Geometric morphometrics and machine learning as tools for the identification of sibling mosquito species of the Maculipennis complex (Anopheles).

Geometric morphometrics allows researchers to use the specific software to quantify and to visualize morphological differences between taxa from insect wings. Our objective was to assess wing geometry to distinguish four Anopheles sibling species of the Maculipennis complex, An. maculipennis s. s., An. daciae sp. inq., An. atroparvus and An. melanoon, found in Northern Italy. We combined the geometric morphometric approach with different machine learning alghorithms: support vector machine (SVM), random forest (RF), artificial neural network (ANN) and an ensemble model (EN). Centroid size was smaller in An. atroparvus than in An. maculipennis s. s. and An. daciae sp. inq. Principal component analysis (PCA) explained only 33% of the total variance and appeared not very useful to discriminate among species, and in particular between An. maculipennis s. s. and An. daciae sp. inq. The performance of four different machine learning alghorithms using procrustes coordinates of wing shape as predictors was evaluated. All models showed ROC-AUC and PRC-AUC values that were higher than the random classifier but the SVM algorithm maximized the most metrics on the test set. The SVM algorithm with radial basis function allowed the correct classification of 83% of An. maculipennis s. s. and 79% of An. daciae sp. inq. ROC-AUC analysis showed that three landmarks, 11, 16 and 15, were the most important procrustes coordinates in mean wing shape comparison between An. maculipennis s. s. and An. daciae sp. inq. The pattern in the three-dimensional space of the most important procrustes coordinates showed a clearer differentiation between the two species than the PCA. Our study demonstrated that machine learning algorithms could be a useful tool combined with the wing geometric morphometric approach.

Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan.

BACKGROUND: In spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Plasmodium falciparum is the dominant malaria parasite in sub-Saharan Africa. However, Plasmodium vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax circumsporozoite protein (Pvcsp) allele diversity. METHODS: Mosquito samples were collected from Central Sudan (Rural Khartoum and Sennar) and Eastern Sudan (New Halfa, Kassala state) using pyrethrum spray catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissected to determine the presence of natural infection of malaria parasites. In addition, the rest of the samples were investigated for the presence of Pvcsp gene using nested-PCR. RESULTS: A total of 1037 adult anopheline mosquitoes were collected from New Halfa (N = 467), Rural Khartoum (N = 132), and Sennar (N = 438). Morphological and molecular identification of the collected mosquitoes revealed the presence of Anopheles arabiensis (94.2%), Anopheles funestus (0.5%), and Anopheles pharoensis (5.4%). None of the dissected mosquitoes (N = 108) showed to be infected with malaria parasite. Overall P. vivax infectivity rate was 6.1% (63/1037) by Pvcsp nested PCR. Co-dominance of An. arabiensis and An. pharoensis is reported in Sennar state both being infected with P. vivax. CONCLUSION: This study reported P. vivax infection among wild-caught anopheline mosquitoes in Central and Eastern Sudan. While An. arabiensis is the most abundant vector observed in all study areas, An. funestus was recorded for the first time in New Halfa, Eastern Sudan. The documented Anopheles species are implicated in Pvcsp allele diversity. Large-scale surveys are needed to identify the incriminated vectors of P. vivax malaria and determine their contribution in disease transmission dynamics.

Identification of mosquitoes (Diptera: Culicidae) from Mexico State, Mexico using morphology and COI DNA barcoding.

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico.

Mating-regulated atrial proteases control reinsemination rates in Anopheles gambiae females.

Anopheles gambiae mosquitoes are the most important vectors of human malaria. The reproductive success of these mosquitoes relies on a single copulation event after which the majority of females become permanently refractory to further mating. This refractory behavior is at least partially mediated by the male-synthetized steroid hormone 20-hydroxyecdysone (20E), which is packaged together with other seminal secretions into a gelatinous mating plug and transferred to the female atrium during mating. In this study, we show that two 20E-regulated chymotrypsin-like serine proteases specifically expressed in the reproductive tract of An. gambiae females play an important role in modulating the female susceptibility to mating. Silencing these proteases by RNA interference impairs correct plug processing and slows down the release of the steroid hormone 20E from the mating plug. In turn, depleting one of these proteases, the Mating Regulated Atrial Protease 1 (MatRAP1), reduces female refractoriness to further copulation, so that a significant proportion of females mate again. Microscopy analysis reveals that MatRAP1 is localized on a previously undetected peritrophic matrix-like structure surrounding the mating plug. These data provide novel insight into the molecular mechanisms shaping the post-mating biology of these important malaria vectors.

Identification key to the Anopheles mosquitoes of South America (Diptera: Culicidae). III. Male genitalia.

BACKGROUND: Accurate identification of the species of Anopheles Meigen, 1818 requires careful examination of all life stages. However, morphological characters, especially those of the females and fourth-instar larvae, show some degree of polymorphism and overlap among members of species complexes, and sometimes even within progenies. Characters of the male genitalia are structural and allow accurate identification of the majority of species, excluding only those in the Albitarsis Complex. In this key, based on the morphology of the male genitalia, traditionally used important characters are exploited together with additional characters that allow robust identification of male Anopheles mosquitoes in South America. METHODS: Morphological characters of the male genitalia of South American species of the genus Anopheles were examined and employed to construct a comprehensive, illustrated identification key. For those species for which specimens were not available, illustrations were based on published illustrations. Photographs of key characters of the genitalia were obtained using a digital Canon Eos T3i attached to a light Diaplan Leitz microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure. RESULTS: An illustrated key to South American species of Anopheles based on the morphology of the male genitalia is presented, together with a glossary of morphological terms. The male genitalia of type-specimens of previously poorly documented species were also examined and included in the key, e.g. Anopheles (Anopheles) tibiamaculatus (Neiva, 1906) which has a unique quadrangular-shaped aedeagus with an apical opening. CONCLUSIONS: Male genitalia of South American species of Anopheles possess robust characters that can be exploited for accurate species identification. Distortion that can occur during the dissection and mounting process can obstruct accurate identification; this is most evident with inadvertent damage or destruction of unique features and interferes with correctly assigning shapes of the features of the ventral claspette. In some species, the shape, and anatomical details of the aedeagus also need to be examined for species identification. For members of the Myzorhynchella Series, both ventral and dorsal claspettes possess multiple characteristics that are herein used as reliable characters for species identification.

Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). IV. Adult females.

BACKGROUND: Morphological identification of adult females of described species of the genus Anopheles Meigen, 1818 in South America is problematic, but necessary due to their differing roles in the transmission of human malaria. The increase in the number of species complexes uncovered by molecular taxonomy challenges accurate identification using morphology. In addition, the majority of newly discovered species have not been formally described and in some cases the identities of the nominotypical species of species complexes have not been resolved. Here, we provide an up-to-date key to identify Neotropical Anopheles species using female external morphology and employing traditionally used and new characters. METHODS: Morphological characters of the females of South American species of the genus Anopheles were examined and employed to construct a species/group identification key. Photographs of key characters were obtained using a digital Canon Eos T3i, attached to a microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure. RESULTS: A morphological identification key to the adult females of species of the genus Anopheles described in South America is presented. Definitions and illustrations of the key characters are provided to facilitate use of key. CONCLUSIONS: Identification of species of the genus Anopheles based on female morphology is challenging because some key characters can be variable and overlapping among species. In addition, the majority of key characters are linked to color and shape of scales, their distribution on the head, scutum, abdomen, maxillary palpi, labium and legs, and pattern of pale and dark scales on dorsal and ventral surfaces of the wing veins. Thus, it is understandable that a specimen needs to be in good condition to be accurately identified. Morphologically similar species, such as those of the Konderi, Oswaldoi, Nuneztovari, Benarrochi and Albitarsis Complexes, and the Triannulatus and Strodei Groups, among others, cannot be accurately identified using characters included in the key. Further investigation will be required to exploit morphological characteristics for identification of members of those complexes, with formal description of new species.

Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). II. Fourth-instar larvae.

BACKGROUND: Accurate species identification of South American anophelines using morphological characters of the fourth-instar larva is problematic, because of the lack of up-to-date identification keys. In addition, taxonomic studies, employing scanning electron microscopy of the eggs and DNA sequence data, have uncovered multiple complexes of morphologically similar species, and resulted in the resurrection of other species from synonymy, mainly in the subgenus Nyssorhynchus. Consequently, the identification keys urgently need to be updated to provide accurate morphological tools to identify fourth-instar larvae of all valid species and species complexes. METHODS: Morphological characters of the fourth-instar larvae of South American species of the genus Anopheles were examined and employed to elaborate a fully illustrated identification key. For species for which no specimens were available, illustrations were based on published literature records. RESULTS: A fully illustrated key to the fourth-instar larvae of South American species of the genus Anopheles (Diptera: Culicidae) is presented. Definitions of the morphological terms used in the key are provided and illustrated. CONCLUSIONS: Morphological identification of South American Anopheles species based on the fourth-instar larvae has been updated. Characters of the spiracular apparatus were determined useful for the identification of morphologically similar species, in the Strodei Group and some taxa in the Myzorhynchella Section. The single versus branched abdominal seta 6-IV used to differentiate Myzorhynchella species from other Nyssorhynchus species was shown to be variable in Myzorhynchella species. Also, the abdominal setae 1-IV,V of Anopheles atacamensis and Anopheles pictipennis were shown to be slightly serrate at the edges. Recognition of this character is important to avoid inaccurate identification of these species as members of the subgenus Anopheles.

Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). I. Introduction.

BACKGROUND: The worldwide genus Anopheles Meigen, 1918 is the only genus containing species evolved as vectors of human and simian malaria. Morbidity and mortality caused by Plasmodium Marchiafava & Celli, 1885 is tremendous, which has made these parasites and their vectors the objects of intense research aimed at mosquito identification, malaria control and elimination. DNA tools make the identification of Anopheles species both easier and more difficult. Easier in that putative species can nearly always be separated based on DNA data; more difficult in that attaching a scientific name to a species is often problematic because morphological characters are often difficult to interpret or even see; and DNA technology might not be available and affordable. Added to this are the many species that are either not yet recognized or are similar to, or identical with, named species. The first step in solving Anopheles identification problem is to attach a morphology-based formal or informal name to a specimen. These names are hypotheses to be tested with further morphological observations and/or DNA evidence. The overarching objective is to be able to communicate about a given species under study. In South America, morphological identification which is the first step in the above process is often difficult because of lack of taxonomic expertise and/or inadequate identification keys, written for local fauna, containing the most consequential species, or obviously, do not include species described subsequent to key publication. METHODS: Holotypes and paratypes and other specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública (FSP-USP), Museo de Entomología, Universidad del Valle (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution (USNMC) were examined and employed to illustrate the identification keys for female, male and fourth-instar larvae of Anopheles. RESULTS: We presented, in four concurrent parts, introduction and three keys to aid the identification of South American Anopheles based on the morphology of the larvae, male genitalia and adult females, with the former two keys fully illustrated. CONCLUSIONS: Taxonomic information and identification keys for species of the genus Anopheles are updated. The need for further morphology-based studies and description of new species are reinforced.

Live In Vivo Imaging of Plasmodium Invasion of the Mosquito Midgut.

The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new vertebrate host. Previous confocal studies with fixed infected midguts showed that ookinetes traverse midgut epithelial cells and cause irreversible tissue damage. Here, we investigated the spatiotemporal dynamics of ookinete midgut traversal and the response of midgut cells to invasion. A novel mounting strategy was established, suitable fluorescent dye combinations were identified and protocols optimized to label mosquito tissues in vivo, and live imaging protocols using confocal microscopy were developed. Tracking data showed that ookinetes gliding on the midgut surface travel faster and farther than those that remain in the lumen or those that have invaded the epithelium. Image analysis confirmed that parasite invasion and cell traversal occur within a couple of minutes, while caspase activity in damaged cells, indicative of cellular apoptosis, and F-actin cytoskeletal rearrangements in cells extruded into the gut lumen persist for several hours. This temporal difference highlights the importance of hemocyte-mediated cellular immunity and the mosquito complement system to mount a coordinated and effective antiplasmodial response. This novel in vivo imaging protocol allowed us to continuously observe individual ookinetes in live mosquitoes within the gut lumen and during cell traversal and to capture the subsequent cellular responses to invasion in real time for several hours, without loss of tissue integrity.IMPORTANCE Malaria is one of the most devastating parasitic diseases in humans and is transmitted by anopheline mosquitoes. The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new host. Here, we developed a new strategy to visualize Plasmodium ookinetes as they traverse the mosquito midgut and to follow the response of damaged epithelial cells by imaging live mosquitoes. Understanding the spatial and temporal aspects of these interactions is critical when developing novel strategies to disrupt disease transmission.

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia.

BACKGROUND: Understanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity. RESULTS: Eighteen distinct species of Anopheles were molecularly identified in a 1 km2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus, An. karwari, An. peditaeniatus, An. vagus, An. barbirostris, An. tessellatus, An. nigerrimus, An. crawfordi, An. maculatus, An. flavirostris and An. kochi. Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama (An. barbirostris and An. vagus) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%. CONCLUSIONS: Karama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit of malaria elimination.

Molecular and morphological evidence of sibling species in Anopheles baileyi Edwards (Diptera: Culicidae) in Bhutan and Thailand.

This paper reports the results of a molecular and morphological study of Anopheles baileyi in Bhutan and Thailand. Phylogenetic analyses of ribosomal (ITS2) and mitochondrial DNA (COI) sequences revealed the presence of four genetically distinct clades, three in Bhutan (Clades I, II and III) and one in Thailand (Clade IV). Most of the larvae in the Bhutanese clades differed from those in the Thai clade in having seta 4-C branched, whereas it is single in the latter. The adults of each clade showed variation of wing markings and overlapping characters. The combination of characteristics of thoracic setae 1,2-P and abdominal seta 3-I was found to be useful for distinguishing the larvae. Pupae were inseparable. We provisionally recognize mosquitoes of Clades I, II, III and IV as members of a sibling species complex, the Baileyi Complex, denoted as species A, B, C and D, respectively. Species A is most likely the type form of An. baileyi s.s. because it was found adjacent to the type locality (Yatung, Tibet), and the others are unrecognized species.

Effects of larval exposure to sublethal doses of Bacillus thuringiensis var. israelensis on body size, oviposition and survival of adult Anopheles coluzzii mosquitoes.

BACKGROUND: Application of the larvicide Bacillus thuringiensis var. israelensis (Bti) is a viable complementary strategy for malaria control. Efficacy of Bti is dose-dependent. There is a knowledge gap on the effects of larval exposure to sublethal Bti doses on emerging adult mosquitoes. The present study examined the effect of larval exposure to sublethal doses of Bti on the survival, body size and oviposition rate in adult Anopheles coluzzii. METHODS: Third-instar An. coluzzii larvae were exposed to control and sublethal Bti concentrations at LC20, LC50 and LC70 for 48 h. Surviving larvae were reared to adults under standard colony conditions. Thirty randomly selected females from each treatment were placed in separate cages and allowed to blood feed. Twenty-five gravid females from the blood-feeding cages were randomly selected and transferred into new cages where they were provided with oviposition cups. Numbers of eggs laid in each cage and mortality of all adult mosquitoes were recorded daily. Wing lengths were measured of 570 mosquitoes as a proxy for body size. RESULTS: Exposure to LC70Bti doses for 48 h as third-instar larvae reduced longevity of adult An. coluzzii mosquitoes. Time to death was 2.58 times shorter in females exposed to LC70Bti when compared to the control females. Estimated mortality hazard rates were also higher in females exposed to the LC50 and LC20 treatments, but these differences were not statistically significant. The females exposed to LC70 concentrations had 12% longer wings than the control group (P < 0.01). No differences in oviposition rate of the gravid females were observed between the treatments. CONCLUSIONS: Exposure of An. coluzzii larvae to sublethal Bti doses reduces longevity of resultant adults and is associated with larger adult size and unclear effect on oviposition. These findings suggest that anopheline larval exposure to sublethal Bti doses, though not recommended, could reduce vectorial capacity for malaria vector populations by increasing mortality of resultant adults.

Wing morphometric variability of the malaria vector Anopheles (Cellia) epiroticus Linton et Harbach (Diptera: Culicidae) for the duration of the rainy season in coastal areas of Samut Songkhram, Thailand.

In Thailand, Anopheles (Cellia) epiroticus Linton et Harbach (Diptera: Culicidae) is the secondary vector of human malaria along coastal regions. While there are some studies of phenotypic variability and population structure of A. epiroticus, more information on morphological variation would enhance epidemiological understanding of medically important mosquito vectors. This research examined morphological variation at three different distances from coastlines of Samut Songkhram Province, Thailand, using landmark-based geometric morphometrics. Wing shape of A. epiroticus was significantly different in the area 0.2 km away from the sea compared to areas 2 and 4 km away from the sea (p < 0.05). Phenotypic variability in wing shape is associated with distance from the sea. Morphological variations in the area closest to the sea were most pronounced, showing a relationship between A. epiroticus and the ecosystem that affects wing geometry. These results provide important information to understand morphological variation of A. epiroticus in coastal areas.

Molecular and morphological evidence for sibling species within Anopheles (Anopheles) lindesayi Giles (Diptera: Culicidae) in Bhutan.

This paper reports the results of a comparative molecular and morphological study of An. lindesayi collected from various districts of Bhutan and An. l. cameronensis from Thailand, compared with GenBank accessions and publications for An. l. japonicus from Japan, South Korea and China, An. l. pleccau from Taiwan, and An. lindesayi from India. Phylogenetic analyses based on ribosomal (ITS2) and mitochondrial (COI) DNA sequences using the Maximum Likelihood method revealed five genetically distinct clades (A, B, C, D and E) in Bhutan. Specimens in Clade A correspond to the original description of An. lindesayi, particularly in wing markings, the pattern of basal pale scales on the hindfemur and the single seta 4-C of larvae, and their COI sequences were closely related to one Indian sequence. Larvae of Clades B, C, D and E are similar in having seta 4-C branched rather than single. The adults of Clades C, D and E (B not available) are distinguishable from those of Clade A and other subspecies. Specimens of Clade C are unique in having a long pale spot on wing vein R and the subcosta, scattered pale scales on several veins and a dark spot at the tip of vein R2. The adults of Clades D and E are similar in having a dark spot at the tip of vein R2 and no scattered pale scales on all other veins. We provisionally recognize mosquitoes of Clades A, B, C, D and E as species A, B, C, D and E, respectively, of the Lindesayi Complex. Species A is An. lindesayi sensu stricto and the others are unnamed species. Concomitantly, the previous concept of the "Lindesayi Complex", which included An. lindesayi, An. menglangensis, An. nilgiricus and An. wellingtonianus, is now recognized as the Lindesayi Subgroup of the Lindesayi Group (Anopheles Series, subgenus Anopheles) with the five sibling species of An. lindesayi comprising a more apposite Lindesayi Complex within the subgroup.

Microanatomy of the American Malaria Vector Anopheles aquasalis (Diptera: Culicidae: Anophelinae) Midgut: Ultrastructural and Histochemical Observations.

The mosquito gut is divided into foregut, midgut, and hindgut. The midgut functions in storage and digestion of the bloodmeal. This study used light, scanning (SEM), and transmission (TEM) electron microscopy to analyze in detail the microanatomy and morphology of the midgut of nonblood-fed Anopheles aquasalis females. The midgut epithelium is a monolayer of columnar epithelial cells that is composed of two populations: microvillar epithelial cells and basal cells. The microvillar epithelial cells can be further subdivided into light and dark cells, based on their affinities to toluidine blue and their electron density. FITC-labeling of the anterior midgut and posterior midgut with lectins resulted in different fluorescence intensities, indicating differences in carbohydrate residues. SEM revealed a complex muscle network composed of circular and longitudinal fibers that surround the entire midgut. In summary, the use of a diverse set of morphological methods revealed the general microanatomy of the midgut and associated tissues of An. aquasalis, which is a major vector of Plasmodium spp. (Haemosporida: Plasmodiidae) in America.

Smallest Anopheles farauti occur during the peak transmission season in the Solomon Islands.

BACKGROUND: Malaria transmission varies in intensity amongst Solomon Island villages where Anopheles farauti is the only vector. This variation in transmission intensity might be explained by density-dependent processes during An. farauti larval development, as density dependence can impact adult size with associated fitness costs and daily survivorship. METHODS: Adult anophelines were sampled from six villages in Western and Central Provinces, Solomon Islands between March 2014 and February 2017. The size of females was estimated by measuring wing lengths, and then analysed for associations with biting densities and rainfall. RESULTS: In the Solomon Islands, three anopheline species, An. farauti, Anopheles hinesorum and Anopheles lungae, differed in size. The primary malaria vector, An. farauti, varied significantly in size among villages. Greater rainfall was directly associated with higher densities of An. farauti biting rates, but inversely associated with body size with the smallest mean sized mosquitoes present during the peak transmission period. A measurable association between body size and survivorship was not found. CONCLUSIONS: Density dependent effects are likely impacting the size of adult An. farauti emerging from a range of larval habitats. The data suggest that rainfall increases An. farauti numbers and that these more abundant mosquitoes are significantly smaller in size, but without any reduced survivorship being associated with smaller size. The higher malaria transmission rate in a high malaria focus village appears to be determined more by vector numbers than size or survivorship of the vectors.

Material stiffness variation in mosquito antennae.

The antennae of mosquitoes are model systems for acoustic sensation, in that they obey general principles for sound detection, using both active feedback mechanisms and passive structural adaptations. However, the biomechanical aspect of the antennal structure is much less understood than the mechano-electrical transduction. Using confocal laser scanning microscopy, we measured the fluorescent properties of the antennae of two species of mosquito- Toxorhynchites brevipalpis and Anopheles arabiensis-and, noting that fluorescence is correlated with material stiffness, we found that the structure of the antenna is not a simple beam of homogeneous material, but is in fact a rather more complex structure with spatially distributed discrete changes in material properties. These present as bands or rings of different material in each subunit of the antenna, which repeat along its length. While these structures may simply be required for structural robustness of the antennae, we found that in FEM simulation, these banded structures can strongly affect the resonant frequencies of cantilever-beam systems, and therefore taken together our results suggest that modulating the material properties along the length of the antenna could constitute an additional mechanism for resonant tuning in these species.

Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia.

BACKGROUND: Anopheles maculatus, a species of the Maculatus Group of subgenus Cellia (Diptera: Culicidae), is an important vector of human malarial protozoa in Java, Indonesia. However, the identity of this species in Indonesia has been questionable because published reports and records are based mainly on morphological identification, which is unreliable for distinguishing members of the Maculatus Group due to overlapping characters. METHODS: We performed morphological assessments, metaphase karyotype preparations, phylogenetic analyses of ITS2 and cox2 sequence data and cross-mating experiments to determine whether the Javanese form and An. maculatus (s.s.) from Thailand were conspecific. RESULTS: The adults of the Java strain are similar to those of An. maculatus (s.s.), but the larvae and pupae exhibit significant differences. The metaphase karyotype of Javanese specimens includes a long acrocentric X chromosome and a small telocentric Y chromosome, which are distinct from other members of the Maculatus Group. Cross-mating of the Java strain with An. maculatus (s.s.) revealed genetic incompatibility. Phylogenetic analysis of ITS2 and cox2 sequences revealed that the Java strain forms a single clade that is distinct from clades of other members of the group (Kimura 2-parameter, K2P, genetic distances 3.1-19.2% and 1.6-9.6%, respectively). CONCLUSIONS: This study provides evidence that the Javanese form of An. maculatus is not conspecific with An. maculatus (s.s.) and constitutes a previously unrecognized species of the Maculatus Group.

Urbanization as a driver for temporal wing-shape variation in Anopheles cruzii (Diptera: Culicidae).

Anopheles cruzii is the main vector of human and simian malaria in the Brazilian Atlantic Forest. This biome, which is an important hotspot of malaria transmission, has suffered fragmentation and deforestation as a result of urban expansion. Fragmentation and deforestation occur continually in the south of the city of São Paulo, Brazil, and findings of An. cruzii in the peridomicile have consequently become more frequent in this part of the city. Although An. cruzii is of considerable epidemiological importance, the impact of urbanization on the microevolution of this species in this malaria-endemic region has not been investigated to date. In this study, we investigated temporal variation in wing shape and size in An. cruzii populations collected in sylvatic, peri-urban and urban areas over a three-year period. Our results show a slight but significant phenotypic variation in all three populations over the study period. Time was a more powerful driver for wing variation than geographic distance. Temporal wing-shape variation appears to be positively associated with urbanization, suggesting that anthropogenic changes in the environment may be a strong driver for wing-shape variation in An. cruzii. Further studies using genetic markers are needed to assess genetic differentiation in these populations.

Proteomics reveals localization of cuticular proteins in Anopheles gambiae.

Anopheles gambiae devotes over 2% of its protein coding genes to its 298 structural cuticular proteins (CPs). This paper provides new LC-MS/MS data on two adult structures, proboscises and palps, as well as three larval samples - 4th instar larvae, just their terminal segment, and a preparation enriched in their tracheae. These data were combined with our previously published results of proteins from five other adult structures, whole adults, and two preparations chosen for their relatively clean cuticle, the larval head capsules left behind after ecdysis and the pupal cuticles left behind after adult eclosion. Peptides from 28 CPs were recovered in all adult structures; 24 CPs were identified for the first time, 6 of these were members of the TWDL family. Most newly identified proteins came from the larval sources. Based solely on peptide recovery, from our data and from other investigators, most available on VectorBase, there were only 4 CPs that were restricted to a single adult structure. More were restricted to a single metamorphic stage, 14 in larvae, 0 in pupae and 32 in adults. Expression data from our earlier RT-qPCR studies reduces these numbers. Charting restriction of CPs to stage or structure is a step forward in establishing their specific roles.