Large-Scale Cytochrome C Oxidase Subunit I Gene Data Analysis for the Development of a Multiplex Polymerase Chain Reaction Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of the Culicoides Subgenus Avaritia Fox, 1955

Abstract

The emergence of culicoid-transmitted bluetongue and Schmallenberg viruses in several European countries demonstrated the ability of indigenous biting midge species to transmit pathogens. Entomologic research programs identified members of the Obsoletus Group (Culicoides subgenus Avaritia) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors is challenging due to the recent discovery of new genetic variants (haplotypes) of C. obsoletus sensu stricto (s.s.), forming distinct clades. In this study, 4422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenus Avaritia members of the genus Culicoides were analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and clades of the Western Palearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three C. obsoletus clades represent independent species, whereas C. montanus should rather be regarded as a genetic variant of C. obsoletus s.s. Based on these findings, specific primers were designed and validated with DNA material from field-caught biting midges which achieved very high diagnostic sensitivity (100%) when compared to an established reference PCR (82.6%).

Keywords: Avaritia; Culicoides; haplotype; mitochondrial cytochrome c oxidase subunit I (COI); obsoletus group; polymerase chain reaction (PCR); vector.

Figure 1

Inter- and intraspecific pairwise comparison of COI gene DNA sequences between the analyzed taxa of the subgenus Avaritia: Interspecific genetic distances are displayed in the left-bottom half of the matrix and highlighted with graded colors from red (low distance) through yellow (medium distance) to green (high distance). Interspecific pairwise identities in gene sequence are presented in graded colors in the right-upper half of the matrix with the opposite meaning of the colors: red (high similarity)—yellow (medium similarity)—green (low similarity). Intraspecific pairwise identities (Intra) are given as well, using the same color code. Values (in %) were calculated through the comparison of species- and haplotype-specific consensus sequences of respective GenBank entries (n). C. obsoletus clade O1 (obs O1), C. montanus (mont), C. sinanoensis (sina), C. obsoletus clade O3 (obs O3), C. obsoletus clade O2 (obs O2), C. sanguisuga (sang), C. scoticus clade 1 (scot 1), C. abchazicus (abch), C. scoticus clade 2 (scot 2), C. alachua (alach), C. chiopterus (chio) and C. dewulfi (dew).

Figure 2

Proof of function of the multiplex PCR test for the members of the Obsoletus Group, including C. chiopterus and C. dewulfi. Specific forward primers were tested regarding their specificity (singleplex, A–F) and capability for multiplexing (G). The forward primers used were the following: obs1-COI-120F (A,G), obs2-COI-167F (B,G), obs3-COI-230F (C,G), sco-COI-317F (D,G), chi-COI-407F (E,G) and dew-COI-470F (F,G). All primers were used in combination with the universal reverse primer PanCuli-COX1-727R. DNA samples used for PCR validation contained either 10⁶ copies of specific target or 10^7.5 copies of unspecific target (synthetic COI gene). For C. dewulfi and C. chiopterus, equivalent amounts of quantified COI gene amplicon were used. Lane 1: 50 bp ladder (50–500 bp Gene Ruler; Roth, Karlsruhe, Germany), lane 2: no template control, lane 3: C. obsoletus clade O1, lane 4: C. obsoletus clade O2, lane 5: C. obsoletus clade O3, lane 6: C. scoticus clade 1, lane 7: C. chiopterus and lane 8: C. dewulfi.