Accurate identification of disease vectors is crucial when collecting epidemiological data. In mosquitoes, which transmit diseases like malaria, yellow fever, chikungunya, and dengue fever, ...identification mainly relies on the observation of external morphological features at different life cycle stages. This process is tedious and labor‐intensive. In this paper, the utility of Raman spectroscopy to discriminate and classify three mosquito species, namely, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus, is presented. The three species were chosen to represent two subfamilies of medically important mosquitoes, that is, the Anophelinae and the Culicinae. The study is primarily a proof of concept on the potential of Raman spectroscopy in mosquito taxonomy. A dispersive Raman microscope was used to record spectra from the legs (femur and tibia) of fresh anesthetized laboratory‐bred mosquitoes. Broad peaks centered around 1400, 1590, and 2060 cm−1 dominated the spectra. These peaks, attributed to cuticular melanin, were important in mosquito discrimination. Variance threshold (VT) and principal component analysis (PCA) were used for feature selection and feature extraction, respectively. The extracted features were then used to train and test linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA) classifiers. VT/PCA/QDA achieved an overall accuracy of 94%, a sensitivity of 87%, and a specificity of 96%, whereas VT/PCA/LDA attained an accuracy of 85%, a sensitivity of 69%, and a specificity of 90%. The success of these relatively simple classification models on Raman spectroscopy data lays the ground for future development of machine learning models that may include discrimination of cryptic species.
Raman signals of three mosquito species—Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus—were dominated by broad melanin peaks centered around 1400, 1590, and 2060 cm−1. Two models, VT/PCA/LDA and VT/PCA/QDA, successfully discriminated the three mosquito species using melanin Raman bands and achieved classification accuracies of 85% and 94%, respectively.
The rapid spread of vector‐borne diseases demands the development of an innovative strategy for arthropod monitoring. The emergence of MALDI‐TOF MS as a rapid, low‐cost, and accurate tool for ...arthropod identification is revolutionizing medical entomology. However, as MS spectra from an arthropod can vary according to the body part selected, the sample homogenization method used and the mode and duration of sample storage, standardization of protocols is indispensable prior to the creation and sharing of an MS reference spectra database. In the present study, manual grinding of Anopheles gambiae Giles and Aedes albopictus mosquitoes at the adult and larval (L3) developmental stages was compared to automated homogenization. Settings for each homogenizer were optimized, and glass powder was found to be the best sample disruptor based on its ability to create reproducible and intense MS spectra. In addition, the suitability of common arthropod storage conditions for further MALDI‐TOF MS analysis was kinetically evaluated. The conditions that best preserved samples for accurate species identification by MALDI‐TOF MS were freezing at –20°C or in liquid nitrogen for up to 6 months. The optimized conditions were objectified based on the reproducibility and stability of species‐specific MS profiles. The automation and standardization of mosquito sample preparation methods for MALDI‐TOF MS analyses will popularize the use of this innovative tool for the rapid identification of arthropods with medical interest.
Abstract
Background
Identification of malaria vectors is an important exercise that can result in the deployment of targeted control measures and monitoring the susceptibility of the vectors to ...control strategies. Although known to possess distinct biting behaviours and habitats, the African malaria vectors
Anopheles gambiae
and
Anopheles arabiensis
are morphologically indistinguishable and are known to be discriminated by molecular techniques. In this paper, Raman spectroscopy is proposed to complement the tedious and time-consuming Polymerase Chain Reaction (PCR) method for the rapid screening of mosquito identity.
Methods
A dispersive Raman microscope was used to record spectra from the legs (femurs and tibiae) of fresh anaesthetized laboratory-bred mosquitoes. The scattered Raman intensity signal peaks observed were predominantly centered at approximately 1400 cm
−1
, 1590 cm
−1
, and 2067 cm
−1
. These peaks, which are characteristic signatures of melanin pigment found in the insect cuticle, were important in the discrimination of the two mosquito species. Principal Component Analysis (PCA) was used for dimension reduction. Four classification models were built using the following techniques: Linear Discriminant Analysis (LDA), Logistic Regression (LR), Quadratic Discriminant Analysis (QDA), and Quadratic Support Vector Machine (QSVM).
Results
PCA extracted twenty-one features accounting for 95% of the variation in the data. Using the twenty-one principal components, LDA, LR, QDA, and QSVM discriminated and classified the two cryptic species with 86%, 85%, 89%, and 93% accuracy, respectively on cross-validation and 79%, 82%, 81% and 93% respectively on the test data set.
Conclusion
Raman spectroscopy in combination with machine learning tools is an effective, rapid and non-destructive method for discriminating and classifying two cryptic mosquito species,
Anopheles gambiae
and
Anopheles arabiensis
belonging to the
Anopheles gambiae
complex.
A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes (Diptera: Culicidae) from the provinces ...Ontario and New Brunswick. Sequence variation was analysed in a 617-bp fragment from the 5' end of the CO1 region. Sequences of each mosquito species formed barcode clusters with tight cohesion that were usually clearly distinct from those of allied species. CO1 sequence divergences were, on average, nearly 20 times higher for congeneric species than for members of a species; divergences between congeneric species averaged 10.4% (range 0.2-17.2%), whereas those for conspecific individuals averaged 0.5% (range 0.0-3.9%).
For differential identification of sibling species in the Anopheles gambiae Giles complex (Diptera: Culicidae), including simultaneous separation of M and S molecular forms within An. gambiae Giles ...sensu stricto, we describe a PCR‐RFLP method. This procedure is more efficient, faster and cheaper than those used before, so is recommended for large‐scale processing of field‐collected larval and adult specimens to be identified in malaria vector studies.
Adult female mosquitoes within the subgenus Culex (Melanoconion) Theobald (Diptera: Culicidae) are difficult to identify to species using external morphological features. We present two multiplexed ...polymerase chain reaction assays that quickly and accurately identify specimens from the southeastern United States based on sequence differences in the internal transcribed spacers of the ribosomal DNA gene array. One assay identifies all species that occur only in Florida, whereas the second assay identifies species that may occur in other southeastern states. These assays require small amounts of DNA, such as DNA from two sonicated legs, or an individual specimen. These assays also may be run as multiple singleplex reactions to determine the mosquito species composition of virus-positive mosquito pools. Reaction volumes may be as low as 10 µl, which reduces assay cost.
Species within the subgenus Culex (Melanoconion) Theobald are the primary enzootic vectors of viruses in the Venezuelan equine encephalitis complex including Everglades virus, and probable enzootic ...vectors of eastern equine encephalitis and West Nile viruses. Adult females of this subgenus are often difficult or impossible to identify to species based on external morphological characters. The use of female cibarial armature allows for the identification of field-collected adult female specimens of Culex (Melanoconion). The cibarial armatures are described and illustrated for all species from the United States and a key to species using this character is presented.
According to the World Health Organization, dengue is estimated to affects 390 million people a year. In Brazil alone in 2019, 1,544,987 probable dengue cases were reported. The main vectors involved ...in the spread of dengue are the mosquitoes of the species Aedes aegypti and Aedes albopictus. In addition to dengue they also transmit other diseases such as chikungunya, zika fever and yellow fever. One way to help the Zoonosis Centers to better control these dangers would be through a tool that could help in the identification of mosquito species. In this work we propose a methodology to classify mosquitoes by species and sex through the use of images associated with Digital Image Processing techniques. After acquiring mosquito samples, we created three databases, one using a smartphone camera, a macro camera and a Digital Single Lens Reflex camera. The Auto Color Correlogram, Gray Level Co-occurrence Matrix and Hu Moments techniques were applied to extract characteristics from the images, and then we used the Support Vector Machine, Random Forest and K-Nearest Neighbors classifiers. We classified 800 images divided into four classes and three databases. In our experiments, we obtained a maximum accuracy of 99.53%.
Sibling species A and B of Anopheles quadriannulatus (Theobald) are recognized as allopatric members of the Anopheles gambiae Giles complex of Afrotropical mosquitoes (Diptera: Culicidae). Species A ...represents An. quadriannulatus sensu stricto, widespread in southern Africa, whereas An. quadriannulatus species B occurs in Ethiopia. Because of difficulty of identification, distribution of An. quadriannulatus sensu lato remains poorly known. Cytotaxonomy and the standard DNA polymerase chain reaction (PCR) assay do not distinguish between species A and B of An. quadriannulatus. By optimizing the standard PCR assay (Scott et al., 1993) for identification of members of the An. gambiae complex, we identified two discriminant fragments of 153 bp and 900 bp from DNA of An. quadriannulatus species B, whereas only the 153 bp fragment was amplified for species A from South Africa. This modified PCR assay can therefore be used to distinguish between species A and B of An. quadriannulatus s.l. as well as other members of the An. gambiae complex.
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