Morphological characterization of antennal sensilla in Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti adults: a comparative study using scanning electron microscopy.

Some mosquitoes, including species of the genus Toxorhynchites, are known for actively preying on other mosquito larvae, making these predators valuable allies in the fight against vector-borne diseases. A comprehensive understanding of the anatomy and physiology of these potential biological control agents is helpful for the development of effective strategies for controlling vector populations. This includes the antennae, a crucial component in the search for hosts, mating, and selection of oviposition sites. This study utilized scanning electron microscopy to characterize the sensilla on the antennae of adult mosquitoes from two species that are exclusively phytophagous, including Toxorhynchites theobaldi and Toxorhynchites violaceus, as well as Lutzia bigoti, which females are allegedly hematophagous. The types of sensilla in each species were compared, and five basic types of antennal sensilla were identified: trichoid, chaetic, coeloconic, basiconic, and ampullacea. The analysis also found that they were morphologically similar across the three species, regardless of feeding habits or sex. The identification and characterization of basic types of antennal sensilla in T. theobaldi, T. violaceus, and L. bigoti suggest that these structures, which play a crucial role in the behavior and ecology, have common functions across different mosquito species, despite differences in feeding habits or sex.

Scanning Electron Microscopy and Geometric Contour Morphometry for Identifying Eggs of Three Amazonian Species of Mansonia (Diptera: Culicidae).

The eggs of three Amazonian species of the genus Mansonia (Diptera: Culicidae) were analyzed using morphological and morphometric characters. Eggs of Mansonia humeralis Dyar & Knab, 1916 were morphologically different from those of Mansonia titillans (Walker, 1848) and Mansonia amazonensis (Theobald, 1901), which were more similar to each other according to linear and geometric morphometry. A principal component analysis generated from elliptic Fourier contour data (PC1-92.6% and PC2-2.61%) indicated that Ma. amazonensis and Ma. titillans are more similar to each other than either is to Ma. humeralis. Discriminant multivariate analysis was highly accurate with only four classification errors and a 90% accuracy rate. The results indicate that the three Amazonian species can be precisely distinguished in the egg stage and that geometric morphometry based on elliptic Fourier contours is a promising method for distinguishing eggs of species of Mansonia. An identification key based on egg morphology is provided to distinguish the four Neotropical species.

The Basic Plan of the Adult Heart Is Conserved Across Different Species of Adult Mosquitoes, But the Morphology of Heart-Associated Tissues Varies.

The heart is a pivotal organ in insects because it performs a number of different tasks, such as circulating nutrients, hormones, and excreta. In this study, the morphologies of the heart and associated tissues, including pericardial cells (PCs) and alary muscles (AMs), in the hematophagous mosquitoes Anopheles aquasalis Curry (Diptera: Culicidae), Aedes aegypti L. (Diptera: Culicidae), and Culex quinquefasciatus Say (Diptera: Culicidae), and the phytophagous Toxorhynchites theobaldi Dyar & Knab (Diptera: Culicidae) were compared using different microscopy techniques. Mosquito hearts are located across the median dorsal region of the whole abdomen. Paired incurrent openings in the heart wall (ostia) are found in the intersegmental regions (segments 2-7) of the abdomen, while an excurrent opening is located in the terminal cone of Ae. aegypti. The sides of the heart contain PC that are more numerous in An. aquasalis and Th. theobaldi. In these two species, PC form a cord of as closely aggregated cells, but in Ae. aegypti and Cx. quinquefasciatus, PC occur in pairs with two or four PC pairs per intersegmental region. In Th. theobaldi, AM binds to all regions of the heart, whereas in other mosquitoes they only bind in the intersegmental regions. The basic plan of the adult heart was conserved across all the adult mosquitoes investigated in this study. This conserved organization was expected because this organ plays an important role in the maintenance of individual homeostasis. However, the species had different PC and of AM morphologies. These morphological differences seem to be related to distinct physiological requirements of mosquito circulatory system.

Scanning electron microscopy of eggs of Georgecraigius fluviatilis (Lutz) (Diptera: Culicidae, Aedini).

Scanning Electron Microscopy was used to describe the eggs of Georgecraigius fluviatilis (Lutz). Length is 722.8±39.6 µm and width is 177.1±9.8 µm. Diameter of the micropylar disk, surrounded by an irregular flattened collar, is 28 µm. The outer chorionic sculpture consists of cells of irregular shapes, containing tubercles with pitted surface. In the ventral region, tubercles of several diameters are irregularly distributed in chorionic cells, while in the dorsal region one larger tubercle is surrounded by several smaller ones. The eggs appear to lack structures for adhesion, certainly unnecessary due to the habit of laying eggs separately on water surfaces.

A new species of mesonivirus from the Northern Territory, Australia.

Here we describe Casuarina virus (CASV), a new virus in the family Mesoniviridae. This is the first report of a mesonivirus in Australia, which extends the geographical range of this virus family to 3 continents. The virus was isolated in 2010 from Coquillettidia xanthogaster mosquitoes during surveillance in the suburbs of Darwin, the capital of the Northern Territory. Cryo-electron microscopy of the CASV virions revealed spherical particles of 65 nm in size with large club-shaped projections of approximately 15 nm in length. The new virus was most closely related to Alphamesonivirus 1, the only currently recognized species in the family. In 2013 a further 5 putative new mesonivirus species were described: Hana, Méno, Nsé, Moumo and Dak Nong viruses. The evolutionary distance between CASV and two of its closest relatives, Cavally and Hana viruses (Jones-Taylor-Thornton distance of 0.151 and 0.224, respectively), along with its isolation from a different genus of mosquitoes captured on a separate continent indicate that CASV is a new species.

Signal-to-noise ratio estimation on SEM images using cubic spline interpolation with Savitzky-Golay smoothing.

A new technique based on cubic spline interpolation with Savitzky-Golay noise reduction filtering is designed to estimate signal-to-noise ratio of scanning electron microscopy (SEM) images. This approach is found to present better result when compared with two existing techniques: nearest neighbourhood and first-order interpolation. When applied to evaluate the quality of SEM images, noise can be eliminated efficiently with optimal choice of scan rate from real-time SEM images, without generating corruption or increasing scanning time.

Scanning electron microscopy of eggs of Sabethes cyaneus.

Mosquitoes of the Neotropical genus Sabethes, some species of which are yellow fever vectors, most often develop through the immature stages in tree holes. Sabethes eggs have not been previously characterized using scanning electron microscopy. Eggs of Sabethes cyaneus (length: 349.6 +/- 2.7 microm; width: 172.6 +/- 1.14 microm; n = 10) are almost biconical when examined from the top. From a lateral perspective 2 surfaces can be seen. One surface is smooth and more convex, whereas the other is less convex and partially covered by a network from which many fungiform tubercles arise. The micropyle is situated on the smooth surface of the pointed anterior tip and is surrounded by an irregular row of tubercles, some of which are leaf shaped. No structures possibly involved in adhesion to surfaces are visible. When hatching, the egg splits dorsoventrally approximately two-thirds of the length from the anterior end. The tubercles appear to be water repellent, and the more convex/smoother surface is downturned, and this position on water was confirmed by direct observation. The eggs float free on the water surface.

[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.

[English and Russian terminology for the thoracic skeletal structures of mosquitoes (Diptera: Culicidae): a critical review].

The authors analyze different names of thoracic skeletal structures in mosquitoes (Dip- tera: Culicidae), used by various authors (Kirkpatrick, 1925; Edwards, 1932; Stackelberg, 1937; Sazonova, 1958; Belkin, 1962a, b: Maslov, 1967: Gutsevich et al., 1970; Knight, Laffoon, 1970; McAlpine, 1981; Becker et al., 2010). In the abovementioned publications, a small number of skeletal structures, morphologically very important for the identification of species and genera of the family Culicidae, are denoted by multiple synonymic names, while certain completely different skeletal structures are represented under a single homonymous name. Duplicated synonymic names for thoracic sclerites, setae, and scales on sclerites considerably hamper the work of specialists determining species of the family Culicidae. In some cases, this fact results in erroneous diagnostics of species. We provide a detailed description of the thoracic morphology of mosquitoes, pointing to existing synonymic Russian and English names for each skeletal structure and also recommend the use of a single Russian term for each structure.

Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae).

The structure of the pyloric armature, hypothesized to aid in blood-meal digestion or parasite resistance, was compared quantitatively among the following 8 species in 5 genera of adult mosquitoes from the southeastern United States: Aedes albopictus, Aedes japonicus, Aedes triseriatus, Anopheles punctipennis, Culex pipiens s.l., Culex restuans, Orthopodomyia signifera, and Toxorhynchites rutilus. Females differed significantly among species in the structure of spines composing the armature, with Aedes spp. forming one general group, Culex spp. another, and An. punctipennis and Or. signifera a third. Relationships of species based on structural characters of the armature were consistent with recent culicid phylogenies. Although pyloric armature has been noted in mosquitoes and other insects, this is the first quantitative investigation of the mosquito pyloric armature.

Mid-Cretaceous charred fossil flowers reveal direct observation of arthropod feeding strategies.

Although plant-arthropod relationships underpin the dramatic rise in diversity and ecological dominance of flowering plants and their associated arthropods, direct observations of such interactions in the fossil record are rare, as these ephemeral moments are difficult to preserve. Three-dimensionally preserved charred remains of Chloranthistemon flowers from the Late Albian to Early Cenomanian of Germany preserve scales of mosquitoes and an oribatid mite with mouthparts inserted into the pollen sac. Mosquitoes, which today are frequent nectar feeders, and the mite were feeding on pollen at the time wildfire consumed the flowers. These findings document directly arthropod feeding strategies and their role in decomposition.

Ultrastructure and morphometry of the egg of Psorophora albigenu Lutz, 1908 (Diptera: Culicidae).

Eggs of Psorophora albigenu were analyzed by light and scanning electron microscopy. Eggs were laid singly and were black and elliptical in outline. A length of approximately 586.4 ± 10.83 µm, a width of 172.3 ± 8.09 µm (n=30), and an egg index (length/width ratio) of 3.33 were observed. The anterior extremity tapered abruptly from a width of 64.9 µm, while such tapering was more gradual at the posterior extremity from a width of 77.4 µm. The ventral surface of the chorionic coating presented cells with tubular aspect containing tubercles in rows with a density of 9-16 tubercles per cell. These tubercles presented two different sizes, with the largest measuring 7.87 ± 01.58 µm and the smallest measuring 0.71 ± 0.53 µm (n=30) in longitudinal diameter. The micropyle orifice was very evident, with a diameter of 3.36 µm. The morphometric characteristics of Ps. albigenu obtained in this study were distinct from those of other species of the Psorophora genus, including Psorophora varipes which has been considered for a long time to be synonymous with Ps. albigenu.

A comparative study of fat body morphology in five mosquito species.

The insect fat body plays major roles in the intermediary metabolism, in the storage and transport of haemolymph compounds and in the innate immunity. Here, the overall structure of the fat body of five species of mosquitoes (Aedes albopictus, Aedes fluviatilis, Culex quinquefasciatus, Anopheles aquasalis and Anopheles darlingi) was compared through light, scanning and transmission electron microscopy. Generally for mosquitoes, the fat body consists of lobes projecting into the haemocoel and is formed by great cell masses consisting of trophocytes and oenocytes. Trophocytes are rich in lipid droplets and protein granules. Interestingly, brown pigment granules, likely ommochromes, were found exclusively in the trophocytes located within the thorax and near the dorsal integument of Anopheles, which is suggestive of the role these cells play in detoxification via ommochrome storage. This study provides a detailed comparative analysis of the fat body in five different mosquito species and represents a significant contribution towards the understanding of the structural-functional relationships associated with this organ.

A comparative study of fat body morphology in five mosquito species

The insect fat body plays major roles in the intermediary metabolism, in the storage and transport of haemolymph compounds and in the innate immunity. Here, the overall structure of the fat body of five species of mosquitoes (Aedes albopictus, Aedes fluviatilis, Culex quinquefasciatus, Anopheles aquasalis and Anopheles darlingi) was compared through light, scanning and transmission electron microscopy. Generally for mosquitoes, the fat body consists of lobes projecting into the haemocoel and is formed by great cell masses consisting of trophocytes and oenocytes. Trophocytes are rich in lipid droplets and protein granules. Interestingly, brown pigment granules, likely ommochromes, were found exclusively in the trophocytes located within the thorax and near the dorsal integument of Anopheles, which is suggestive of the role these cells play in detoxification via ommochrome storage. This study provides a detailed comparative analysis of the fat body in five different mosquito species and represents a significant contribution towards the understanding of the structural-functional relationships associated with this organ.

Armigeres subalbatus (Diptera: Culicidae) prophenoloxidase III is required for mosquito cuticle formation: ultrastructural study on dsRNA-knockdown mosquitoes.

We previously suggested that Armigeres subalbatus (Coquillett) prophenoloxidase III (As-pro-PO III) might be associated with morphogenesis of larvae and pupae. Because PO and its activation system are present in the insect cuticle, and cuticle formation is a major event during pupal morphogenesis, we used ultrastructural analysis to examine the effects of As-pro-PO III knockdown on the formation of pupal and adult cuticle. Inoculation of As-pro-PO III dsRNA resulted in the incomplete formation of nascent pupal endocuticle and pharate adult cuticle, i.e., significantly fewer cuticular lamellae were deposited, the helicoidal pattern of chitin microfibrils was disorganized, and numerous electron-lucent spaces were present in the cuticular protein matrix. Similar disruptions were observed in the cuticle of adults derived from As-pro-PO III dsRNA-inoculated pupae. It has long been suggested that the quinines, generated by PO-catalyzed oxidation reactions, function as cross-linking agents; therefore, it seems reasonable to suggest that the loss of As-pro-PO III-mediated protein-protein linkages causes morphological abnormalities in the protein matrix. Our findings suggest that As-pro-PO III plays a role in cuticle formation in mosquitoes, a novel function for phenol-oxidizing enzymes.

Reducing scanning electron microscope charging by using exponential contrast stretching technique on post-processing images.

An exponential contrast stretching (ECS) technique is developed to reduce the charging effects on scanning electron microscope images. Compared to some of the conventional histogram equalization methods, such as bi-histogram equalization and recursive mean-separate histogram equalization, the proposed ECS method yields better image compensation. Diode sample chips with insulating and conductive surfaces are used as test samples to evaluate the efficiency of the developed algorithm. The algorithm is implemented in software with a frame grabber card, forming the front-end video capture element.

Oomycetes: Lagenidium giganteum.

Lagenidium giganteum is a facultative parasite of mosquito larvae that initiates infection by production of biflagellate zoospores that selectively recognize and attach to larval cuticle. Following penetration of the cuticle, the parasite proliferates within the host, killing it within 24-60 h. Under optimum conditions the mycelia differentiate to produce asexual and/or sexual reproductive structures that produce zoospores within hours (asexual stage) to amplify the initial infection, or remain dormant for days, months or years (sexual stage), until conditions are conducive to mosquito breeding and spore germination. Recycling following a single application has been documented for up to 8-10 years. Environmental conditions that reduce or eliminate zoospore production, including temperature extremes (less than 16 degrees C or greater than 32 degrees C) and moderate levels of salinity and organic load, preclude use of the parasite for operational mosquito control. Three formulations of L. giganteum have been registered with the USEPA. Widespread use of the parasite will be possible when yields of the sexual stage in liquid culture are increased by a factor of ca. 10(2).

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection.

The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.

Mosquito cells infected with Japanese encephalitis virus release slowly-sedimenting hemagglutinin particles in association with intracellular formation of smooth membrane structures.

Arthropod-borne flaviviruses can grow in both arthropod and mammalian cells. Virion morphogenesis, though well studied in mammalian cells, is still unclear in arthropod cells. Here, we compared a mosquito cell line C6/36 and a mammalian cell line Vero in extracellular virus particles and intracellular ultrastructures triggered by infection with Japanese encephalitis virus (JEV). Sedimentation analyses of virion and slowly-sedimenting hemagglutinin (SHA) particles released by infection with the Nakayama strain revealed that C6/36 cells produced higher envelope (E) antigen levels in the SHA than the virion fraction in contrast to Vero cells that showed the opposite pattern. Specific infectivities per ng of E were similar in both cells, whereas specific hemagglutinating activities in the SHA fraction were lower in C6/36 than Vero cells. The precursor membrane protein was less efficiently cleaved to the membrane protein in SHA particles released from C6/36 than Vero cells. Ultrastructural studies showed more remarkable production of smooth membrane structures (SMSs) in C6/36 than in Vero cells. The differences in sedimentation patterns of extracellular virus particles between Nakayama-infected C6/36 and Vero cells were consistently observed in 5 other strains (Beijing P1, Beijing P3, JaTH-160, KE-093 and JaGAr-O1), except for KE-093-infected C6/36 cells which exhibited the Vero-type sedimentation profile under conditions of open cultivation. By electron microscopy, the production of SMSs from KE-093-infected C6/36 cells under open conditions was markedly less than that under closed conditions where the cells exhibited the C6/36-type sedimentation profile. Thus, intracellular SMS formations were associated with extracellular SHA production in JEV-infected mosquito cells.