Sequencing and Description of the Mitochondrial Genome of Orthopodomyia fascipes (Diptera: Culicidae)

Abstract

The genus Orthopodomyia Theobald, 1904 (Diptera: Culicidae) comprises 36 wild mosquito species, with distribution largely restricted to tropical and temperate areas, most of which are not recognized as vectors of epidemiological importance due to the lack of information related to their bionomy and involvement in the cycle transmission of infectious agents. Furthermore, their evolutionary relationships are not completely understood, reflecting the scarcity of genetic information about the genus. Therefore, in this study, we report the first complete description of the mitochondrial genome of a Neotropical species representing the genus, Orthopodomyia fascipes Coquillet, 1906, collected in the Brazilian Amazon region. Using High Throughput Sequencing, we obtained a mitochondrial sequence of 15,598 bp, with an average coverage of 418.5×, comprising 37 functional subunits and a final portion rich in A + T, corresponding to the control region. The phylogenetic analysis, using Maximum Likelihood and Bayesian Inference based on the 13 protein-coding genes, corroborated the monophyly of Culicidae and its two subfamilies, supporting the proximity between the tribes Orthopodomyiini and Mansoniini, partially disagreeing with previous studies based on the use of molecular and morphological markers. The information generated in this study contributes to a better understanding of the taxonomy and evolutionary history of the genus and other groups of Culicidae.

Keywords: Culicidae; mitogenome; molecular taxonomy; mosquitoes; phylogenetics.

Figure 1

Location map of the municipality of Santa Bárbara do Pará demarcating the collection site of the investigated species. This Figure was created using the software QGIS v.3.10.4 (available at https://qgis.org/en/site/, accessed on 30 March 2024) in conjunction with the IBGE 2022 cartographic database (available at https://www.ibge.gov.br/, accessed on 30 March 2024).

Figure 2

(A) Structural representation of Or. fascipes mtDNA. Internal values indicate the content of nucleotide bases. The blue inner graph indicates the distribution of genomic coverage by region. The blue, red, yellow, and gray blocks indicate the PCGs, tRNAs, rRNAs, and A + T region, respectively. Genes outside and inside the circle have forward and reverse transcription directions, respectively. (B) Heatmap of nucleotide/amino acid identity between taxa of the genus Orthopodomyia, based on the barcode region of the COI gene. The lower and upper triangles contain the percentages of nucleotide and amino acid identity, respectively. The taxon highlighted in red indicates the obtained sequence.

Figure 3

(A) AT% composition, (B) AT-skew, and (C) GC-skew of 16 previously characterized mosquito mtDNAs, including Or. fascipes (highlighted in red). Hierarchical groupings (clusters) of species (y-axis) are established based on the quantity of each metric per region evaluated (x-axis). The scales, particularly AT-skew and GC-skew, consider the real (non-normalized) values obtained in each analysis.

Figure 4

Relative use of synonymous codons of Or. fascipes (highlighted in red) in comparison to other representatives of the Culicinae subfamily. Hierarchical groupings (clusters) of species (y-axis) are established based on the RSCU of each codon (x-axis), with indications regarding the scale values.

Figure 5

(A) ENc-GC3s graph. The red dashed line indicates the curve expected when codon usage bias is affected only by mutation pressure. GC12-GC3 neutrality plots are arranged in the order of mitochondrial complexes: (B) I (NADH), (C) III (Cytb), (D) IV (COX), and (E) V (ATP). In the graphs, each blue point (varying in tone according to the complex) represents a PCG independent of the Culicinae mtDNAs used as a reference in the analyses. PCGs of Or. fascipes are highlighted in red.

Figure 6

(A) dN/dS ratios calculated from pairwise analyses between 17 Culicinae mtDNAs. The ω ratios and PCGs are arranged along the y and x axes, respectively. (B) Representation of the proportion of non-synonymous (dN) (gray strings) and synonymous (dS) (red strings) substitutions that occur in each PCG. This Figure serves as a visual complement to (A), indicating that the accumulation of synonymous substitutions is predominantly greater in all Culicinae PCGs, directly influencing the observed negative (or purifying) selection pressure (ω < 1).

Figure 7

(A) Nucleotide substitution saturation graph of the set of evaluated sequences, considering all PCGs, demonstrating the increase and linear association of transition (Ts) and transversion (Tv) rates (y-axis) in relation to genetic distance (x-axis). (B) Graphical representation of the results obtained from the application of the test proposed by Xia et al. (2003) [65], statistically supported (Iss < Iss.c, p = 0.00001), and showing the low occurrence of nucleotide saturation in the set of sequences evaluated.

Figure 8

(A) Phylogeny reconstructed using the Maximum Likelihood method based on the 13 PCGs of Or. fascipes (highlighted in red) and other taxa available in the GenBank repository (NCBI). Bootstrapping support values (BS) are shown on each node. The colored dots indicate the main reconstructed taxonomic groupings: family Culicidae (black) and subfamilies Culicinae (blue), and Anophelinae (yellow). (B) Maximum Likelihood mapping diagram demonstrating the quality of the phylogenetic signal resulting from the quartet analysis, with 98.2% resolved quartets. (C) Four-cluster likelihood mapping (FcLM) diagram representing four taxon groupings in Culicinae. The analyzed groups are indicated in the topology by the letters “a” (Aedini), “b” (Culicini), “c” (Orthopodomyiini + Mansoniini + Toxorhynchitini + Sabethini), and “d” (Aedeomyiini + Uranotaeniini). The bidirectional arrows indicate different hypotheses regarding the relationships among the four clusters, with their respective colors in the diagram corresponding to the support percentages.