Improving the DNA Barcode Library of Mosquito Species With New Identifications and Discoveries in North-Central Argentina.

The family Culicidae is represented by 244 species in Argentina, many of them with epidemiological importance. DNA barcodes are effective tools for identifying mosquito species, for knowing genetic variability, and for establishing phylogenetic relationships. This work aims to explore mosquito diversity employing different species delimitation approaches and to establish formally a DNA barcode library for the Argentinian mosquito fauna. Barcode fragments of 80 specimens of Argentinian mosquitoes of 28 species of the genera Aedeomyia Theobald (Diptera: Culicidae), Anopheles Meigen (Diptera: Culicidae), Coquillettidia Dyar (Diptera: Culicidae), Culex L. (Diptera: Culicidae), Haemagogus Williston (Diptera: Culicidae), Mansonia Blanchard (Diptera: Culicidae), Nyssorhynchus Blanchard (Diptera: Culicidae), Ochlerotatus Lynch-Arribálzaga (Diptera: Culicidae), Psorophora Robinneau-Desvoidy (Diptera: Culicidae) and Uranotaenia Lynch-Arribálzaga (Diptera: Culicidae) were sequenced. Another 82 sequences were obtained from public databases to establish the phylogenetic relationships using Maximum Likelihood and Bayesian Inference, and the species boundaries based on three approaches (ABGD, GMYC, and mPTP). Sixteen of the 28 species sequenced were recovered as monophyletic, of which 12 were also recognized as molecular operational taxonomic units according to the three methodologies. The disparity between morphology and barcode-based identifications could be explained by synonymy, species complexes occurrence, hybridization, incomplete lineage sorting, or the effect of the geographical scale of sampling. Twenty of the 28 sequenced species are new barcodes for Argentina and 11 are the first for science. This increases from 31 to 52 (12.7 to 21.31%) and from six to 10 (28.57 to 47.62%) the number of species and genera, respectively, with barcode sequences in Argentina. New species records are provided.

Simultaneous sex and species classification of silkworm pupae by NIR spectroscopy combined with chemometric analysis.

BACKGROUND: Most studies only focus on the sex discrimination of silkworm pupae. However, species differentiation of silkworm pupae is also needed in sericulture. To classify the sex and species at the same time, the present study adopts near infrared (NIR) spectroscopy combined with multivariate analysis. RESULTS: First, spectra samples were acquired using an NIR sensor, comprising female and male silkworm pupae from three species. Second, three different variables selection approaches were used, including a successive projections algorithm, competitive adaptive reweighted sampling (CARS) and interval partial least squares (iPLS). Third, identification models were built based on random forest and partial least squares discriminant analysis (PLSDA). The experimental results show that iPLS-PLSDA model (95.24%) gives a high performance when using the one of the three variable selection methods alone. To further increase the performance, the variable selection methods are optimized. The accuracy of the iPLS-CARS-PLSDA model is as high as 98.41%. CONCLUSION: The present study demonstrates that the optimized variable selection method in combination with NIR spectroscopy represents a suitable strategy for sex and species identification of silkworm pupae. © 2020 Society of Chemical Industry.

Efficient Monitoring of Adult and Immature Mosquitoes Through Metabarcoding of Bulk Samples: A Case Study for Non-Model Culicids With Unique Ecologies.

The rapid and economical monitoring of mosquitos is imperative to understanding the dynamics of both disease vectors and nuisance species. In light of technological advances in mosquito sampling and DNA sequencing, health agencies can now utilize the full potential of metabarcoding pipelines for rapid and standardizable surveillance. Here, we describe mosquito spatial and temporal variation, with particular focus on Mansonia Blanchard species, in the Madeira (Rondônia State) and the Ribeira (São Paulo) watersheds, Brazil using metabarcoding of the D2 rDNA marker. Sampling and molecular pipelines were used to evaluate the taxonomic contribution of mosquitos in pools of culicids collected en masse from macrophyte-roots (immatures) and from Mosquito Magnet traps and protected human landings (adults). Results for adult captures are comparable to morphological diagnoses and clarify previously unknown temporal and spatial species turnover. Metabarcoding of immature stages also confirmed the extent of the geographical distribution of some species and each taxon's association with macrophyte species. Given the benefits of metabarcoding, such as taxonomic acuity, high throughput processing, and objectivity, we suggest such techniques should be more fully incorporated into culicid monitoring schemes. The metabarcoding protocol described herein paired with standardized field sampling schemes, when used by mosquito monitoring professionals, offers substantial improvements in terms of practicality, speed and cost.

Identification of Aedes mosquitoes by MALDI-TOF MS biotyping using protein signatures from larval and pupal exuviae.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) biotyping is an innovative strategy, applied successfully for the identification of numerous arthropod families including mosquitoes. The effective mosquito identification using this emerging tool was demonstrated possible at different steps of their life-cycle, including eggs, immature and adult stages. Unfortunately, for species identification by MS, the euthanasia of the mosquito specimen is required. METHODS: To avoid mosquito euthanasia, the present study assessed whether aedine mosquitoes could be identified by MALDI-TOF MS biotyping, using their respective exuviae. In this way, exuviae from the fourth-instar and pupal stages of Aedes albopictus and Aedes aegypti were submitted to MALDI-TOF MS analysis. RESULTS: Reproducible and specific MS spectra according to aedine species and stage of exuviae were observed which were objectified by cluster analyses, composite correlation index (CCI) tool and principal components analysis (PCA). The query of our reference MS spectra database (DB) upgraded with MS spectra of exuviae from fourth-instar larvae and pupae of both Aedes species revealed that 100% of the samples were correctly classified at the species and stage levels. Among them, 93.8% (135/144) of the MS profiles reached the threshold log score value (LSV > 1.8) for reliable identification. CONCLUSIONS: The extension of reference MS spectra DB to exuviae from fourth-instar and pupal stages made now possible the identification of mosquitoes throughout their life-cycle at aquatic and aerial stages. The exuviae presenting the advantage to avoid specimen euthanasia, allowing to perform complementary analysis on alive mosquitoes.

Schizonyxhelea thomsenae (Wirth), description of the pupa and first records from Argentina, Brazil and Peru (Diptera: Ceratopogonidae).

The pupa of Schizonyxhelea thomsenae (Wirth) is described and illustrated from material collected in Misiones province, Argentina. A key for the four known species of pupae of Schizonyxhelea is given, a diagnosis and photographs of the adult are also provided, and the geographic distribution of the species in the Neotropical region is enlarged including first records from Argentina, Brazil and Peru based mainly on adult specimens.

Anopheles larval species composition and characterization of breeding habitats in two localities in the Ghibe River Basin, southwestern Ethiopia.

BACKGROUND: Documentation of the species composition of Anopheles mosquitoes and characterization of larval breeding sites is of major importance for the implementation of larval control as part of malaria vector control interventions in Ethiopia. The aims of this study were to determine the Anopheles larval species composition, larval density, available habitat types and the effects of related environmental and physico-chemical parameters of habitats in the Ghibe River basin of southwestern Ethiopia. METHODS: Anopheles larvae were sampled from November 2014 to October 2016 on a monthly basis and 3rd and 4th instars were identified microscopically to species. The larval habitats were characterized based on habitat perimeter, water depth, intensity of light, water current, water temperature, water pH, water turbidity, distance to the nearest house, vegetation coverage, permanence of the habitat, surface debris coverage, emergent plant coverage, habitat type and substrate type. RESULTS: In total, 9277 larvae of Anopheles mosquitoes and 494 pupae were sampled from borrow pits, hoof prints, rain pools, pools at river edges, pools in drying river beds, rock pools, tire tracks and swamps. Anopheles larval density was highest in pools in drying river beds (35.2 larvae per dip) and lowest in swamps (2.1 larvae per dip) at Darge, but highest in rain pools (11.9 larvae per dip), borrow pits (11.2 larvae per dip) and pools at river edges (7.9 larvae per dip), and lowest in swamps (0.5 larvae per dip) at Ghibe. A total of 3485 late instar Anopheles mosquito larvae were morphologically identified. Anopheles gambiae sensu lato was the primary Anopheles mosquito found in all larval habitats except in swamps. Temperature at the time of sampling and emergent vegetation, were the most important variables for Anopheles mosquito larval density. Anopheles gambiae density was significantly associated with habitats that had smaller perimeters, were sunlit, had low vegetation cover, and a lack of emergent plants. Generally, Anopheles mosquito larval density was not significantly associated with water pH, water temperature, water turbidity, algal content, and larval habitat depth. CONCLUSION: Different species of Anopheles larvae were identified including An. gambiae s.l., the main malaria vector in Ethiopia. Anopheles gambiae s.l. is the most abundant species that bred in most of the larval habitat types identified in the study area. The density of this species was high in sunlit habitat, absence of emergent plants, lack of vegetation near habitat and habitats closer to human habitation. Rainfall plays a great role in determining the availability of breeding habitats. The presence of rain enable to create some of the habitat types, but alter the habitats formed at the edge of the rivers due to over flooding. Controlling the occurrence of mosquito larvae through larval source management during the dry season, targeting the pools in drying river bed and pools formed at the edge of the rivers as the water receded can be very crucial to interrupt the re-emergence of malaria vectors on the onset of rainy season.

Revision of Simulium rufibasis (Diptera: Simuliidae) in Japan and Korea: Chromosomes, DNA, and Morphology.

The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92-4.63%.

A new species of the Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with its phylogenetic relationships with related species in the Simulium asakoae species-group.

A new species of black fly, Simulium (Gomphostilbia) rampae, is described, based on adult male, its pupal exuviae and mature larvae collected from Doi Inthanon National Park, northern Thailand. This new species is placed in the Simulium asakoae species-group, and is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, cone-shaped terminal hook, and cocoon with an anterodorsal projection, and in the larva by the medium-long postgenal cleft. A DNA analysis using COI gene supported its assignment to the S. asakoae species-group and showed its close relationship to S. (G.) udomi Takaoka & Choochote and S. (G.) chiangdaoense Takaoka & Srisuka. This is the fourth member of the S. asakoae species-group recorded from Thailand.

Colonization of Coquillettidia linealis (Skuse) with reference to other Coquillettidia and Mansonia species.

The immature stages of Coquillettidia and Mansonia mosquitoes are cryptic and spend the duration of their development attached to the tissues of subsurface aquatic plants. This obligatory association makes them difficult to collect and has precluded detailed investigation of the biology of Coquillettidia linealis, a species of significant pest and vector status in Australia, as well as other species in the genus Coquillettidia in Australia and elsewhere. This paper describes the first successful establishment and maintenance of a colony of Cq. linealis. Blood-seeking wild adults were collected and induced to oviposit under laboratory conditions, where methods were developed to rear larval populations to adult maturity in a colony that was maintained through eight successive generations. Colonies of Coquillettidia xanthogaster and Coquillettidia sp. nr crassipes were also established and reared through at least six generations and five generations, respectively, while two species of Mansonia, Mansonia uniformis and Mansonia septempunctata, were also reared successfully for six and two generations, respectively.

Lutzia (Metalutzia) chiangmaiensis n. sp. (Diptera: Culicidae), Formal Name for the Chiang Mai (CM) Form of the Genus Lutzia in Thailand.

The Chiang Mai (CM) form of the genus Lutzia, subgenus Metalutzia, in Thailand is diagnosed and formally named Lt. chiangmaiensis Somboon & Harbach, n. sp. The species is contrasted with related species, and information is provided on its bionomics and distribution.

Diversity and ecology of black flies in tea plantation area of Puncak Bogor, Indonesia.

Black flies fauna in Indonesia has been studied extensively, however their ecology is still remaining unexplored. The study was carried out by collecting the larvae and pupae black flies in tea plantation area, Puncak Bogor. The collection sites were the streams located in 3 differents type of altitude, i.e. (1) the natural forest area (1200 m above the sea level) (P1); (2) the tea plantation area (900-1200 m asl) (P2); and (3) the housing area of the official tea plantation (900 m asl) (P3). The collection of black flies were done beweekly from July 2012 to April 2013. The result showed that 12 species of black flies were recorded which belong to one genus, Simulium s.l. and three subgenera (Nevermannia, Gomphostilbia and Simulium s.str). In P1, the area with high elevation (natural forest), it was found the most number of black flies species (11 species). In the streams around and in the tea plantation area (P2), it was found 7 species, and in P3, the official housing area, it was recorded 8 species. The analysis satistically showed that P1 was dominated by larvae and pupae of S. (S.) argyrocinctum, P2 by larvae and pupae of S. (N.) feuerborni, and P3 by S. (S.) argyrocinctum and S. (N.) feuerborni. The distribution of these species was different according to the elevation of the areas.

Description of immature stages of Thanatophilus sinuatus (Coleoptera: Silphidae).

Necrophagous beetles of genus Thanatophilus are well recognized as a group of beetles with a high potential utility in forensic entomology. They can be used to estimate postmortem interval (PMI) or validate the value for other groups of insects commonly encountered on human remains, like blowflies (Calliphoridae). However, reliable tools for instar and species identification of their larvae are needed as such information is crucial for allowing accurate PMI estimate. One of the most common species of the genus Thanatophilus in Europe is Thanatophilus sinuatus. This species occurs frequently on human remains and its larvae feed on decaying tissues throughout their development. Therefore, the larvae could become useful bioindicators for forensic entomology, although their current description does not allow reliable instar or species identification. Our goal was to provide morphological characters for species and instar identification of all larval stages of T. sinuatus. The larvae were obtained from laboratory rearing under controlled conditions (20 °C and 16:8 h of light/dark period). Qualitative and quantitative morphological instar and species-specific characters are described and illustrated. Additionally, we report observations of biological and developmental lengths for all stages of the species. We also compared these morphological characters with recent description of T. rugosus and provided an identification key of these two similar and often co-occurring species. We noticed that some characters for instar identification were shared between T. sinuatus and T. rugosus and were confirmed by comparison with larvae of T. dentigerus that they can be applied to other species of the genus.

Characterization and Identification of Puparia of Hydrotaea Robineau-Desvoidy, 1830 (Diptera: Muscidae) From Forensic and Archaeological Contexts.

Flies and beetles are the main components of the entomofauna colonizing a body after death. Following the recognition of constant and predictable colonization patterns and the knowledge about the dependence of the insect development to temperature, a new discipline, forensic entomology, has provided information useful to reconstruct criminal events. Funerary archaeoentomology has also applied the same rationale in archaeological contexts. Puparia represent a large fraction of the insect remains that can be found associated with a cadaver, especially when the body is mummified or in the advanced stages of decomposition. Puparium identification is still a problematic topic due to the lack of identification keys and, in several cases, a lack of diagnostic feature descriptions. Here, we focus the attention on some Hydrotaea Robineau-Desvoidy (Diptera, Muscidae) puparia from forensic and archaeological contexts. Puparia of Hydrotaea capensis (Wiedemann), Hydrotaea ignava (Harris), Hydrotaea aenescens (Wiedemann), Hydrotaea similis Meade, Hydrotaea pilipes Stein, and Hydrotaea dentipes (Fabricius) are here detailed and illustrated. Posterior spiracles, anal plate, and intersegmental spines have been considered as good diagnostic characters for the identification of these puparia.

Direct Analysis in Real Time-Mass Spectrometry and Kohonen Artificial Neural Networks for Species Identification of Larva, Pupa and Adult Life Stages of Carrion Insects.

Species determination of the various life stages of flies (Order: Diptera) is challenging, particularly for the immature forms, because analogous life stages of different species are difficult to differentiate based on morphological features alone. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) combined with supervised Kohonen Self-Organizing Maps (SOM) enables accomplishment of species-level identification of larva, pupa, and adult life stages of carrion flies. DART-HRMS data for each life stage were acquired from analysis of ethanol suspensions representing Calliphoridae, Phoridae, and Sarcophagidae families, without additional sample preparation. After preprocessing, the data were subjected to a combination of minimum Redundancy Maximal Relevance (mRMR) and Sparse Discriminant Analysis (SDA) methods to select the most significant variables for creating accurate SOM models. The resulting data were divided into training and validation sets and then analyzed by the SOM method to define the proper discrimination models. The 5-fold venetian blind cross-validation misclassification error was below 7% for all life stages, and the validation samples were correctly identified in all cases. The multiclass SOM model also revealed which chemical components were the most significant markers for each species, with several of these being amino acids. The results show that processing of DART-HRMS data using artificial neural networks (ANNs) based on the Kohonen SOM approach enables rapid discrimination and identification of fly species even for the immature life stages. The ANNs can be continuously expanded to include a larger number of species and can be used to screen DART-HRMS data from unknowns to rapidly determine species identity.

A New Species of the Simulium (Simulium) argentipes Species-Group (Diptera: Simuliidae) From Taiwan, and Its Phylogenetic Relationships With Four Related Species.

A new black fly species, Simulium haiduanense Takaoka, Low & Huang (Diptera: Simuliidae), is described on the basis of females, males, pupae, and mature larvae from Taiwan. This new species is placed in the Simulium argentipes species-group of the subgenus Simulium (Diptera: Simuliidae) and is characterized by the yellowish female legs, ovipositor valves rounded apically and with its inner margin concave, claw with a small subbasal tooth, male style without a basal protuberance, pupal gill with eight filaments, corbicular cocoon, and larval abdomen lacking paired protuberances. It represents the first record of the S. argentipes species-group from Taiwan. Taxonomic notes are given to separate this new species from all eight species in the same species-group. The phylogenetic relationships of this new species with four related species are presented.

A New Gall Midge Species of Asphondylia (Diptera: Cecidomyiidae) Inducing Flower Galls on Clinopodium nepeta (Lamiaceae) From Europe, Its Phenology, and Associated Fungi.

A new gall midge, Asphondylia nepetae sp. n. Viggiani (Diptera: Cecidomyiidae), causing flower gall on Clinopodium nepeta (L.) Kuntze (Lamiaceae), is described from Europe. The morphological characteristics of adult, larvae, and pupa are described and illustrated. Molecular approach (by sequencing 28S-D2, ITS2, and COI) confirmed that A. nepetae is a distinct species. The development of the gall is always associated with the presence of the fungus Botryosphaeria dothidea (Moug.: Fr.) Ces. and De Not. (Botryosphaeriales: Botryosphaeriaceae). The new species can complete several generations per year, on the flowers of the same host plant and its adults emerge from late spring to autumn. Pupae overwinter inside peculiar flower galls in a state of quiescence. The impact of the pest is highly variable with a percentage of flowers infested that ranged between 3 and 57.5% in the sampled years. Insect mortality was, at least in part, due to parasitoids that attack the young stages of the midge. Among them, the dominant species was Sigmophora brevicornis (Panzer) (Chalcidoidea: Eulophidae).

A New Species of the Simulium (Simulium) striatum Species Group (Diptera: Simuliidae) from Thailand, and Its Differentiation from Two Related Species Based on a Fast-Evolving Nuclear Gene.

Simulium (Simulium) phraense sp. nov. (Diptera: Simuliidae) is described from females, males, pupae, and larvae from Thailand. This new species is placed in the Simulium striatum species group and is most similar to Simulium (Simulium) nakhonense Takaoka & Suzuki (Diptera: Simuliidae) from Thailand among species of the same species group but is barely distinguished from the latter species by lacking annular ridges on the surface of the pupal gill filaments. The fast-evolving nuclear big zinc finger (BZF) gene has successfully differentiated this new species from its allies, S. (S.) nakhonense and Simulium (Simulium) chiangmaiense Takaoka & Suzuki (Diptera: Simuliidae) of the S. striatum species group. The BZF gene sequences show that this new species is more closely related to S. (S.) nakhonense than to S. (S.) chiangmaiense, further supporting its morphological classification.

A New Species of Simulium (Asiosimulium) (Diptera: Simuliidae) from Thailand.

Simulium (Asiosimulium) saeungae sp. nov. (Diptera: Simuliidae) is described based on females, males, pupae, and mature larvae collected from Nan Province, Northern Thailand. It is characterized by the medium-long cerci in the female, enlarged hind basitarsus, and broad ventral plate with its posterior margin not deeply concave in the male, arborescent pupal gill with 42-56 filaments in the pupa and smaller number of primary rays of the labral fan (30-33) in the larva. This is the fifth species of the subgenus Asiosimulium, the second smallest among 10 subgenera in the Oriental Region. Taxonomic notes are given to distinguish this new species from the three known species from Thailand and one from Nepal.

Identification of immature stages of phlebotomine sand flies using MALDI-TOF MS and mapping of mass spectra during sand fly life cycle.

The aim of the study was to evaluate the potential of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for the species identification of sand flies at different developmental stages and map changes in their protein profiles during the course of whole life cycle. Specimens of six different species from laboratory colonies at larval and pupal stages were examined using MALDI-TOF MS. The protein profiles of larvae were stable from the L2 to L4 developmental stages and clearly distinguishable at the species level. In a validation study, 123 larvae of the six species were queried against reference database resulting in 93% correct species identification (log score values higher than 2.0). The spectra generated from sand fly pupae allow species identification as well and surprisingly, in contrast to biting midges and mosquitoes, they did not change during this developmental stage. For adults, thorax was revealed as the optimal body part for sample preparation yielding reproducible spectra regardless age and diet. Only variations were uncovered for freshly engorged females profiles of which were affected by blood signals first two days post bloodmeal. The findings demonstrate that in addition to adult species differentiation MALDI-TOF MS may also serve as a rapid and effective tool for species identification of juvenile stages of phlebotomine sand flies.

Morphology and Polytene Chromosomes of a New Species of Simulium (Trichodagmia) (Diptera: Simuliidae) from the Espinhaço Mountains of Brazil.

The species richness of black flies in the Simulium (Trichodagmia) orbitale (Diptera: Simuliidae) species group is greatest in southern Brazil, where 9 of 19 species are found. A new species in the S. orbitale group was collected during a survey of black flies in Minas Gerais state, Brazil, in the Espinhaço Mountains, an area rich in endemic taxa. The new species is morphologically similar to Simulium guianense Wise (Diptera: Simuliidae), the main vector of the causal agent of onchocerciasis in Brazil and Venezuela. It can be identified in the male by the unpatterned scutum and slightly concave ventral plate with a prominent median projection, in the female by the bilobate anal lobe, in the pupa by the 12 slender gill filaments with nonsclerotized apices, branching in a rake-like pattern, and in the larva by the elongated abdominal segments V-VIII and ventrolaterally lobulate segment IX. The polytene chromosomes have one unique fixed inversion in the IS arm, relative to all other Brazilian members of the group, confirming the morphological evidence of a distinct species.