Molecular identification and wing variations among malaria vectors in Akure North Local Government Area, Nigeria

Members of the Anopheles gambiae complex and Anopheles funestus group are significant vectors of the malaria parasite Plasmodium species in the Afro-tropical region of the world. Molecular identification and variation in the wing were studied among female An. Gambiae complex and An. funestus group, to investigate morphological variations in the wing of local vectors populations of adult female mosquitoes found in five different locations in Akure North Local Government Area of Ondo State (Oba—Ile, Igoba, Isinigbo, Ita—Ogbolu and Iju). The variations in the wing character were found in the 3rd main dark spot area (Pre-apical dark spot—character 8) on the coastal region (Vein region I) of Anopheles gambiae complex wing; with two types (A and B) of wings identified with An. gambiae complex in the study area. Molecular study shows that all the wing type A are Anopheles gambiae s.s., they represent 53.39% of the total An. gambiae complex in the study area. Some of the Anopheles gambiae s.s. (28.30%) and all An. arabiensis (18.30%) were found with wing type B. Among 750 individual Anopheles mosquito species identified using Polymerase Chain Reaction (PCR method), 433 samples representing 57.73% were An. gambiae s.s. while 97 (12.93%) samples were An. arabiensis. Anopheles leesoni was the only member of the An. funestus group identified in the study area. Anopheles leesoni mosquitoes identified in the study location were 182, representing 24.27% of the total Anopheles mosquito species identified using the molecular method. Anopheles gambiae s.s., An. arabiensis, and An. leesoni are only Anopheles mosquito species responsible for malaria transmission in the study area. Anopheles leesoni was the only member of the An. funestus group identified in the study area.

www.nature.com/scientificreports/ infection is a significant public health problem in Africa. In Africa, the primary vector of malaria parasites belongs to Anopheles gambiae complex and Anopheles funestus group [3][4][5] . Anopheles gambiae complex contains eight members with similar morphological features, especially among the closest sibling species Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles coluzzii; the primary vector of malaria parasites in Africa 5 . Among the member of the Anopheles funestus group, Anopheles funestus s.s. has been reported as an important vector of malaria parasites in the Afro-tropical region. Another member of the Anopheles funestus group, Anopheles leesoni, whose source of blood meal is animal, has been reported predominating in human dwelling and feeding on human blood 6,7 . The existence of species complexes in Anopheles vectors has led to difficulties in precisely identifying sibling species (isomorphic species) and subspecies (morphologically, cytologically, and polymorphic races) members that possess identical morphology or minimal morphological distinction 8,9 . The modern-day taxonomy seems to have solved the problems associated with complexes species known to be groups of morphologically indistinguishable species that are genetically different and differ significantly in vectorial potential. There is limitation in the uses of morphological approaches in Anopheles species identification. This could cause discrepancy and result in identification problem 10 . The number of sensilla coeloconica, the value of the papal index, and the eggs' shape can differentiate Anopheles species complexes. Though, none of these characters gave complete discriminant as the morphological characters of adults are variable and overlap in many instances.
Identifying vectors that belong to species complexes has long been a stumbling block in malaria epidemiology and control 11 . Thus, this research aims to identify the vector of malaria parasites using molecular techniques and variation in morphological characters of the wing in five major settlements in Akure North Local Government Area of Ondo State, Nigeria.

Morphological study of the adult female Anopheles mosquito wing. Upon adult emergence,
Anopheles mosquitoes were identified using standard morphological characters keys supplied in Gunathilaka 10 , Nagpal and Sharma 13 . Species identification was done based on taxonomic keys 14 . The wing characters of Afrotropical Anopheles mosquitoes responsible for transmitting malaria parasites was considered following the morphological characters and wing spots nomenclature provided by Gillies and Coetzee 14 .
DNA extraction. Before the DNA extraction, the individual mosquito samples were store in 70% ethanol and refrigerated. The genomic deoxyribonucleic acid (DNA) was extracted from the thorax and abdomen tissues of individual mosquitoes using genomic DNA purification kit for animal tissue provided by Jena Bioscience.
Amplification of extracted DNA. The amplification of extracted DNA was carried out following the pro-  15 . The prepared PCR master mix of 12.5 μl was added into each 200 μl tube, after which 1.0 μl of extracted DNA was added to each of the tubes. Each tube was loaded into the PCR machine, and an appropriate program was selected. The PCR product was allowed through the first PCR cycle for initial denaturation at 95 °C for 2 min, after which it underwent denaturation at 95 °C for 30 s. The products were annealing at 55 °C for 30 s, followed by product extension at 72 °C for 40 s. Appropriate program for Anopheles funestus amplification process was selected following the protocol described by Solis BioDyne for a Polymerase Chain Reaction. The DNA cocktail used for species identification of Anopheles funestus group contains 4.0 μl pre-mix, 6.6 μl ddH 2  Data analysis and interpretation. The bands that appear on the gel were documented and scored according to Anopheles specific species base pairs. Data were subjected to statistical analysis using SPSS Version 26. The population of Anopheles mosquitoes was compared among the study locations using Chi-square analysis.

Molecular identification of Anopheles gambiae complex. Out of 550 morphological identified
Anopheles gambiae complex, 530 individual Anopheles gambiae which represent 96.36% were positive while 20 (3.64%) of the collected samples were negative ( Table 1). The main specific Anopheles gambiae complex species that was present in the identified samples were Anopheles gambiae s. s. and Anopheles arabiensis. Molecular analysis confirmed that 78.27% were Anopheles gambiae s.s., the DNA bands of this species was molecularly identified on the gel image with 390 base pair. About 17.64% of samples were confirmed to be Anopheles arabiensis, this was identified with 315 base pair on the gel image ( Fig. 1). Both identified species were well distributed across the five study locations. The highest number of Anopheles gambiae s.s. was found among the mosquito samples of Anopheles gambiae complex collected from Oba-Ile with 89.90%, while the least number of Anopheles gambiae s. s. was recorded from Igoba (73.64%) and Isinigbo (75.46%). The number of Anopheles arabiensis mosquito from these five locations were small compare to Anopheles gambiae s. s., the percentage of Anopheles arabiensis molecularly identified from Igoba, Isinigbo and Itaogbolu were 23.64%, 20.91% and 18.18% respectively. The least were from Oba-Ile (9.09%). There was significant difference (Chi-square = 12.18; df = 4; P > 0.05) between these two species of Anopheles gambiae complex present in the study area.
Variation in Anopheles gambiae complex wing types. The two distinctive wing types of Anopheles gambiae complex collected from the study area were studied, this was done alongside with the molecular confirmation of the specific species. These two types of the wing were presented earlier on Fig. 2A and B. The variations in the wing character were found in the 3 rd main dark spot area (Pre-apical dark spot-character 8) on coastal region (Vein region I). The most common type of the wing in the population of An. gambiae complex studied were those with the interrupted pale spot (Character 9; Fig. 2A) fused with proceeding pale spot of the sub-coastal pale spot. In the other type, pale interruption was found within the pre-apical dark spot (Character 8; Fig. 2B). The result of the molecular analysis confirmed that all the samples with wings having a pale interruption proceed with the sub-coastal pale spot (wing type A) are An. gambiae s. s, which represented 53.39% of the total number of positive samples for An. gambiae complex. Among the wing type B (those with pale interruption found within the pre-apical dark spot), 60.73% of the samples was Anopheles gambiae s. s., this represented 28.30% of the total number of positive samples for An. gambiae complex while 39.27% were Anopheles arabiensis/this represented 18.30% of total number of positive samples for An. gambiae complex. There was significant difference (Chi-square = 24.39; df = 4; P < 0.05) in population of the Anopheles gambiae complex mosquitoes across the location and between the type A and type B wing ( Table 2).

Molecular identification of Anopheles funestus group. Among the 200 samples of morphologically
identified as belonging to Anopheles funestus, 182 (91.0%) of the studied samples were Anopheles leesoni (a member of funestus group) and 9.0% failed the amplify (Table 3). A representative of agarose gel image showing the DNA fragment bands were presented on Fig. 3. Anopheles leesoni was identified on the gel at 146 base pair.

Discussion
The molecular identification of malaria vectors in the study location confirmed three important species of Anopheles mosquitoes responsible for malaria transmission in the study area. Two of these malaria vectors belong to the complex of Anopheles gambiae (An. gambiae s. s. and An. arabiensis) while An. leesoni was the only member of An. funestus group found in the study area. These three species of Anopheles mosquitos are major malaria parasite Table 1. Distribution of molecular identified Anopheles gambiae complex in the study area. Chi-square (χ 2 ) = 12.18; df = 4; P = 0.14 (P > 0.05).  Rozendaal 15 , the identified species of Anopheles mosquitoes in the area are the vector of public health importance due to their anthropophilic behaviour. An. gambiae s.s. is the most abundant in the studied locations and the most important within the member of Anopheles gambiae complex. Fahmy et al. 16 and Kabbale et al. 17 have described An. gambiae s. s. as the largest anthropophilic among Anopheles mosquito species due to their preference for human blood, and they are primarily common around human dwellings. The preference of An. gambiae s.s. for human blood and their endophagic behaviour makes this insect the most important vector of malaria parasites in Afro-tropical regions where they are more abundant. The present research work supports the finding of Kabbale et al. 17 . In the research carried out by Hamza et al. 18 , An. arabiensis was reported to be predominant over An. gambiae s.s. but this was different from what was reported in this present study, which described An. gambiae s.s. more predominant and widespread compared to An. arabiensis in all study locations. Anopheles gambiae s.s. and An. arabiensis occur sympatrically in the same breeding habitat. The two forms (A and B) of wings of identified An. gambiae s.s. and An. arabiensis was presented earlier in Fig. 2a and b. This present study revealed that all the species with the wing type A are molecularly identified as An. gambiae s.s., this character could be used in rapid field identification of members of An. gambiae complex in the study location. In their study, Sanford et al. 19 described the importance of wing characters of the two molecular forms of An. gambiae s.s. which include the measurement of morphometric characters of the sympatric species distribution from their study areas. The molecular identification of the An. gambiae complex mosquito samples with the wing type B revealed two species, which are An. gambiae s.s. and An. arabiensis. This result shows that using wing type B in sorting out the species member of An. gambiae complex might lead to species discrimination. In the study of the wings and molecular identification of these vectors, the result of this present study has shown that An. gambiae s.s. can have both types of wings (type A and B). Anopheles gambiae with wing type A are more predominant compare to those wing type B in the study locations. The outcome of the present study has revealed that some of the species of An. gambiae s.s. and An. arabiensis share wing type B. The only method for separating both species is by using the molecular approach.
In this present study, the only member of An. funestus group confirmed in the study location was An. leesoni. Anopheles leesoni has been reported as a zoophilic member of the An. funestus group 20 . Among the member of An. funestus group, An. funestus s.s. is the only member of this group that is anthropophilic and responsible for the transmission of malaria parasites in Africa, other members of this group prefer animal blood. Anopheles leesoni, An. parensis and An. rivulorum, which are primarily zoophilic, have been reported by Temu et al. 6  www.nature.com/scientificreports/ feeding on human blood (anthropophilic) and found infected with Plasmodium falciparum and transmission of the malaria parasites. All the An. leesoni identified in this present study, was found within the human dwellings; this contradicts the earlier report of Gillies and De Meillon 20 , which reported that An. leesoni found close to cattle ranches and feeding mainly on cattle. Kamau et al. 21 reported An. leesoni is found close to human dwellings in their study in Kenya. In Nigeria, a West Africa region of Africa, endophilic and anthropophagic behaviour of An. leesoni has been reported by Awolola et al. 22 . Kent and Norris 23 have reported a collection of An. leesoni already fed on either goat or cattle around human dwellings in Zambia; this suggested that An. leesoni have the tendency of feeding on both human (anthropophilic) and animal (zoophilic) blood and also feed indoor (endophagic) and outdoor (exophagic).

Conclusions
The molecular identification has revealed that Anopheles gambiae s.s. can have different type of wing (A and B). Further study is needed to assess whether individual with different types of wings have different competency in malaria transmission. Presence of An. leesoni in the study area shows that the vector could also contribute to the burden malaria infection in the study locations, however, further exploration is needed toward this end.

Data availability
All analysed data involved in this study are included in this manuscript.  Table 3. Molecular identification of Anopheles funestus group in the study area. χ 2 = 557.72; df = 4; P = 0.01.