of mosquito biodiversity, since no more studies of culicids were carried out in this region since 1971, even though they are important vectors of human and/or animal pathogens. Understanding their geographical distribution will provide us with the tools to predict possible risks of VBD that could be influenced by land use and climate changes (Johnson et al., 2008; Gorris et al., 2021).