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.

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.

Mosquitoes cloak their legs to resist insecticides.

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.

Variant Ionotropic Receptors are Expressed in the Antennae of Anopheles sinensis (Diptera: Culicidae).

Mosquitoes transmit many harmful diseases that seriously threaten public health. The mosquito's olfactory system is of great significance for host selection. Inotropic receptors (IRs) and olfactory receptors (ORs) have been demonstrated to be capable of odorant molecular recognition. Analyzing the molecular principles of mosquito olfaction facilitates the development of prevention and therapy techniques. Advances in the understanding of IRs have been seriously inadequate compared to those of ORs. Here, we provide evidence that 35 Anopheles sinensis IR (AsIR) genes are expressed, 7 of which are in the antennae and 2 have expression levels that are upregulated with a blood meal. A homologous analysis of the sequences showed that AsIRs are a subfamily of ionotropic glutamate receptors (iGLURs). This is the first that time IRs have been identified in Anopheles sinensis in vitro. The ultrastructure of the antennae supports the theory that diverse sensilla are distributed in the antennae. The results here may facilitate the revelation of the regulation mechanism in AsIRs, which could mitigate the transmission of diseases by mosquitoes.

The Midgut Muscle Network of Anopheles aquasalis (Culicidae, Anophelinae): Microanatomy and Structural Modification After Blood Meal and Plasmodium vivax (Haemosporida, Plasmodiidae) Infection.

The mosquito midgut is divided into two regions named anterior midgut (AMG) and posterior midgut (PMG). The midgut expands intensely after the blood ingestion to accommodate a large amount of ingested food. To efficiently support the bloodmeal-induced changes, the organization of the visceral muscle fibers has significant adjustments. This study describes the spatial organization of the Anopheles aquasalis (Culicidae, Anophelinae) midgut muscle network and morphological changes after bloodmeal ingestion and infection with Plasmodium vivax (Haemosporida, Plasmodiidae). The midgut muscle network is composed of two types of fibers: longitudinal and circular. The two types of muscle fibers are composed of thick and thin filaments, similar to myosin and actin, respectively. Invagination of sarcoplasm membrane forms the T-system tubules. Sarcoplasmic reticulum cisternae have been observed in association with these invaginations. At different times after the bloodmeal, the fibers in the AMG are not modified. A remarkable dilation characterizes the transitional area between the AMG and the PMG. In the PMG surface, after the completion of bloodmeal ingestion, the stretched muscle fibers became discontinued. At 72 h after bloodmeal digestion, it is possible to observe the presence of disorganized muscle fibers in the midgut regions. The Plasmodium oocyst development along the basal layer of the midgut does not have a significant role in the visceral musculature distribution. This study provides features of the visceral musculature at different blood feeding times of An. aquasalis and shows important changes in midgut topography including when the mosquitoes are infected with P. vivax.

Detection of Plasmodium berghei infected Anopheles stephensi using near-infrared spectroscopy.

BACKGROUND: The proportion of mosquitoes infected with malaria is an important entomological metric used to assess the intensity of transmission and the impact of vector control interventions. Currently, the prevalence of mosquitoes with salivary gland sporozoites is estimated by dissecting mosquitoes under a microscope or using molecular methods. These techniques are laborious, subjective, and require either expensive equipment or training. This study evaluates the potential of near-infrared spectroscopy (NIRS) to identify laboratory reared mosquitoes infected with rodent malaria. METHODS: Anopheles stephensi mosquitoes were reared in the laboratory and fed on Plasmodium berghei infected blood. After 12 and 21 days post-feeding mosquitoes were killed, scanned and analysed using NIRS and immediately dissected by microscopy to determine the number of oocysts on the midgut wall or sporozoites in the salivary glands. A predictive classification model was used to determine parasite prevalence and intensity status from spectra. RESULTS: The predictive model correctly classifies infectious and uninfectious mosquitoes with an overall accuracy of 72%. The false negative and false positive rates were 30 and 26%, respectively. While NIRS was able to differentiate between uninfectious and highly infectious mosquitoes, differentiating between mid-range infectious groups was less accurate. Multiple scans of the same specimen, with repositioning the mosquito between scans, is shown to improve accuracy. On a smaller dataset NIRS was unable to predict whether mosquitoes harboured oocysts. CONCLUSIONS: To our knowledge, we provide the first evidence that NIRS can differentiate between infectious and uninfectious mosquitoes. Currently, distinguishing between different intensities of infection is challenging. The classification model provides a flexible framework and allows for different error rates to be optimised, enabling the sensitivity and specificity of the technique to be varied according to requirements.

Morphometric and morphological appraisal of the eggs of Anopheles stephensi (Diptera: Culicidae) from India.

BACKGROUND & OBJECTIVES: Anopheles stephensi is one of the most important urban malaria vectors in India and contribute about 12% of total malaria cases. An. stephensi has three ecological variants; type, intermediate and mysorensis that can be differentiated on the basis of differences in number of ridges on egg float and on the basis of spiracular indices. Because of its anthropophilic nature the 'type' form is an efficient malaria vector. In the present study, the egg surface morphometry and morphology of An. stephensi 'type' form was studied and detail distinguish- ing characters were recorded for its correct identification. METHODS: Eggs of An. stephensi 'type' form were studied by scanning electron microscopy (SEM) after sputter- coating with gold. In total 23 egg characters were analysed morphologically and morphometrically, which included egg attributes, deck attributes, ventral tubercles, micropyle and float attributes. RESULTS: The dorsal surface of the egg of 'type' form was curved while the ventral surface was concave and both anterior and posterior ends were blunt. The average length and width of egg was 473.94 + 11.18 and 154.69 + 2.66 µm respectively. The number of float ribs observed was 20.33 ± 0.33. The maximum length of float was found to be 246.57 + 15.27 µm, whereas maximum width was 87.16 + 3.83 µm. INTERPRETATION & CONCLUSION: The present study has generated some important data which is specific to An. Stephensi 'type' form and provided significant morphological and morphometric standards for its correct identification. This information could be useful in differentiation of An. stephensi 'type' form from other ecological forms of the same species as well as other species of Anopheles.

Three-dimensional structures of the tracheal systems of Anopheles sinensis and Aedes togoi pupae.

Mosquitoes act as a vector for the transmission of disease. The World Health Organization has recommended strict control of mosquito larvae because of their "few, fixed, and findable" features. The respiratory system of mosquito larvae and pupae in the water has a weak point. As aquatic organisms, mosquito larvae and pupae inhale atmosphere oxygen. However, the mosquito pupae have a non-feeding stage, unlike the larvae. Therefore, detailed study on the tracheal system of mosquito pupae is helpful for understanding their survival strategy. In this study, the three-dimensional (3D) structures of the tracheal systems of Anopheles sinensis and Aedes togoi pupae were comparatively investigated using synchrotron X-ray microscopic computed tomography. The respiratory frequencies of the dorsal trunks were also investigated. Interestingly, the pupae of the two mosquito species possess special tracheal systems of which the morphological and functional features are distinctively different. The respiratory frequency of Ae. togoi is higher than that of An. sinensis. These differences in the breathing phenomena and 3D structures of the respiratory systems of these two mosquito species provide an insight into the tracheal systems of mosquito pupae.

Scanning electron microscopy of antennal sensilla of the eight Anopheles species of the Hyrcanus Group (Diptera: Culicidae) in Thailand.

Antennal sensilla were first investigated in the eight medically and veterinary important Anopheles mosquito species (Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae (= Anopheles lesteri), Anopheles peditaeniatus, Anopheles pursati, and Anopheles sinensis) of the Hyrcanus Group in Thailand, using scanning electron microscopy (SEM). Four types of sensilla, including sensilla chaetica (large and small), sensilla trichodea (sharp- and blunt-tipped), sensilla basiconica or grooved pegs (types I, II, and III), and sensilla coeloconica (large and small), were observed on the female antennae of the eight species. The greatest number of sensilla found along the flagellum of all the Anopheles species consisted of sensilla trichodea. Grooved pegs type II were not found on the antennae of An. peditaeniatus. Interestingly, clusters of 10-15 grooved pegs type III, with blunt-tipped and unevenly grooved-lengthwise sensilla, and a sunken group of 7-12 grooved pegs type III, with slightly curved and point-tipped sensilla, were found distally on flagellomeres 3-7 of An. argyropus and An. peditaeniatus, respectively. In addition, the key for species identification, based on fine structure and morphometrics of antennal sensilla among the eight species, was constructed and differentiated successfully. However, in order to focus intensively on the exact function of these sensilla, further electrophysiological study is needed in understanding their significant role in mosquito behavior, especially when these insects seek hosts for transmitting pathogens to humans.

Hemolymph circulation in insect flight appendages: physiology of the wing heart and circulatory flow in the wings of the mosquito Anopheles gambiae.

The wings of insects are composed of membranes supported by interconnected veins. Within these veins are epithelial cells, nerves and tracheae, and their maintenance requires the flow of hemolymph. For this purpose, insects employ accessory pulsatile organs (auxiliary hearts) that circulate hemolymph throughout the wings. Here, we used correlative approaches to determine the functional mechanics of hemolymph circulation in the wings of the malaria mosquito Anopheles gambiae Examination of sectioned tissues and intravital videos showed that the wing heart is located underneath the scutellum and is separate from the dorsal vessel. It is composed of a single pulsatile diaphragm (indicating that it is unpaired) that contracts at 3 Hz and circulates hemolymph throughout both wings. The wing heart contracts significantly faster than the dorsal vessel, and there is no correlation between the contractions of these two pulsatile organs. The wing heart functions by aspirating hemolymph out of the posterior wing veins, which forces hemolymph into the wings via anterior veins. By tracking the movement of fluorescent microspheres, we show that the flow diameter of the wing circulatory circuit is less than 1 µm, and we present a spatial map detailing the flow of hemolymph across all the wing veins, including the costa, sub-costa, ambient costa, radius, media, cubitus anterior, anal vein and crossveins. We also quantified the movement of hemolymph within the radius and within the ambient costa, and show that hemolymph velocity and maximum acceleration are higher when hemolymph is exiting the wing.

Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology.

BACKGROUND: The malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India. METHODS: The adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics. RESULTS: Currently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6-7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E. CONCLUSIONS: The present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.

Morphological changes in the spiracles of Anopheles gambiae s.l (Diptera) as a response to the dry season conditions in Burkina Faso (West Africa).

BACKGROUND: Survival to dry season conditions of sub-Saharan savannahs is a major challenge for insects inhabiting such environments, especially regarding the desiccation threat they are exposed to. While extensive literature about insect seasonality has revealed morphologic, metabolic and physiological changes in many species, only a few studies have explored the responses following exposure to the stressful dry season conditions in major malaria vectors. Here, we explored morphological changes triggered by exposure to dry season conditions in An. gambiae s.l. mosquitoes by comparing females reared in climatic chambers reflecting environmental conditions found in mosquito habitats during the rainy and dry seasons in a savannah area of Burkina Faso (West Africa). RESULTS: Using scanning electron microscopy (SEM) and confocal imaging, we revealed significant changes in morphological features of the spiracles in females An. gambiae s.l. exposed to contrasted environmental conditions. Hence, the hairs surrounding the spiracles were thicker in the three species when raised under dry season environmental conditions. The thicker hairs were in some cases totally obstructing spiracular openings. Specific staining provided evidence against contamination by external microorganisms such as bacteria and fungi. However, only further analysis would unequivocally rule out the hypothesis of experimental artifact. CONCLUSION: Morphological changes in spiracular features probably help to limit body water loss during desiccating conditions, therefore contributing to insect survival. Differences between species within the An. gambiae complex might therefore reflect different survival strategies used by these species to overcome the detrimental dry season conditions in the wild.

Reproductive biology in Anophelinae mosquitoes (Diptera, Culicidae): Fine structure of the female accessory gland.

The morphology and ultrastructure of female accessory reproductive glands of Anopheles maculipennis s.s., Anopheles labranchiae and Anopheles stephensi were investigated by light and electron microscopy. The reproductive system in these species is characterized by two ovaries, two lateral oviducts, a single spermatheca and a single accessory gland. The gland is globular and has a thin duct which empties into the vagina, near the opening of the spermathecal duct. Significant growth of the accessory reproductive gland is observed immediately after blood meal, but not at subsequent digestion steps. At ultrastructural level, the gland consists of functional glandular units belonging to type 3 ectodermal glands. The secretory cells are elongated and goblet shaped, with most of their cytoplasm and large nucleus in the basal part, close to the basement lamella. Finely fibrous electron-transparent material occupies the secretory cavity that is in contact with the end of a short efferent duct (ductule) emerging from the gland duct. The present study is the first detailed description of female accessory gland ultrastructure in Anophelinae and provides insights into the gland's functional role in the reproductive biology of these insects.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors.

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Anopheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Temporal and spatial expression of cuticular proteins of Anopheles gambiae implicated in insecticide resistance or differentiation of M/S incipient species.

BACKGROUND: Published data revealed that two of the 243 structural cuticular proteins of Anopheles gambiae, CPLCG3 and CPLCG4, are implicated in insecticide resistance and a third, CPF3, has far higher transcript levels in M than in S incipient species. We studied the distribution of transcripts for these three genes in the tissues of An. gambiae and the location of the proteins in the cuticle itself to gain information about how these cuticular proteins contribute to their important roles. Our data are consistent with CPLCG3/4 contributing to a thicker cuticle thus slowing penetration of insecticides and CPF3 possibly having a role in the greater desiccation tolerance of the M form. METHODS: Using RT-qPCR, we established the temporal expression of the genes and by in situ hybridization we revealed the main tissues where their mRNAs are found. Electron microscopy immunolocalization, using secondary antibodies labeled with colloidal gold, allowed us to localize these proteins within different regions of the cuticle. RESULTS: The temporal expression of these genes overlaps, albeit with higher levels of transcripts from CPF3 in pharate adults and both CPLCG3 and CPLCG4 are higher in animals immediately after adult eclosion. The main location of mRNAs for all three genes is in appendages and genitalia. In contrast, the location of their proteins within the cuticle is completely different. CPF3 is found exclusively in exocuticle and CPLCG3/4 is restricted to the endocuticle. The other CPF gene expressed at the same times, CPF4, in addition to appendages, has message in pharate adult sclerites. CONCLUSIONS: The temporal and spatial differences in transcript abundance and protein localization help to account for An. gambiae devoting about 2% of its protein coding genes to structural cuticular proteins. The location of CPLCG3/4 in the endocuticle may contribute to the thickness of the cuticle, one of the recently appreciated components of insecticide resistance, while the location of CPF3 might be related to the greater desiccation resistance of the M form.

"Super e-noses": Multi-layer perceptron classification of volatile odorants from the firing rates of cross-species olfactory receptor arrays.

Current electronic noses, or e-noses, that employ insect odorant receptors (Ors) as their sensory front end are potentially limited by the fact that the Ors come from a single species. In addition, a realistic e-nose also demands low numbers of Ors at its sensory front end due to the difficulties of receptor/sensor integration and functionalisation. In this work, we report the first investigations of a `Super E-Nose' that incorporates Ors from both the model organism Drosophila melanogaster fruit fly (DmOr) and the mosquito, Anopheles gambiae (AgOr). Furthermore, we report how an Artificial Neural Network (ANN), in the form of a hybrid double hidden layer Multi-Layer Perceptron (MLP), can be used to determine the optimal Ors that provide the best prediction performance in the classification of unknown odorants into their respective chemical class. Our findings demonstrate how 3-Or arrays consisting of DmOr only, AgOr only, or cross-species DmOr-AgOr combinations correctly classified all unknown odorants of the validation set. In addition, we report that all 3-Or combinations perform equally well as the complete 74 DmOr-AgOr array. Thus, the results of this work support further investigation into cross-species `Super E-noses' coupled with hybrid MLPs for the classification of unknown odorants.

[Updated inventory of mosquitoes (Diptera: Culicidae) of the island of La Réunion, Indian Ocean]. / Inventaire actualisé des moustiques (Diptera : Culicidae) de l'île de La Réunion, océan Indien.

A literature analysis coupled with new entomological surveys conducted between 2009 and 2012 led to changes in the list of mosquito species present on the island of La Réunion. Using morphological criteria, Orthopodomyia arboricollis is replaced by Or. reunionensis. On the basis of morphometrical and genetic criteria, Culex univittatus is replaced by Cx. neavei. Cx. poicilipes, which was already reported missing 40 years ago, has not been found again. Anopheles arabiensis is confirmed as the only species of the Gambiae complex present on the island. Thus, twelve species are currently known. For each of them, elements of taxonomic, biological and medical interest are listed. An. arabiensis is a major vector of human Plasmodium (last case of indigenous malaria in 1967). In the Indian Ocean, Aedes albopictus and Ae. aegypti both are competent for transmitting dengue and chikungunya viruses. In Africa, Cx. quinquefasciatus transmits Wuchereria bancrofti and Cx. neavei transmits the Sindbis virus; both species also transmit the West Nile virus. Cx. tritaeniorhynchus is the major vector of Japanese Encephalitis virus in Asia. Two species are endemic (Ae. dufouri and Or. reunionensis), the ten other ones are also found in Madagascar and on the African continent (An. coustani, An. arabiensis, Ae. fowleri, Cx. tritaeniorhynchus, Cx. neavei, Cx. insignis, Lutzia tigripes), with three of them having also a cosmopolitan distribution (Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus). Among the twelve recorded taxa, eight species are anthropophilic, three are supposedly zoophilic and one is a predatory species. No new invasive anthropophilic species did settle on the island. Updated identification keys of larval and adult stages are proposed.

Susceptibility of mosquito and lepidopteran cell lines to the mosquito iridescent virus (IIV-3) from Aedes taeniorhynchus.

Mosquito iridescent viruses (MIV) are members of the genus Chloriridovirus that currently contains only the type IIV-3 from Aedestaeniorhynchus. The complete genome of invertebrate iridescent virus -3 (IIV-3) has been sequenced and the availability of a tissue culture system would facilitate functional genomic studies. This investigation, using quantitative PCR and electron microscopy, has determined that the mosquito cell lines Aedes aegypti (Aag2), Aedes albopictus (C6/36) and Anopheles gambiae (4a3A) as well as the lepidopteran cell line from Spodoptera frugiperda (SF9) are permissive to IIV-3 infection. However, IIV-3 infection remained longer in Aag2 and C6/36 cells. Virus produced in C6/36 cell line was infectious to larvae of A. taeniorhynchus by injection and per os. Ultrastructural examination of 4a3A and SF9 cells infected with IIV-3 revealed an unusual feature, where virions were localized to mitochondria. It is speculated that containment with mitochondria may play a role in the lack of persistence in these cell lines.

[Analysis of the spatial organization of the XL chromosome attachment site in nurse cell nuclei of the malaria mosquito Anopheles atroparvus].

The spatial position of the site of XL chromosome attachment to the nuclear envelope of ovarian nurse cells relative to the oocyte has been analyzed in the malaria mosquito Anopheles atroparvus. The XL chromosome attachment sites in the oocyte-nurse cell system of this species have been demonstrated to be orderly arranged, with the attachment sites in two out of three nurse cells in the same layer identically oriented relative to the oocyte.

Redescription of Anopheles (Nyssorhynchus) antunesi Galvão & Amaral and description of a new species of the Myzorhynchella Section (Diptera: Culicidae) from Serra da Mantiqueira, Brazil.

Anopheles (Nyssorhynchus) pristinus Nagaki & Sallum, n. sp. of the Myzorhynchella Section is described based on morphological characters of adult females, males, fourth-instar larvae, pupae and male genitalia. Anopheles (Nyssorhynchus) antunesi Galvão & Amaral is characterized to fix its identity and distinguish it from An. pristinus. The eggs of An. antunesi are described for the first time. Molecular characterization employing sequences of the COI mitochondrial gene and the ITS2 region of ribosomal DNA are provided for each species. An. antunesi and An. pristinus are compared with morphologically similar species of the Myzorhynchella Section. The results of the present study suggest that the new species has been misidentified as both An. antunesi and Anopheles lutzii Cruz. An. antunesi and An. pristinus are sympatric, occurring at high altitudes in Serra da Mantiqueira, Southeastern Brazil.