Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies

Abstract

Background: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread.

Methods: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape.

Results: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2-7 intra-individual (mean 4.7) and 16-34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil.

Conclusions: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.

Keywords: Aedes albopictus; Brazil; Cloning; Disease vector; Molecular haplotyping; Sequencing; South-western Europe; Wing geometric morphometry; mtDNA cox1; rDNA 5.8S-ITS-2.

Fig. 1

a Position of 16 landmarks and b contour digitized on Ae. albopictus wings for landmark-based and outline-based geometric morphometric analysis, respectively. Scale = 1 mm

Fig. 2

Phylogenetic networks of Ae. albopictus haplotypes based on mtDNA cox1 sequences. a Median network with south-western Europe and Brazil populations. The area of each haplotype is proportional to the total sample. Small red-filled circle represents intermediate haplotype not present in the sample. Mutational steps between haplotypes are represented by line length. b TCS network with different localities and countries in Asia, Europe, and the Americas. Circles are proportional to the number of samples represented for each haplotype. Slashes on branches between nodes indicated mutations. The colors represented in the legend correspond with those represented by the haplotype network. Haplotype information detailed in Additional file 1: Table S1

Fig. 3

Numerical and graphical distribution of Ae. albopictus 5.8S-ITS-2 rDNA unique and shared haplotypes between countries and populations according to DnaSP, H haplotype

Fig. 4

TCS network among identified 5.8S-ITS-2 haplotypes of Ae. albopictus from south-western Europe and Brazil according to PopART. Circles are proportional to the number of samples represented for each haplotype. Slashes on branches between nodes indicated mutations. The colors represented in the legend correspond with those represented by the haplotype network. The smallest black nodes indicate unobserved haplotypes (median vectors)

Fig. 5

a Variation of centroid size and b wing perimeter (converted from pixels to mm) of females and males of Ae. albopictus from south-western Europe and Brazil. Each box shows the group median that separates the 25th and 75th quartiles and range. Each blue bar represents one specimen

Fig. 6

a Superposition of the mean landmark configurations and b outlines of Ae. Albopictus from south-western Europe and Brazil. Left, females (F); right, males (M)

Fig. 7

a Landmark-based and b outline-based discriminant analysis of Ae. albopictus from south-western Europe and Brazil. Factor map of the two canonical factors (CFs) derived from final shape variables for females (left) and males (right). Each point represents a specimen. The horizontal axis is the first CF; the vertical axis is the second CF. In brackets, percentage of contribution