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.

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.

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.

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.

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.

Estudo taxonômico sobre Anopheles (Nyssorhynchus) strodei Root (Diptera: Culicidae): uma abordagem morfológica e molecular / Taxonomic studies on Anopheles (Nyssorhynchus) strodei Root (Diptera: Culicidae) a morphological and molecular approach

Estudo taxonômico sobre Anopheles (Nyssorhynchusy strodei Root (Diptera: Culicidae). Uma abordagem morfológica e molecular. 2010. 139p. Tese de Livre docência. Faculdade de Saúde Pública da Universidade de São Paulo. Anopheles albertoi Unti e Anopheles arthuri Unti são retiradas da sinonímia com Anopheles strodei Root, e uma forma morfologicamente distinta, adiante designada Anopheles CP, do Complexo Strodei de Anopheles (Nyssorhynchus) é caracterizada. As genitálias masculinas de An. arthuri e An. albertoi são descritas e ilustradas pela primeira vez. Anopheles strodei, An. arthuri e An. albertoi foram, inicialmente, separadas com base em imagens dos ovos, obtidas em microscópio eletrônico de varredura e, em seguida, cada tipo de ovo foi associado com caracteres diagnósticos da genitália masculina. A identificação de Anopheles CP foi baseada em caracteres morfológicos da genitália masculina, caracterizados e ilustrados no presente trabalho. Os resultados das análises filo genéticas, utilizando dados de seqüências gênicas, foram mais claros sem a inclusão de grupos externos. Neste caso, utilizando tanto os dados do gene nuclear White, como do gene White combinado com o gene mitocondrial COI, as quatro espécies incluídas no estudo foram, claramente, separadas. Quando Anopheles quadrimaculatus Say e Anopheles stephensi Liston foram incluídas como grupos externos, os dados combinados dos genes White e COI recuperaram o monofiletismo de An. strodei e An. albertoi, enquanto a posição taxonômica de An. arthuri não foi bem resolvida. A única seqüência de Anopheles CP apareceu separada dos outros grupos, em todas as análises. A análise Bayesiana dos dados do ITS2 e aquelas realizadas utilizando informações das estruturas secundárias, empregando as estratégias de bootstrap em distância e o "profile neighbor joining", ambas implementadas no programa ProfDistS, demostraram o monofiletismo de An. albertoi, An. arthuri e Anopheles CP.

Morphological aspect of the midgut of anopheles aquasalis (Curry, 1932) Insecta: Diptera / Aspecto morfológico del intestino medio de anopheles aquasalis (Curry, 1932) Insecta: Diptera

The midgut of adult female Anopheles aquasalis presents a narrow anterior or thoracic region and a distensible posterior or abdominal region constituted by the epithelium formed by a cell layer whose apical portion presents microvilli and the basal portion, a basal labyrinth. The thoracic region revealed heterogeneous cellular staining affinity mainly by the presence of acidic components. The ultrastructural aspect showed columnar cells with the presence of the vesicle, mitochondria, endoplasmic reticulum and secreting cells. The abdominal region of the midgut revealed an irregular epithelium whose cells presented a basophilic cytoplasm and acidophil granules. It was also found secreting and/or basal cells with narrow cytoplasm. The ultrastructural observation of this region demonstrated cells with evident nucleus, endoplasmic reticulum and mitochondria. Larger vesicles and small granules were found distributed throughout the cytoplasm. The basal lamina that supports the epithelium presented a generally irregular aspect and the muscle fibers have longitudinal and circular organization and were found separating the epithelium from the haemocel. This study will contribute to analyses on the vector mosquito-parasite interaction mechanism in this specimen.

La seccion media del intestino de la hembra de Anopheles aquasalis presenta una estrecha region anterior o toráxica y una region posterior o abdominal constituida por el epitelio formado por una camada de células cuya porcion apical presenta microvilosidades y la porcion basal presenta un laberinto basal. La region toráxica reveló afinidad de tintura celular principalmente para componentes acídicos. El aspecto ultra estructural mostró células columnares con la presencia de la vesícula, mitocondrias, retículo endoplasmático y células secretoras. La region abdominal del intestino medio reveló un epitelio irregular con células con citoplasma basófilo y granulos acidófilos. También se encontraron células secretoras y/o básales con citoplasma estrecho. La observacion ultra estructural de la region mostró células con núcleos, retículo endoplasmático y mitocondrias evidentes. Vesículas largas y granulos pequeños fueron encontrados distribuidos por todo el citoplasma. La lámina basal que apoya el epitelio presentó un aspecto irregular y las fibras musculares tienen organizacion longitudinal y circular y separan el epitelio del hemocele. Este estudio contribuirá al análisis del mecanismo de interaccion entre el mosquito y el parásito en este espécimen.

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis.

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope.

Comparative morphometric and morphological studies of eggs under scanning electron microscope (SEM) were undertaken in the three strains of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and Form C (Chiang Mai and Mae Hong Son strains). Morphometric examination revealed the intraspecific variation with respect to the float width [36.77 +/- 2.30 microm (Form C: Chiang Mai strain) = 38.49 +/- 2.78 microm (Form B: Chiang Mai strain) = 39.06 +/- 2.37 microm (Form B: Phet Buri strain) > 32.40 +/- 3.52 microm (Form C: Mae Hong Son strain)] and number of posterior tubercles on deck [2.40 +/- 0.52 (Form B: Phet Buri strain) = 2.70 +/- 0.82 (Form B: Chiang Mai strain) < 3.10 +/- 0.32 (Form C: Chiang Mai strain) = 3.20 +/- 0.42 (Form C: Mae Hong Son strain)], whereas the surface topography of eggs among the three strains of two karyotypic forms were morphologically similar.

Ultrastructure of the eggs of two species of Anopheles (Anopheles) Meigen (Diptera, Culicidae)

Ultraestrutura dos ovos de duas espécies de Anopheles (Anopheles) Meigen, 1818 (Diptera, Culicidae). A ultraestrutura dos ovos de duas espécies morfologicamente semelhantes da Série Arribalzagia, Anopheles (A.) costai Fonseca & Ramos, 1939 and A. (A.) mediopunctatus (Lutz, 1903), é descrita e ilustrada usando-se microscopia eletrônica de varredura.. Embora sejam morfologicamente semelhantes nos estádios adulto, estruturas da genitalia masculina, larva de quarto estádio e pupa, os ovos destas duas especies são distintos. Os ovos de A. costai e A. mediopunctatus são comparados com os de A. forattinii Wilkerson & Sallum, 1999. Apresenta-se a ilustração incompleta do ovo de A. forattinii para comparação com os ovos das outras duas espécies.

Interaction of insect trypanosomatids with mosquitoes, sand fly and the respective insect cell lines.

Interaction experiments between hematophagous insects and monoxenous trypanosomatids have become relevant, once cases of human infection involving these protozoa have been reported. Moreover, investigations related to the interaction of insects with trypanosomatids that harbour an endosymbiotic bacterium and thereby lack the paraflagellar rod structure are important to elucidate the role of this structure in the adhesion process. In this work, we compared the interaction of endosymbiont-bearing trypanosomatids and their aposymbiotic counterpart strains (without endosymbionts) with cell lines of Anopheles gambiae, Aedes albopictus and Lutzomyia longipalpis and with explanted guts of the respective insects. Endosymbiont-bearing strains interacted better with insect cells and guts when compared with aposymbiotic strains. In vitro binding assays revealed that the trypanosomatids interacted with the gut epithelial cells via flagellum and cell body. Flagella attached to the insect gut were enlarged, containing electrondense filaments between the axoneme and flagellar membrane at the point of adhesion. Interactions involving the flagellum lacking paraflagellar rod structure were mainly observed close to tight junctions, between epithelial cells. Endosymbiont-bearing trypanosomatids were able to colonise Aedes aegypti guts after protozoa feeding.

Anopheles albitarsis embryogenesis: morphological identification of major events.

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180 degrees rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

The eggs of Anopheles galvaoi and Anopheles evansae, two species of the subgenus Nyssorhynchus.

The ultrastructure of the eggs of Anopheles (Nyssorhynchus) galvaoi Causey, Deane, and Deane and Anopheles (Nyssorhynchus) evansae (Brethes) are described and illustrated with scanning electron micrographs. The eggs of these species are similar to those of Anopheles (Nyssorhynchus) aquasalis Curry, Anopheles (Nyssorhynchus) oswaldoi (Peryassu), and Anopheles (Nyssorhynchus) konderi Galvão and Damasceno in having floats long, widely joined posteriorly on the dorsal surface, the frill encircling the anterior end of the egg, and the crown absent. A few distinctive characters to distinguish An. evansae from An. galvaoi are given.

Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.

Taxonomic study of species formerly identified as Anopheles mediopunctatus and resurrection of An. costai (Diptera: Culicidae).

Anopheles (Anopheles) mediopunctatus (Lutz) and Anopheles (Anopheles) costai Fonseca & Ramos are redescribed with illustrations of the male genitalia and larval and pupal stages. The pupa of An. costai has paired lateral projections on the wing case, a feature also known in members of the Umbrosus Group from Southeast Asia. An. costai is resurrected from the synonymy of An. mediopunctatus based on features of the male genitalia, larva, and pupa, and An. bonneorum Fonseca & Ramos (emended from bonnei) is considered to be a new synonym of An. costai. It is noted that the author of An. mediopunctatus is Lutz, not Theobald, as cited in most literature references.

Ultrastructure of eggs of Anopheles rondoni, Anopheles lutzii, and Anopheles parvus, three species of the subgenus Nyssorhynchus.

The ultrastructures of the eggs of Anopheles (Nyssorhynchus) rondoni (Neiva & Pinto), Anopheles (Nyssorhynchus) lutzii Cruz, and Anopheles (Nyssorhynchus) parvus (Chagas) are described and illustrated with scanning electron micrographs. The egg of Anopheles rondoni is similar in several respects to those of other species of the Argyritarsis Section. The egg of An. lutzii is similar to that of Anopheles antunesi Galvão and Amaral in having floats widely joined anteriorly on the ventral side, and the anterior end barely visible beyond the floats. The egg of An. parvus is remarkable in possessing an anterior fingerlike structure that bears several lobed tubercles at the apex. The fingerlike structure and the micropyle are within the prominent anterior crown formed by the frill. The egg of An. parvus has floats with the anterior pole uppermost, which is an unusual position for Anopheles.

Description of the egg of Anopheles (Anopheles) intermedius (Peryassu, 1908) (Diptera: culicidae) by scanning electron microscopy

Os ovos de An. intermedius foram descritos e ilustrados por Costa Lima (1929). Este autor, baseando-se nos desenhos de Peryassu (1908) para An. maculipes, chamou atencao para o fato do ovo desta especie ser semelhante ao de An. maculipes. Posteriormente, Causey et al. (1944), estudando os ovos de An. intermedius e An. maculipes ao estereomicroscopio, diferenciou-os por caracteres da franja...

Estudo exocorial de Anopheles (Nyssorhynchus) albitarsis s.l. com a utilizaçäo de microscópio eletrônico de varredura e sistema de análise de imagens / Chorionic sculpture of Anopheles (Nyssorhynchus) albitarsis s.l. with the utilization of the scanning electron microscope and the image analysis system

Coleta fêmeas de Anopheles (Nyssorhynchus) albitarsis s.l. do Vale do Ribeira, Estado de Säo Paulo. Identifica as populaçöes A e B, mede seus ovos, fotografa seus exocórios no microscópio eletrônico de varredura e as fotografias foram analisadas utilizando-se o sistema especialista BioScan OPTIMAS©, v. 4.10, para análise de imagens em microcomputador padräo IBM-PC, de modo a identificar possíveis diferenças entre os exocórios das referidas populaçöes. Demonstra haver diferença estatisticamente significante quanto à largura e à razäo comprimento/largura dos ovos analisados. Entretanto, o estudo do aspecto exocorial no microscópio eletrônico de varredura e no sistema de análise de imagens näo apresentou dados que levassem à diferenciaçäo das populaçöes

Scanning electron microscopic observations of Anopheles albimanus (Diptera: Culicidae) eggs.

To investigate the existence of subspecies of Anopheles albimanus Wiedeman in southern Mexico, the egg morphology of specimens obtained from several field populations and from insectary-adapted colonies of uniform pupal phenotype was examined. Scanning electron microscopic observations have shown that the eggs of An. albimanus are polymorphic in respect to the size and shape of their floats, but not in their ornamentation. Four types of eggs were found. Differences in the proportion of the various morphological types were statistically significant, although proportions of egg types were variable among individuals within the same population. These observations are suggestive of distinctive populations and warrant further studies using more sensitive methods to investigate sibling species in An. albimanus sensu lato.