Wing Interferential Patterns (WIPs) and machine learning for the classification of some Aedes species of medical interest

Hematophagous insects belonging to the Aedes genus are proven vectors of viral and filarial pathogens of medical interest. Aedes albopictus is an increasingly important vector because of its rapid worldwide expansion. In the context of global climate change and the emergence of zoonotic infectious diseases, identification tools with field application are required to strengthen efforts in the entomological survey of arthropods with medical interest. Large scales and proactive entomological surveys of Aedes mosquitoes need skilled technicians and/or costly technical equipment, further puzzled by the vast amount of named species. In this study, we developed an automatic classification system of Aedes species by taking advantage of the species-specific marker displayed by Wing Interferential Patterns. A database holding 494 photomicrographs of 24 Aedes spp. from which those documented with more than ten pictures have undergone a deep learning methodology to train a convolutional neural network and test its accuracy to classify samples at the genus, subgenus, and species taxonomic levels. We recorded an accuracy of 95% at the genus level and > 85% for two (Ochlerotatus and Stegomyia) out of three subgenera tested. Lastly, eight were accurately classified among the 10 Aedes sp. that have undergone a training process with an overall accuracy of > 70%. Altogether, these results demonstrate the potential of this methodology for Aedes species identification and will represent a tool for the future implementation of large-scale entomological surveys.

www.nature.com/scientificreports/(Skuse, 1895).Aedes albopictus, a vector of public health importance, has encompassed a rapid change in its global distribution due to the worldwide trade in second-hand tires, often containing stagnant water, making it an ideal place for eggs and larvae, and its easiness to adapt to new environments, even in a temperate climate.This expansion offers opportunities for arboviruses (viruses transmitted by arthropods) to circulate in new areas, becoming a common cause of epidemics in Ae. aegypti-free countries.In addition to the mentioned species, at least seven others are indigenous mosquitoes on more than two continents (https:// www.world atlas.com/ conti nents accessed on 01/24/2023).
For the entomological survey, identifying potential vectors belonging to the Aedes genus is typically based on intrinsic and extrinsic morphological criteria with morphological keys of determination as a tool.This is a time-consuming task that requires expertise and training.The discriminative morphological identification of adult Ae. albopictus is readily amenable by looking at bold black shiny scales and distinct silver-white scales on the palpus and tarsi 4 .Additionally, the dorsal scutum is black, with a distinguishing white stripe in the center, beginning at the dorsal surface of the head and continuing along the thorax.This character is nevertheless present in the specimen of the Scutellaris groups belonging to the Stegomyia subgenus, roughly 40 species.It cannot as perse be considered a diagnostic character.It is a medium-sized mosquito (2.0-10.0mm; males are on average 20% smaller than females).Differential morphology of males from females includes the plumose antennae and modified mouthparts for nectar feeding.Dark scales cover the abdominal tergites.Legs are black with white basal scales on each tarsal segment.The abdomen narrows into a point characteristic of the genus Aedes.Adult Ae. aegypti can be recognized by white marks on the legs and a marking lyre on the thorax.In areas with multiple sympatric Aedes species with or without medical importance, these distinctions according to morphological characteristics are only sometimes easily discriminative.In addition, it is common for older adult specimens to have missed or damaged body parts or characters (e.g., scales, legs) that are essential for accurate identifications.Specimens with damage in critical regions for diagnostic characters separating vector species from closely related non-vector ones further puzzled entomological surveys.
Machine learning models, especially convolutional neural networks, can classify objects by identifying visible and non-visible features to the naked eye 5 .They are of choice and have been extensively used for insect identification involving whole insect image recognition.They demonstrate astonishing accuracy on a wide range of Arthropods [6][7][8] , including Culicidae [9][10][11] .Features related to animal behavior (e.g., flying and walking trajectories, postures, etc.) 12 , allowing at-distance identification of alive specimens, have also been tested.Models based on insect morphology imaging of immobilized insects, an approach close to the entomological expertise deployed to identify insects that have inter-genus inter-species high morphological similarities, require a considerable number of data for training each Genus/species to learn the features and gain validation accuracy 10,13,14 .Databases needed to train such models on whole insect recognition are filled with pictures of several poses, dorsal-ventral, etc., to collect taxonomic discrimant characters 6,[15][16][17] .
Wing Interference Patterns (WIPs) have received attention for their taxonomic potential [18][19][20] .The thin-film interference occurring on the wings' transparent membrane allows the formation of a colored pattern.These WIPs significantly vary among specimens belonging to different species but moderately between representatives of the same species or between sexes.Unlike the angle-dependent iridescence effect of a flat film, the newton color series displayed is proportional to the thickness of the wing membrane at any given point, wing structures acting as diopters ensuring the WIPs appear essentially non-iridescent 19 .In previous papers, we have shown the value of WIPs for Glossina and Anopheles classification 21,22 .
In the context of global climate change and the emergence of zoonotic infectious diseases, identification tools with field application are required to strengthen efforts in the entomological survey of arthropods with medical interest 23,24 .Therefore, implementing new and affordable methods to accurately identify mosquitoes of the Aedes genus is a prerequisite for an entomological survey.To this aim, we have explored the accuracy and reliability of Wing Interferential Patterns (WIPs) to accurately identify Aedes specimens and classify them using a deep learning (DL) procedure.

Aedes selection and storage
The first WIPs reference collection of Culicidae gathers samples belonging to the Aedes genus using well-established Aedes albopictus laboratory breeds.Specimens were also selected in the ARIM collection (https:// arim.ird.fr/) of IRD (Institut de Recherche pour le Développement).In addition, samples collected in natura whose identification was performed by trained entomologists at the time of their catch with available regional identification keys were also included in the database.The description of the samples used in this study is given in Table 1, and the worldwide distribution of samples included in the study is illustrated in Fig. 1.A complete list of the Diptera dataset used in this study and of species included in the database with their associated class is available in the supplementary data and the figshare server (Supdata: ID of species included in the study).

Image acquisition and database construction
The procedures applied to capture WIPs were the same as those described for Glossina sp.WIPs acquisition 21 .Wings were dissected and deposited on a glass slide, adding a small cover slide.The picture was taken with a Keyence™ VHX 1000 microscope, using the VH-Z20r camera and a Keyence VH K20 adapter, allowing an illumination incidence of 10°.The High Dynamic Range (HDR) function was used for all photos.All pictures were enlarged to a maximal occupancy, making the size of the wing not a discriminating parameter for insect species identification.The numerical parameters settled are the same as described and detailed in the publication on Glossina spp. 21

Collected dataset, image pre-processing, and dataset splitting for training/learning and validation
A rich annotated image dataset, including 494 pictures of 24 Aedes species, is available (figshare: https:// figsh are.com/s/ 597f4 e2f2d 8206e 09603).Under-sampled Aedes species (less than ten samples/pictures) were discarded for training to prevent overfitting.All processed images were resized to 256 and 116 pixels for width and height, respectively.Pixel values were normalized within the (0, 1) range.
The dataset was then prepared for k-fold cross-validation, with k = 5 (Fig. 1).K-fold cross-validation is a classic approach to evaluate the robustness of a machine learning method, including Deep Learning ones.Mainly, for this study, the dataset was randomly shuffled and partitioned into k equal-size subsets with similar class distributions.A separately learned classifier was evaluated for each subgroup using kth of the whole dataset for validation and the remaining k−1 as training data (see Fig. 2 for illustration).This strategy allowed for measuring the mean accuracy of 5 distinct models and is the most accurate neural network performance estimation method 25 .
This strategy allowed for measuring the mean accuracy of the five distinct classifiers.Among all existing machine learning methods, Deep Convolutional Neural Networks and their different architectures have shown in the last decade to be the most adapted for image classification.Compared to classic shallow methods (Support Vector Machine, Random Forest, and Boosting-based approaches for the main ones), they do not need handcrafted features as input of the learning process: the selection of the best features is intrinsic to the method itself and is particularly well adapted to the WIPs scenario.A pipeline overview of the complete training procedure using CNN is shown in Fig. 3.

Training of the neural network (CNN)
The data used to develop the Deep Learning model includes Culicidae and non-Culicidae specimens 26 .The methodology used was initially developed and tested on Tetse flies 21 .Briefly, the original CNN architecture MobileNet 27 , ResNet 28 , and YOLOv2 29 architecture were deemed for automatically classifying Aedes sp. with the abovementioned dataset.Compared to classic Deep Learning, ours is more compact to cope with the specificity of our dataset in terms of size; therefore, thinner image recognition and classification architecture were developed to consider its reduced size.The first one is inspired by MobileNet, which takes advantage of depth-wise convolution 27 .We propose to work with only one depth-wise convolution per layer of the CNN architecture to reduce the complexity and the number of extracted features.In addition, batch normalization was set to speed up and stabilize the training process 30 .
In addition to this first compact CNN architecture based on MobileNet, two interconnected layers like VGG 31 for YOLOv2 were applied with a DarkNet-19 architecture 29 , as performed on tsetse flies samples 21 .As this architecture tends to over-fit the training set (meaning a lack of generalization of the performance when data other than the training data set are considered), we test two reduced architectures, i.e., using 1 or 2 scales less than the original network.For clarity, we called them DarkNet-9 (8 convolution layers and 1 classification layer) and DarkNet-14 (13 convolution layers and 1 classification layer).We also reproduced the ResNet18 architecture from He and collaborator 28 and trained it from random initialization.Even if this architecture seems too "deep" (may lead to overfitting) compared to our other architectures, the intrinsic properties of ResNet18, residual connections, allow convergence of the training procedure 21 .We used a standard approach (shallow approach) based on extracting SURF descriptors (an efficient implementation of the classic SIFT descriptors), a Bag of Features (BoF) representation using a 4000 codewords dictionary, and an SVM with a standard polynomial kernel similar to it was proposed in Sereno et al. 32 .For each task, we only use one fully connected layer with the softmax activation to predict the probability that an image belongs to the correct class.We train our networks using Stochastic Gradient Descent (SGD) with a learning rate of 10 2 and a momentum of 0:9 for 30 epochs.The method was developed on a workstation with a quad-core CPU 3.0 GHz and 16Go RAM.Information on the training options, accuracy, and sensitivity, as well as the code, are available at https:// github.com/ marce nsea/ dipte ra-wips/ commit/ 12f39 ab500 a3f82 0cfb8 17c67 ef25c 58094 2301d.

Training and classification
We explored the training classifier accuracy on the Aedes dataset and on datasets of Culicidae that do not belong to the Aedes genus (non-Aedes) and from mosquitoes that do not belong to the Culicidae family (non-Culicidae) as negative samples.We trained the CNN on such a combination to improve the model's accuracy.The database initially filled with a total of 494 pictures of Aedes spp.WIPs pictures.Since we cannot fill the database with all  www.nature.com/scientificreports/Aedes species acting as primary vectors of viruses, parasites, or bacteria with a medical interest, we first focused on a restricted set of species (Table 1).Using this pictures-set, we ascertained the accuracy of the process to discriminate the Aedes genus (Meigen, 1818) from other Culicidae (Meigen, 1818), including Anophelinae (Grassi, 1900), Culicinae (Meigen, 1818), and non-Culicidae samples members (e.g., Psychodidae, Glossini-dae…).From our dataset and process, the automatic classification process accuracy is rather good and higher than 95% (Table 2).We also evaluated the DL process's accuracy at the subgenus level (Table 3).The Aedes genus encompasses 79 subgenera, with only 14 gathering species documented to act as primary vectors of viruses, parasites, and bacteria of medical interest.As mentioned in the introductory section, the subgenus Ochlerotatus was "raised" to the level of the genus in 2000 1 .Considering that a large number of the scientific literature refers to the subgenus Ochlerotatus, we refer to this taxonomic nomenclature to carry out our analysis 33 .Our Aedes dataset does not represent the overall diversity at the subgenera level.Nevertheless, the computed accuracy for the identification process is high (> 85%) for the Stegomyia and Ochlerotatus subgenera, with a classification accuracy of 97.0 and 86.7%, respectively.Still, it fails to address the Finlaya subgenus accurately.Such low accuracy might be due to the small sample size available to train and test the process for Finlaya.Therefore, efforts in implementing the database with enough specimens representative of the Finlaya and other subgenera must be made to address our methodology's accuracy in addressing the classification process at the subgenus taxonomic level.Overall, our methods are the first based on deep learning with the versatility of automatically handling samples at the subgenus level, which might be of interest to speed and facilitate the identification of specimens of Aedes species of medical interest.
Finally, the reliability of the DL model to accurately classify WIPs pictures of 10 Aedes species was calculated, and the results are presented in Table 4. Variable level of accuracy is recorded, ranging from not accurate (0.00%) to perfect classification (100.00%).A perfect accuracy (100.00%) is achieved for 50% of Aedes species whose WIPs pictures are filled in the dataset.More than 75% accuracy in classification is recorded for 7 Aedes species, but the DL methods failed to assign 2 Aedes species correctly (Ae.echinus and Ae.mariae) (Table 4).Even if populations of Ae. albopictus bear a high variability independently of whether the populations are native or invasive 34,35 , we record a high classification accuracy (Table 4).The DL approach based on WIPs can manage the diversity of specimens sampled in two distant geographic areas, France and La Réunion.Aedes aegypti exists in two subspecies or forms: the domestic ecotype, Ae. aegypti aegypti found outside Africa and the presumed ancestral Ae. aegypti formosus occurring in sub-Saharan Africa in sympatry with Ae. aegypti aegypti in some ecologies 36,37 .Our samples of Ae. aegypti originating from sub-Saharan Africa were not identified at the subspecies level.They may be a mix of the two ecotypes with subtle variations in their WIPs.This might be the underlying factor triggering the lower accuracy (72.7%) of the identification process of Ae. aegypti in our dataset, as compared to the accuracy recorded for Ae.albopictus (95.8%) classification.This deserves to be investigated.Nevertheless, the overall calculated specific recognition of samples belonging to Aedes remains satisfactory.

Misclassified pictures
Inspection of the machine learning model weaknesses helps identify underlying problems.This can be performed via a review of the miss-predicted images.This will provide insights into what makes a photo hard to classify for the model.Our data set takes advantage of having to be filled with pictures of samples collected in natura or from laboratory colonies but also preserved for an extended period or damaged.This large panel of images taken with the same microscope under the same light condition, preparation process, and preprocessing methodologies will help to delineate factors (e.g., age, sample origin, sample preservation…) affecting the classification process.In Fig. 4, selected examples are presented.Deep learning models rely more on textures than shapes.A more extensive training set can avoid pitfalls linked to photo or sample quality, prevent confusing set-ups when taking pictures, and improve the accuracy of the automated classification.A guideline can be added to the application to advise participants to make good images of Culicidae samples.
Besides misclassified pictures, we generated activations maps pointing to which image region the architectures use, according to the feature extraction layers of the DCNN (Fig. 5).
These activation maps illustrate that the membrane and the vein structure are not targeted by the DCNN and that features activated concentrate in the lower part of the picture, particularly on the color pattern, when learning was successful Fig. 5 and high accuracy in the classification (Accuracy > 76%), while when classification www.nature.com/scientificreports/accuracy is low (30%), features extracted spread all over the wing.In all cases, neither the wing shape nor the venation are features targeted by DCNN.

Discussion
With robust vector surveillance, emerging vector-borne disease threats can be addressed.During an entomological survey, most routine identification involves skilled personnel that use diagnostic criteria, like the size, shape, or texture of specimens or the presence or absence of certain key features.Since the morphological identifications of medically important Aedes species is complex and meticulous, requiring highly skilled specialists, an alternative is to use computer vision that, like mosquito taxonomists, relies on visual (morphological) characters to assign a definitive taxon name.A framework based on a deep neural network was developed to extract mosquito anatomy from images.The process was tested on 23 Culicidae belonging to the Aedes, Anopheles, and Culex Genus 9 , but only 3 Aedes species (Ae.aegypti, Ae. infirmatus, and Ae.taeniorhynchus).Nevertheless, manipulating mosquitoes to acquire and extract morphological characters is complicated and damages specimens.A real-time pipeline was set but has only been tested for discrimination of Ae. aegypti and Ae.albopictus 15 .Finally, deep convolutional network approaches using whole-body pictures of field-caught mosquitoes have shown promise, demonstrating astonishing accuracy, even classifying unknown/unidentified species before further identification 10 .Nevertheless, we can anticipate that morphology-based methods might suffer from the same limitation encountered by taxonomists for cryptic species/subspecies delineations, for Aedes species belonging   to the Neomelanoconion identification whose require examination of internal organs, and as well as the misidentification of damaged specimens.Besides the mentioned methods, some characteristics like flight tone and wing beat, which play a role in mosquito reproduction [38][39][40] , were used for the automated identification assay of Ae. albopictus and Ae.triseriatus 39,40 , and more recent development permits an "at a distance" discrimination between Ae. albopictus, Ae. aegypti and Culex sp. 41,42.Here, we prove the classification accuracy with WIPs is above 75% for 8 of the ten species investigated in our study.Morphometry analysis of insects' wings has demonstrated interest in vector mosquitoes and sibling or cryptic species [43][44][45] identification.Nevertheless, this requires intact wings and extensive preprocessing to mount them without deformation.All that severely impedes its application for entomological survey purposes, but it is still attractive in taxonomic and populational studies.Our methodology would promise that Aedes species identification can be accurately performed with minimal training, cost, and time.In addition, wings are relatively robust insect organs that limit the risks of damaging them.In addition, they can be easily stored at room temperature and be carried with minimal caution.
Alternative methodologies not relying on morphological characteristics were implemented.These alternatives are DNA barcoding and protein profiling by MALDI-TOF, but they can also involve the wing beat features.MALDI-TOF has been applied to a restricted number of Aedes species at the egg stage, i.e., Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. koreicus, Ae. phoeniciae, and Ae.triseriatus, 4 have medical importance 46,47 .It was also set up to identify the second larval stage to pupae of Ae. albopictus and Ae.aegypti 48 and on the fourth instar larvae 49 but with higher accuracy for late larval instar 50 .Its efficiency for field-trapped or colony-reared insect adults is limited.It includes species having (Ae.albopictus, Ae. vigilax, Ae. vexans, Ae. notoscriptus, Ae. polynesiensis) or not (Ae.fowleri, and Ae.dufouri) a medical importance [51][52][53][54][55] .However, the interoperability of the MALDI-TOF methodology requires a standardized procedure in the conservation of samples and the choice of the mosquito body part of adult specimens 56 .For MS identification, standardization of procedures for preparation and reproducibility between instruments and homemade databases will be desirable [57][58][59][60] .Contrary to MALDI-TOF or DNA-barcoding methodologies, our method is not designed to identify Aedes at larval stages.Nevertheless, a survey on adult mosquitoes is an alternative to larval surveys because it addresses the vector life stage responsible for transmission and thus adds information on vector density 61 .Infrared spectroscopy (NIR and MIR) can detect changes in the mosquito cuticle by quantifying light absorbed 62 .The discriminative capability of such methodology at the species level has been investigated on a restricted number of Aedes species having a medical interest (Aedes aegypti, Ae. albopictus, Ae. japonicus, and Ae.triseriatus) 63 .The main interest of this methodology relies on its capacity to grade the age of natural Anopheles spp.populations when coupled with a Deep learning approach 64 .DNA barcoding is also an alternative to morphological identification methods.A quest for "Aedes" as a keyword in the barcode of Life system (BOLD) database indicates 27,458 records with sequences representative of 328 species (http:// v4.bolds ystems.org/ accessed on 21 of July 2022) 65 .During proactive surveillance of invasive and/or medically and veterinary important species, genetic identification by PCR is costly, destructive, and time-consuming 66 , a limitation that does not carry our method.Prompt and correct identification of exogenous species is required to monitor entry points under the International Health Regulations (IHR) to limit the dispersal and establishment of new vector species.In this case, even entomologists experienced in identifying local mosquito fauna may experience difficulty accurately identifying damaged specimens.We provide evidence that WIPs analysis coupled with Deep Learning for discriminative identification competency to accurately classify Aedes species at various taxonomic levels (genus, subgenus, and species).The competency to accurately classify Aedes samples at the subgenus level is of interest to medical entomologists and public health services since a restricted number of Aedes subgenus encompass species of medical interest.In this study, among Aedes species accurately classified (Accuracy > 75%), four are of medical interest (Ae.albopictus, Ae. aegypti, Ae. luteocephalus, and Ae.polynesiensis).Therefore, we can envision that a database of WIPs encompassing proven Aedes vectors at a global or regional scale would allow remote territories to address the vector status of a specimen.Increasing the area of vector surveillance is of high interest, even at a continental scale.Community science relying on the participation of non-expert people was therefore applied for mosquitoes 67,68 .Unfortunately, the quality of entomological data can be poor in terms of identification accuracy.Deep learning approaches have offered opportunities to overcome this limitation and have been developed to follow Ae.albopictus in Europe, from pictures taken by citizen 11 .However, this methodology wasn't tested in high Aedes species diversity areas.Unfortunately, our method, as it stands, cannot be translated for this purpose because of the cost associated with microscopy and the need for high-quality pictures.Nevertheless, because wings are tiny and light, they can quickly be sent for identification purposes with this methodology.A global comparison between the various methods developed for Aedes identification is given in Table 5.
Current global vector surveillance systems are unstandardized, burdensome on public health systems, and threatened by the worldwide scarcity of entomologists.The Deep learning analysis of Aedes WIPs resulting in robust classification could empower non-experts to identify disease vectors accurately and rapidly in the field.This is of interest in areas where large-scale and/or proactive surveys must be performed, like the Pacific area, where more than 20,000 islands must be surveyed 56,69 .This situation would make exogenous species introduction unnoticed.Nevertheless, the methods need now to be further investigated by evaluating, even qualitatively, whether the proposed approach could be usable in real-life scenarios regarding several important criteria: cost (including infrastructure, material, training, and cost associated with technically skilled personnel), computational resources, analyzing time, sample destructiveness, diversity, and taxonomic level (species, sibling species, cryptic species, sub-species) of the classification.All these will increase vector surveillance's programmatic capability and capacity with minimal training and cost.

Figure 2 .
Figure 2. Schematic representation of the dataset splitting for learning (red) and testing (orange) from Cannet et al. 21.

Figure 3 .
Figure 3. Schematic representation of the pipeline process developed for Glossina identification using the Convolutional Neural Network (CNN) approach from Cannet et al. 21.

Figure 5 .
Figure 5. Activation maps for Aedes with a high confidence value (A) and low confidence value (B) on the prediction.Warm colors (yellow and red) of the activation point to wing regions where local features are essential for the decision.

Table 5 .
Synthetic view of advantages and limitations of some identification methodologies for medically important arthropods in the framework of pro-active or large-scale entomological surveys.Number of Aedes species tested.*The technical cost includes sample preparation (DNA, protein extraction, slides, etc.) and expenses linked to the need for a skilled professional and the presence of dedicated infrastructure for running experiments.

A Aedes spp. in our database Med int* Or Year N Country code Morphological identification by trained entomologists
.

Table 1 .
List of named Aedes species and description of samples included in the dataset (A) and of Aedes species having a medical interest but not included in our dataset (B).

Table 2 .
Test for accuracy of the DL (Deep Learning) process for the Aedes (Meigen, 1818) genus identification.N, number of pictures in the test subset.Accuracy data (%) in bold.

Table 3 .
Test for accuracy (%) of the DL classification process at the subgenus taxonomic level.N, number of pictures in the test subset, Accuracy in %.

Table 4 .
Accuracy in % of the DL process for Aedes spp.classification.Significant values are in[italics].N, number of images in the test subset.