Molecular characterization and phylogenetic analysis of collected mosquitoes (Diptera: Culicidae) from Northcentral Nigeria using mitochondrial COI and ribosomal IGS gene regions

Abstract

Background

Mosquitoes are important vectors of disease-causing organisms such as filarial worms, malaria parasites, and arboviruses endemic to sub-Saharan Africa including Nigeria. Malaria is a disease caused by a plasmodium parasite, transmitted by the bite of infected mosquitoes and is no doubt a public health concern. There is limited information on the genetic diversity of mosquitoes in Nigeria. This is necessary because information about the genetic diversity of mosquitoes is a very important step towards vector control and management with the aim to mitigate or eliminate burden resulting from malaria and other diseases caused by mosquitoes. In the present study, we investigated the genetic variability and relatedness of mosquitoes based on the DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) and ribosomal intergenic non-coding spacer gene regions (IGS). Mosquitoes were collected from five different states in Northcentral Nigeria, they were morphologically identified using standard keys and genomic DNA was extracted. The specific regions of interests were amplified, and the PCR products were then sequenced.

Results

PCR was able to successfully amplify the expected amplicon sizes of COI and IGS sequences (710 and 169) base pairs, respectively. For COI sequence, pairwise genetic distance between mosquito species ranged from 0.00 to 0.17 in the COI sequences. The pairwise genetic distance among Culex, Aedes and Anopheles species in the IGS sequences ranged from 0.000 to 0.118. Phylogenetic analysis of the sequences of the mitochondrial cytochrome oxidase subunit showed that there was genetic diversity amongst the different mosquito species sampled. It effectively showed marked differences between Culicine and Anopheline mosquitoes.

Conclusions

The ribosomal IGS primers used for this study only amplified Anopheles spp. However, it revealed that there is diversity among the Anopheles gambiae and Anopheles arabiensis samples collected. This study concludes that the mitochondrial COI and ribosomal IGS gene regions are reliable markers for mosquito genetic diversity study and will surely yield a reliable result for molecular diversity assessment of mosquito species.

Background

Mosquitoes belong to the family Culicidae which is comprised of three sub families which include Toxorhynchitinae, Anophelinae and Culicinae. Toxorhynchitinae, which has only one genus Toxorhynchites, is not of any medical importance because it feeds on nectar rather than blood (Collins & Blackwell, 2000). Mosquitoes serve as major vectors of several diseases because it feeds on blood and transmits viral, bacterial, protozoan and other diseases (Becker et al., 2010). Mosquitoes occur in a wide range of both aquatic and terrestrial habitats and have different morphological and behavioural adaptations to these habitats (Becker et al., 2010). Culicine mosquitoes like Aedes spp. and Culex spp. are important carriers of human pathogens (Molina-Cruz et al., 2013). Anopheline mosquito is the major vector of human malaria. Identifications of mosquito species are based on main morphological features such as legs, antennae, wings and position of the abdomen. However, many mosquitoes exist as species complexes, such as Culex pipiens L. sensulato (s.l.) (Cornel et al., 2012) and Anopheles gambiae s.l. (Scott et al., 1993), limiting their identification based on morphology alone. Previous mosquito diversity studies in Nigeria have mainly employed morphology (Afolabi et al., 2019; Oduola et al., 2013) with few studies on molecular analysis to identify the different mosquito species (Lamidi et al., 2017; Msugh-Ter et al., 2017).

Molecular approaches to support morphological data provide better insight into the genetic diversity of mosquito species (Kumar et al., 2007) and may play a vital role designing and executing effective strategies in the malaria vector control strategies. Different molecular markers have been used for characterization, diversity and population genetic analysis of mosquitoes. Mitochondrial DNA analysis has proven to be a useful and reliable marker for population genetics, intraspecific phylogeography, and systematics (Zink & Barrowclough, 2008). Mosquitoes have been successfully differentiated genetically using the mitochondrial cytochrome c oxidase I (COI) gene region (Hebert et al., 2003). Krüger et al. (2014) have previously used COI gene region for molecular identification of Aedes geniculatus. Anopheles daciae mosquitoes have characterized in Germany using ITS2 sequence analysis (Weitzel et al., 2012). Genetic diversity and molecular characterization of mosquitoes in North-Central Nigeria using ribosomal DNA ITS2 and mitochondrial 16S-DNA sequences have been reported (Iyiola et al., 2020). The IGS region is a reliable molecular marker in studying genetic and phylogenetic divergence between closely-related of Anopheles mosquito species (Sharma & Chaudhry, 2010).

Variations in larval environment may contribute to insecticide susceptibility of Anopheles mosquitoes (Owusu et al., 2017). Taken into consideration genetic modifications that can be induced due environmental influences, the high abundance and widespread presence of invasive species reported across the world, there is a need to provide a baseline information on genetic diversity of mosquitoes in North-central Nigeria. This is vital because precise, consistent and reliable identification of mosquitoes using DNA-based approach is important and will go a long way to provide a baseline data required in malaria vector control strategies and war against malaria vector in Nigeria. Our central hypothesis is that due to constant genetic mutation, there could be invasive mosquito species in Nigeria which could be a setback to sustainable approaches for surveillance, prevention and control of vector-borne diseases. Therefore, this study investigated genetic diversity study of mosquitoes using a molecular approach. We investigated species composition and genetic diversity between and within mosquito species in North-Central Nigeria using the mitochondria COI and ribosomal DNA intergenic spacer IGS gene regions. Data from this study will provide a robust assessment of mosquito species’ distributions and molecular diversity in Northcentral Nigeria.

Materials and methods

Sample collection

Mosquito larvae were collected from different mosquito breeding sites like abandoned earthen ponds, gutters, stagnant water, water storage containers, abandoned car tyres, etc. in five states in North-Central Nigeria. The states include Benue, Kogi, Kwara and Niger States and Abuja, the Federal capital of Nigeria. The samples were collected from April 2018 to March 2019. Samples were transported to the laboratory and separated into different genera based on the presence or absence of breathing tubes and the length of breathing tubes. They were then kept in bowls labelled based on location and genera. Upon emergence into adult, the mosquitoes were collected using aspirator and put in a labelled 1.5 ml Eppendorf tubes and preserved dry on desiccated silica gel for further analysis.

Morphological identification

Morphological identification of mosquitoes collected was carried out with the aid of a dissecting microscope using standard morphological keys as described by Gillies and Coetzee (1987).

DNA extraction

Genomic DNA was extracted from the whole body of the mosquitoes using Zymo Research Insect Mini prep kit with slight modifications to the manufacturer’s protocol. The isolated genomic DNA (gDNA) was checked using NanoDrop 2000c to check for the quantity and purity of the samples. The genomic DNA extracted was also run on 1% agarose prepared with 1X TBE buffer at 80 V for 1 h to confirm the presence of DNA.

PCR amplification of the mitochondrial cytochrome oxidase subunit I (COI) region

The mitochondrial cytochrome oxidase subunit I (COI) gene region was amplified using primers stated in Table 1. PCR amplification was carried out using 5X Hot FIREPol Blend Master Mix with 7.5 mM MgCl₂ from Solis BioDyne Estonia. A 25 µL reaction mixture was prepared using 5 μL of 5X Hot FIREPol, 0.25 μL each of both primers, 14.5 μL of nuclease free water and 5 μL of template DNA. The PCR was run using the following conditions: initial denaturation at 95 ° for 15 min, denaturation at 95 ° for 30 s, annealing at 42 ° for 1 min, elongation at 72 ° for 1 min for 30 cycles and final elongation at 72 ° for 10 min. 5µL of each reaction mixture was run on 1.8% DNA agarose gel with 1X TBE running buffer and stained with ethidium bromide stain. Electrophoresis was conducted at 80 V, 150 mA for 1 h 30 min. The gel was viewed under a UV transilluminator.

Table 1 Primer sequences from COI and IGS sequences used for detection of polymorphism in the Mosquito populations

PCR amplification of the intergenic spacer (IGS) region

The primers used for the amplification of this region were adapted from Ajamma et al. (2016a, b). The primers details are provided in Table 1.

PCR amplification was carried out using 5X Hot FIREPol Blend Master Mix with 7.5 mM MgCl₂ from Solis BioDyne Estonia. A 25 µL reaction mixture was prepared using 5 μL of 5X Hot FIREPol, 0.25 μL each of both primers, 14.5 μL of nuclease free water and 5 μL of template DNA. The PCR was run using the following conditions: initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 50 °C for 1 min, elongation at 72 °C for 1 min for 30 cycles and final elongation at 72 °C for 5 min. 5µL of each reaction mixture was run on 2% DNA agarose gel with 1X TBE running buffer and stained with ethidium bromide stain. Electrophoresis was conducted at 90 V, 150 mA for 90 min. The gel was viewed under UV light in a gel documentation system.

Phylogenetic analysis

The PCR mixtures were sent to Inqaba Biotech, South Africa for sequencing. Sequences in ABI format was subjected to trimming, editing and base culling were carried out using Sequencher 5.4.6. The sequences were exported to notepad. These text files were aligned using the ClustalW multiple alignment feature on BioEdit and the aligned sequences were saved as FASTA files, later opened on MEGA7 and phylogeny was inferred using maximum likelihood tree with 1000 bootstrap replications.

Results

Species composition

We determined species composition of mosquitoes collected from Niger, Kogi, Benue, Kwara states and Abuja in North-central Nigeria. A total of 2,100 mosquito samples were collected and they included Culex, Anopheles and Aedes species. Culex spp. were the most abundant of all the mosquitoes species collected during this study (Table 2). Aedes spp. were found in Benue, Kwara and Kogi States. Anopheles spp. were the abundant mosquito species collected from Benue State.

Table 2 Number of mosquitoes collected from different sites in North-Central Nigeria

Molecular analysis and PCR amplification

DNA agarose gel images of the PCR amplicons of the mitochondrial IGS and COI gene regions, respectively, after electrophoresis (Figs. 1, 2). The IGS and COI gene regions of all the extracted DNA of the mosquito samples were amplified successfully using primers stated in Table 1. PCR mixtures were loaded on 1.8% agarose and run at 90 V for 90 min. It was then viewed with UV transilluminator and photographed. Lane M is the 100 bp DNA ladder, while lane 21–45 is the PCR amplicons of different mosquito samples. The expected and amplified PCR fragment sizes were 169 and 710 (base pairs) for IGS and COI gene regions, respectively, (Figs. 1 and 2).

Fig. 1

Amplification profile of mosquito species with primers from the IGS gene region. M = DNA markers (size 100 bp), 1–20: Mosquito samples

Fig. 2

Amplification profile of mosquito species with primers from the COI gene region. M = DNA markers (size 100 bp), 21–45: Mosquito samples

Phylogenetic analysis

Dendogram of the intergenic spacer region sequences of the Anopheles spp. amplified for this study is shown in Fig. 3. The phylogenetic tree constructed is a Maximum-Likelihood tree with 1000 bootstrap replications according to Tamura-Nei method. Green circles in Fig. 3 represent the samples sequenced in this study and those in red boxes are Anopheles spp. sequences retrieved from the NCBI database. Culex pipiens pipiens sequence, denoted in blue diamond, also retrieved from the NCBI database was used to represent the outgroup. Anopheles gambiae haplotype in this study clustered together with the sequence available at the GenBank database and was isolated from Guinea Bissau (Ascension number KX828857.1). The sequences are supported by a bootstrap value of 68 at the node. Likewise, the Anopheles arabiensis also clustered with the sequences gotten from Brazil but was supported by a bootstrap of 65 at the node. (Fig. 3). Dendogram of the mitochondrial cytochrome oxidase subunit I of the mosquito species is shown in Fig. 4. The lemon circles denote the mosquito samples sequenced for this study, the lilac triangles represent the sequences collected from the NCBI database and the red diamond represents the outgroup used to root the tree. Some Culex pipiens sequence in this study showed divergence with those from Turkey, while others clustered with the sequences from Kenya. Culex watti and Culex antennatus sequences from this study also clustered with same species from Kenya and they are greatly supported with a bootstrap > 90 (Fig. 4). Data from analysis of evolutionary divergence between COI and IGS gene sequences of some mosquito species used in this study are as indicated in Tables 3 and 4, respectively. DNA nucleotide sequences were deposited at the GenBank database for future reference, and they were assigned with Genbank accession numbers OK257522-OK257529 for COI and OK236349-OK236355 for IGS.

Fig. 3

Molecular phylogenetic analysis of the intergenic spacer (IGS) region of Anopheles spp. by Maximum Likelihood method inferred by Tamura-Nei method after 1000 replications. Culex pipiens pipiens was used as an outlier

Fig. 4

Molecular phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I (COI) region of mosquito species by Maximum Likelihood method inferred by Tamura-Nei method after 1000 replications. Periplaneta americana was used as an outlier

Table 3 Estimates of evolutionary divergence between mitochondrial COI sequences of some mosquitoes used in this study

Table 4 Estimates of evolutionary divergence between the IGS region sequences of some Anopheles species used in this study

Estimates of evolutionary divergence between the COI sequences obtained from the Culex and Anopheles species are shown in Table 3. As expected between mosquito species, the estimated pairwise genetic distance was observed between Anopheles gambiae and Culex pipiens species in the COI sequences from mitochondrial DNA at 0.17.

In Table 4, the DNA nucleotide sequence of Anopheles gambiae collected from Ilorin, Kwara State is genetically different from Anopheles gambiae collected from Abuja and Niger state of Nigeria with a pairwise genetic distance value of 0.009. Anopheles arabiensis collected from Yandev, Benue State, Nigeria is genetically different within species with a pairwise genetic distance value of 0.009. The highest pairwise genetic distance was 0.118 was observed between Anopheles gambiae and Anopheles arabiensis.

Discussion

In this study, 2,100 mosquito samples were collected and Anopheles sp has the highest proportion in Benue state with non in Kogi state. Culex sp was found to have highest species composition in Niger states. Similarly, Aedes sp was found to have the highest abundance  in Kogi state with  no Aedes reported in both Niger state and Abuja. The presence of mosquitoes in the studied locations could be due to overpopulation of these areas which may have led to improper waste and sewage disposal which provides a suitable habitat for the Culex mosquito species. Mosquitoes’ abundance could be related to urbanization, over-abundance of human activities as well as other anthropogenic activities that has led to poor waste disposal, poor sanitary levels, uncontrolled run-offs, etc. which has created a habitat for breeding of these mosquitoes. This result is consistent with data from  a study carried out in Benin City by Aigbodion and Uyi (2013) which reported that Culex and Aedes had higher abundance over Anopheles mosquitoes. Mosquitoes collected from Benue states showed a higher relative abundance of Anopheles species. It is reasonable to suggest that relative species abundance of Anopheles may likely lead to prevalence of malaria especially among infants, pregnant women and immuno-compromised individuals in this area if no vector control measures are put in place. This observation is consistent with the report of a study carried out by Aju-Ameh et al. (2017) where the highest Anopheles mosquitoes were recorded in Gboko, Benue state, Nigeria. In Ilorin, Kwara State, three mosquito genera were recorded and Culex spp was the most prevalent. However, a study by Ajao and Adeleke (2014) had the highest abundance and this was different from the report from a study conducted by Amaechi et al. (2018), where Anopheles gambiae had the highest abundance.

The intergenic spacer (IGS) primers used for this study were only able to distinguish between Anopheles spp. The intergenic region produced amplicon size of 169 bp. Sequence variations were observed in the DNA based-nucleotide sequence of the IGS gene region. Nucleotide bases substitutions of both transition and transversion types were observed when the sequenced DNA was aligned. Twelve (12) variable sites were observed among all the species which included 5 transversion and 7 transitions. Between the An. gambiae and An. arabiensis, 11 variable sites were observed, with A-G transitions being the most common. A single variable site of G-T and A-T transversion was also observed within the species of An. gambiae and An. arabensis, respectively. However, for COI sequence, we observed that evolutionary individual divergence between species ranged from 0.000 to 0.009 (Table 3). The pairwise genetic distances were determined for Anopheles species. Low genetic distance value was observed. This observation indicates that the Anopheles diverge more recently. This also showed that there is low individual divergence among the Anopheles species of mosquitoes collected in this study. This is consistent with previous study of Hamza et al. (2014) which reported high genetic similarities and low individual divergence within the An. gambiae individuals collected from Sudan. From the phylogenetic trees, it was observed that the region could differentiate the different mosquito subfamilies with Anophelinae occupying the basal position. This region could also differentiate the different mosquito species genera which is in correlation with a study of Ajamma et al. (2016a, b) where it was reported that COI was able to distinguish between members of Mansonia, Culex and Aedes. The phylogenetic tree constructed from IGS gene region is shown in Fig. 3. It identified two Anopheles species complex which are An. gambiae and An. arabiensis and were able to separate between the two species of Anopheles as they both form a separate clade on the phylogenetic tree with the species of An. gambiae clustering together and that of An. arabiensis also clustered together (Fig. 3). This is supported by the study of Ajamma et al. (2016a, b) where it was reported that IGS gene region was able to successfully distinguish between Anopheles mosquito species. Anopheles gambiae haplotype in this study clustered together with the sequence retrieved from GenBank database and was isolated from Guinea Bissau (Ascension number KX828857.1). The sequences are supported by a bootstrap value of 68 at the node. Likewise, the Anopheles arabiensis also clustered with the sequences gotten from Brazil but was supported by a bootstrap of 65 at the node. (Fig. 3). This is an evidence of identical and clearly established phylogenetic relationship among the Anopheles species used in this study Guinea Bissau anopheles retrieved from Genbank. The phylogenetic tree in Fig. 4 showed that the different Culex species, namely C. pipiens, C. watti and C. antennatus, formed separate clades of similar species clustering together and supported by a good bootstrap value. C. pipiens from this study clustered with C. pipiens from Kenya and Turkey, while C. watti from this study clustered with the one from Kenya. This revealed evidence of identical and clearly established phylogenetic relationship among the Culex species used in this study due to clustering of similar species on the same nodes. Similarly, An. gambiae from this study clustered with Uganda anopheles retrieved from Genbank database. There is no doubt that these show evidence of similarity in the mtDNA sequence of Culex species from our study and those from Kenya, Turkey and Anopheles. It could be hypothesized that the mosquito populations collected and sequenced are probably from the same origin with those from other parts of the world, although there is no evidence of their dispersal routes. This observation is supported by previous studies which suggested that Culex is not a monophyletic group (Rozo-Lopez & Mengual, 2015). There were also low genetic variations in the DNA sequences of mosquito species both within and between species. DNA nucleotide base substitutions (transition and transversion) were observed among the mosquito species studied. In all the sequenced COI samples, there was 230 variable sites the highest being A → T transversions within the Culex pipiens sequences, there were three variable sites which is G ↔ A transition, there were 22 variable sites between the three Culex antennatus species which include 14 transversions and 8 transitions. There are also 173 variable sites among all the Culex species sequenced. These results are consistent with previous study of Shouche and Patole (2000) where they also reported a high A-T type transversion in both 16S rRNA and COI sequences.

Conclusions

This study concludes that the reliability of the use of ribosomal IGS and mitochondrial COI gene regions for molecular diversity study and precise identification mosquito species in North-Central Nigeria because they can provide sufficient information on the mosquito species diversity present in this region. Both regions were able to show the evolutionary relationship amongst the mosquito species as well as infer the molecular divergence existing between and among them. Further investigation into the evolutionary relationship and divergence among species complexes with larger sample size is therefore recommended and this could be achieved through DNA barcoding and population genetic structure of mosquito species in North-central  Nigeria. We therefore suggest increased researches on applications of genetics and population genomics in mosquito control strategies to effectively wage war against malaria.