The field of morphometrics is developing quickly. Recent advances have enabled geometric techniques to be applied to many zoological problems, particularly those involving epidemiologically-relevant mosquitoes. Herein, we briefly introduce geometric morphometric (GM) techniques and then review selected groups of mosquitoes (Culicidae) to which those techniques have been applied. In most of the reviewed cases, GM was capable of satisfactorily discriminating among the tested groups primarily when the studies considered differences within and among species, sexual dimorphism, treatments and the separation of laboratory strains. Although GM approaches have developed quite rapidly, some caution must be taken during data processing for a reliable biometrical approach, such as allometry and asymmetry analyses, scale removal and wing clarification staining for landmark digitization. We also critically forecast directions in this field and discuss how the creation of image databases should enhance species identification in culicids.

Mosquitoes are responsible for the transmission of important infectious diseases, causing millions of deaths every year and endangering approximately 3 billion people around the world. As such, precise identification of mosquito species is crucial for an understanding of epidemiological patterns of disease transmission. Currently, the most common method of mosquito identification relies on morphological taxonomic keys, which do not always distinguish cryptic species. However, wing geometric morphometrics is a promising tool for the identification of vector mosquitoes, sibling and cryptic species included. This study therefore sought to accurately identify mosquito species from the three most epidemiologically important mosquito genera using wing morphometrics. Twelve mosquito species from three epidemiologically important genera (Aedes, Anopheles and Culex) were collected and identified by taxonomic keys. Next, the right wing of each adult female mosquito was removed and photographed, and the coordinates of eighteen digitized landmarks at the intersections of wing veins were collected. The allometric influence was assessed, and canonical variate analysis and thin-plate splines were used for species identification. Cross-validated reclassification tests were performed for each individual, and a Neighbor Joining tree was constructed to illustrate species segregation patterns. The analyses were carried out and the graphs plotted with TpsUtil 1.29, TpsRelw 1.39, MorphoJ 1.02 and Past 2.17c. Canonical variate analysis for Aedes, Anopheles and Culex genera showed three clear clusters in morphospace, correctly distinguishing the three mosquito genera, and pairwise cross-validated reclassification resulted in at least 99% accuracy; subgenera were also identified correctly with a mean accuracy of 96%, and in 88 of the 132 possible comparisons, species were identified with 100% accuracy after the data was subjected to reclassification. Our results showed that Aedes, Culex and Anopheles were correctly distinguished by wing shape. For the lower hierarchical levels (subgenera and species), wing geometric morphometrics was also efficient, resulting in high reclassification scores.

BackgroundSexual dimorphism in animals has been studied from different perspectives for decades. In 1874 Darwin hypothesized that it was related to sexual selection, and even after nearly 140 years, when additional empirical data has become available and the subject has been investigated from a contemporary viewpoint, this idea is still supported. Although mosquito (Culicidae) wings are of great importance as they play a sex-specific role, little is known about wing sexual dimorphism in these pathogen-vector insects. Detection and characterization of wing sexual dimorphism in culicids may indirectly enhance our knowledge of their epidemiology or reveal sex-linked genes, aspects that have been discussed by vector control initiatives and developers of genetically modified mosquitoes.MethodsUsing geometric morphometrics, we carried out a comparative assessment of wing sexual dimorphism in ten culicid species of medical/veterinary importance from genera Culex, Aedes, Anopheles and Ochlerotatus collected in Brazil.ResultsDiscriminant analysis revealed significant sexual dimorphism in all the species studied, indicating that phenotypic expression of wing shape in mosquitoes is indeed sex-specific. A cross-validated test performed to reclassify the sexes with and without allometry yielded very similar results. Mahalanobis distances among the ten species showed that the species had different patterns of shape sexual dimorphism and that females are larger than males in some species.ConclusionWing morphology differed significantly between species. The finding of sexual dimorphism in all the species would suggest that the wing geometry of Culicidae is canalized. Although sexual dimorphism is prevalent, species-specific patterns occur. Allometry was not the main determinant of sexual dimorphism, which suggests that sexual selection or other evolutionary mechanisms underlie wing sexual dimorphism in these insects.

No abstract

Scientific research into the epidemiology of dengue frequently focuses on the microevolution and dispersion of the mosquito Aedes aegypti. One of the world’s largest urban agglomerations infested by Ae. aegypti is the Brazilian megalopolis of Sao Paulo, where >26,900 cases of dengue were reported until June 2015. Unfortunately, the dynamics of the genetic variability of Ae. aegypti in the Sao Paulo area have not been well studied. To reduce this knowledge gap, we assessed the morphogenetic variability of a population of Ae. aegypti from a densely urbanised neighbourhood of Sao Paulo. We tested if allelic patterns could vary over a short term and if wing shape could be a predictor of the genetic variation. Over a period of 14 months, we examined the variation of genetic (microsatellites loci) and morphological (wing geometry) markers in Ae. aegypti. Polymorphisms were detected, as revealed by the variability of 20 microsatellite loci (115 alleles combined; overall Fst = 0.0358) and 18 wing landmarks (quantitative estimator Qst = 0.4732). These levels of polymorphism are higher than typically expected to an exotic species. Allelic frequencies of the loci changed over time and temporal variation in the wing shape was even more pronounced, permitting high reclassification levels of chronological samples. In spite of the fact that both markers underwent temporal variation, no correlation was detected between their dynamics. We concluded that microevolution was detected despite the short observational period, but the intensities of change of the markers were discrepant. Wing shape failed from predicting allelic temporal variation. Possibly, natural selection (Qst>Fst) or variance of expressivity of wing phenotype are involved in this discrepancy. Other possibly influential factors on microevolution of Ae. aegypti are worth searching. Additionally, the implications of the rapid evolution and high polymorphism of this mosquito vector on the efficacy of control methods have yet to be investigated.

Aedes aegypti is the most important vector of dengue viruses in tropical and subtropical regions. Because vaccines are still under development, dengue prevention depends primarily on vector control. Population genetics is a common approach in research involving Ae. aegypti. In the context of medical entomology, wing morphometric analysis has been proposed as a strong and low-cost complementary tool for investigating population structure. Therefore, we comparatively evaluated the genetic and phenotypic variability of population samples of Ae. aegypti from four sampling sites in the metropolitan area of São Paulo city, Brazil. The distances between the sites ranged from 7.1 to 50 km. This area, where knowledge on the population genetics of this mosquito is incipient, was chosen due to the thousands of dengue cases registered yearly. The analysed loci were polymorphic, and they revealed population structure (global F(ST)=0.062; p<0.05) and low levels of gene flow (Nm=0.47) between the four locations. Principal component and discriminant analyses of wing shape variables (18 landmarks) demonstrated that wing polymorphisms were only slightly more common between populations than within populations. Whereas microsatellites allowed for geographic differentiation, wing geometry failed to distinguish the samples. These data suggest that microevolution in this species may affect genetic and morphological characters to different degrees. In this case, wing shape was not validated as a marker for assessing population structure. According to the interpretation of a previous report, the wing shape of Ae. aegypti does not vary significantly because it is stabilised by selective pressure.

Although native to the tropical and subtropical areas of Southeast Asia, Aedes albopictus is now found on five continents, primarily due to its great capacity to adapt to different environments. This species is considered a secondary vector of dengue virus in several countries. Wing geometric morphometrics is widely used to furnish morphological markers for the characterisation and identification of species of medical importance and for the assessment of population dynamics. In this work, we investigated the metric differentiation of the wings of Ae. albopictus samples collected over a four-year period (2007-2010) in São Paulo, Brazil. Wing size significantly decreased during this period for both sexes and the wing shape also changed over time, with the wing shapes of males showing greater differences after 2008 and those of females differing more after 2009. Given that the wings play sex-specific roles, these findings suggest that the males and females could be affected by differential evolutionary pressures. Consistent with this hypothesis, a sexually dimorphic pattern was detected and quantified: the females were larger than the males (with respect to the mean) and had a distinct wing shape, regardless of allometric effects. In conclusion, wing alterations, particularly those involving shape, are a sensitive indicator of microevolutionary processes in this species

Wing diagnostic characters for Culex quinquefasciatus and Culex nigripalpus (Diptera, Culicidae). Culex quinquefasciatus and Culex nigripalpus are mosquitoes of public health interest, which can occur sympatrically in urban and semi-urban localities. Morphological identification of these species may be difficult when specimens are not perfectly preserved. In order to suggest an alternative taxonomical diagnosis, wings of these species were comparatively characterized using geometric morphometrics. Both species could be distinguished by wing shape with accuracy rates ranging from 85-100%. Present results indicate that one can identify these species relying only on wing characters when traditional taxonomical characters are not visible.
Caracteres alares diagnósticos de Culex quinquefasciatus and Culex nigripalpus (Diptera, Culicidae). Culex quinquefasciatus e Culex nigripalpus são mosquitos de interesse à saúde pública e que podem ocorrer em simpatria em ambientes urbanos e semi-urbanos. A identificação morfológica dessas espécies pode ser difícil nos casos em que os espécimes não estão bem preservados. Com o intuito de sugerir um método diagnóstico taxonômico, asas de indivíduos dessas espécies foram caracterizadas comparativamente utilizando-se morfometria geométrica. Ambas as espécies puderam ser distinguidas pela forma alar com índices de confiabilidade entre 85-100%. Os resultados presentes indicam que é possível identificar essas espécies baseando-se apenas no formato alar, nos casos em que os caracteres taxonômicos tradicionais não são visíveis