Morphometric comparison of the midgut epithelial cells in male and female Aedes aegypti L. (Insecta, Diptera).

Midgut epithelial cells of male and female Aedes aegypti, 3 days after emergence, were compared morphometrically. The results of the present investigation concerning the female, are in good agreement with those of a previous study (Hecker et al., 1974), demonstrating that morphometric investigation of midgut epithelia in A. aegypti can successfully be reproduced, and that the mosquito strain used did not show quantitative morphological changes due to laboratory rearing. In males, the cells of the anterior (A) and posterior part (P, 'stomach') of the midgut differ in their quantitative composition. Higher values are found for the microvilli and for the basal labyrinth in the A-part. On the other hand a higher volume density of the mitochondria is present in the P-part. No significant differences are found in the A-part between males and females. Significant differences, however, are present in the P-part. Distinctly more rer in the female stomach can be correlated with the synthesis of enzymes for blood digestion, which are absent in the male. In addition, the more complex functions of the female P-part are also reflected by higher values for other organelles and membrane systems (e.g. mitochondria, basal labyrinth).     

Morphometric parameters of the midgut cells of Aedes aegypti L. (Insecta,Diptera) under various conditions

Summary Previous morphometric or biochemical investigations have yielded different data on the distribution of free and membrane-bound ribosomes in midgut cells of Aedes aegypti. In the present paper ribosomal distribution has been morphometrically analysed to determine whether different mosquito strains, different food and different narcosis used in these previous studies, and/or methodological errors, could account for the different results.Most of the cellular parameters in the stomach epithelium of female A. aegypti, strain Rockefeller, and their changes during blood digestion, are comparable to those measured for another Aedes strain (Segemaganga, Hecker and Rudin 1979), and are generally similar to those of Anopheles stephensi (Hecker 1978). Proteolytic activity against casein is similar for both Aedes strains with a maximum activity being registered around 30 h after a blood meal. During digestion of human serum there is no increase in the ratio of membranebound to free ribosomes, and no significant increase in the surface area of the rough endoplasmic reticulum or of the number of bound or free ribosomes. Proteolytic activity is distinctly lower than during blood digestion. Immobilization of mosquitoes prior to dissection by ether narcosis or by shaking in a test tube has no significant influence on cellular parameters in females fed on sugar solution and investigated 3days after emergence.It is concluded that the differences in ribosomal parameters previously obtained by morphometrical (Hecker and Rudin 1979) and biochemical (Gander et al. 1980) methods, can only partly be explained by the selection of different food for the mosquitoes, and must also have been caused by methodological inadequacies.

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Zusammenfassung Frühere morphometrische und biochemische Untersuchungen erbrachten teilweise unterschiedliche Resultate betreffend Verteilung freier und membrangebundener Ribosomen in Mitteldarmzellen von Aedes aegypti. In der vorliegenden Arbeit wurde morphometrisch untersucht, ob diese Unterschiede bedingt waren durch die Verwendung verschiedener Mückenstämme, unterschiedlichen Futters und verschiedener Narkosemethoden durch die beiden Arbeitsgruppen, oder ob methodische Einflüsse dafür verantwortlich waren.Die meisten Zellparameter im Magenepithel von A. aegypti, Stamm Rockefeller, wie auch ihre Änderungen während der Verdauung eines Blutmahls, entsprachen den für einen andern Aedes-Stamm (Segemaganga, Hecker und Rudin 1979) gemessenen Werten und stimmten im allgemeinen mit denjenigen für Anopheles stephensi (Hecker 1978) überein. Die proteolytische Aktivität gegen Casein war bei beiden Stämmen gleich mit einem Aktivitäts-Maximum um 30h nach Blutmahl. Bei der Verdauung von menschlichem Serum konnte keine Zunahme des Verhältnisses von membrangebundenen zu freien Ribosomen, keine signifikante Oberflächenvergrößerung des rauhen endoplasmatischen Retikulums und keine signifikante Erhöhung der Zahl gebundener und freier Ribosomen gemessen werden. Die Proteaseaktivität war deutlich schwächer als während der Verdauung von Blut. Betäubung der Mücken vor der Sektion mit Aether oder durch Schütteln in Reagenzgläsern ergab im Vergleich keinen signifikanten Einfluß auf die Zellparameter von Zuckerwasser-gefütterten Weibchen, die drei Tage nach dem Schlüpfen untersucht wurden.Unterschiede in den Ribosomenparametern, die mit morphometrischen Methoden (Hecker und Rudin 1979) einerseits und biochemischen (Gander et al. 1980) andererseits untersucht wurden, konnten nur teilweise durch die Wahl unterschiedlichen Futters für die Mücken durch die beiden Arbeitsgruppen erklärt werden. Es müssen zusätzlich methodische Einflüsse für diese Unterschiede verantwortlich sein.

     

Functional morphology of the midgut of Aedes aegypti L. (Insecta,Diptera) during blood digestion

Morphometric analysis of the epithelial lining of the stomach of A. aegypti suggests that digestion of the first blood meal in the stomach of this species can be viewed as a series of phases that can be correlated with physiological data from the literature. In phase Ia (0-10 h after blood meal [abm]) the whorls of the rough endoplasmic reticulum unfold, the Golgi zones increase, and the basal labyrinth is enlarged. This coincides with processes of synthesis and secretion (e.g., peritrophic membrane, esterases and lipases) and transport by the stomach epithelium. In phase Ib (10-20 habm) the cellular parameters measured further increase, indicating high synthetic and secretory activities (e.g., digestive enzymes). In phase Ic (20-30 habm) cell structures involved in synthesis and secretion still exhibit high values coinciding with maximal activity of proteases in the gut. Enhanced surface area of microvilli, prominent lipid inclusions, and appearance of glycogen deposits in the gut epithelium suggest increased absorption, storage, and transport functions of the stomach cells. In phase II (30-36 habm) structural alteration points to a gradual shift from synthesis and secretion to absorption, partial storage, and transport of nutrients. In phase III (36-72 habm) the cellular apparatus is reduced concomitant with the ending of the digestive cycle. Lipid inclusions and glycogen deposits disappear from the stomach epithelium.     

Normal versus alpha-amanitin induced cellular dynamics of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera) in response to blood feeding.

In midgut epithelial cells (stomach) of untreated female A. aegypti an increase in the surface area of the rough endoplasmic reticulum (rer) and in the ratio of membrane-bound to free ribosomes is morphometrically measured during digestion of the first blood meal. This can be correlated with the synthesis and release of digestive proteases. The dynamics of the ribosomes in A. aegypti are similar to those in A. stephensi. 3 ng alpha-amanitin per mosquito prevent normal blood digestion, the proliferation of the rer and the increase in the ratio of bound to free ribosomes. On the other hand, some synthesis of new ribosomes takes place.     

Variability in the flight muscles of field and laboratory strains of the mosquito,Aedes aegypti (L.) (Diptera : Culidae)

The distribution of indirect flight muscle fibres was examined in histological sections of females from 3 unrelated field populations of Aedes aegypti (L.) (Diptera : Culicidae) and compared with that in an inbred laboratory strain. The number of muscle fibres, the frequency of branching of fibres and the degree of bilateral asymmetry were very similar in all 3 field populations. By comparison, the number of muscle fibres in the laboratory strain was more variable and the degree of branching and asymmetry greater. This suggests that in nature the flight muscles are subject to stabilizing selection for an optimal phenotype.     

Fine structure of tarsal sensilla of Aedes aegypti (L.) (Diptera: Culicidae)

The tarsi of all three pairs of legs of both sexes of Aedes aegypti (L.) bear spine sensilla, five types of hair sensilla, which are designated A, B, C1, C2 and C3, and campaniform sensilla. Type A and B hairs, spines, and cam-paniform sensilla are innervated by one neuron with a tubular body, a characteristic of cuticular mechanoreceptors. In particular the hairs and spines are tactile receptors and the campaniform sensilla are proprioceptors. The C1, C2, and C3 hair sensilla have the morphological features of contact chemoreceptors. Type C1 and C3 hairs are innervated by five and four neurons, respectively, which extend to the tip of the hair. Type C2 is innervated by five neurons, one of which terminates at the base of the hair in a tubular body while the remaining four extend to the tip of the hair. The role of the type C hairs in oviposition behavior, nectar feeding, and recognition of conspecific females is discussed. Presumed efferent neurosecretory fibers occur near the spine and hair sensilla.     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

Development and ageing of the salivary glands of adult female Aedes aegypti (L.) and Aedes togoi (Theobald) mosquitoes (Diptera : Culicidae)

Histological sections of the salivary glands of adult female Aedes aegypti and Aedes togoi (Diptera : Culicidae), varying in age from less than 1 day to 48 days after emergence were examined by light microscopy. Following emergence, the glands underwent morphological differentiation and also enlarged as secretory product accumulated, especially in the posterior regions. Development was complete within about 3 days (A. aegypti) or 7 days (A. togoi). Ageing, which began between 10 and 15 days after emergence in both species, predominantly affected the posterior regions of the glands. Regression, involving loss of stored secretion and cytological evidence of reduced protein synthesis, was accompanied by degenerative changes including substantial cytoplasmic vacuolation and increased prominence of large cyst-like structures. Although, in general, both regression and degeneration increased with time, there was marked variation between one gland and another in the speed with which ageing changes progressed.     

Secretion in the male accessory glands of Aedes aegypti (L.) (Diptera : Culicidae)

The objective of this work is to determine the nature, the mode of synthesis, and release of the secretion of accessory glands of the male mosquito, Aedes aegypti (Diptera : Culicidae). Cytological studies revealed that each of the 2 glands has 2 morphological types of regionally specialized secretory cells, each type differing in the nature, and the release of its products. The anterior region (AR) cells secrete small (0.3–1.3 μm) electron-lucent granules, which during the peak period of synthesis seemed to transport the flocculent and fibrous granular substance to the apical cytoplasm of the cell by means of binary fusions. This cytoplasm, when filled with secretory granules, pinches off and falls freely into the lumen of the gland, thus, exhibiting a macroapocrine mode of secretion. The posterior region (PR) cells, however, elaborate large (mean diameter 2 μm) electron-dense granules, which are released into the lumen essentially by rupture of the cell membrane at the apical end. In males mated to depletion, the volume of the gland is reduced by 67%; there is no subsequent recovery of secretory capacity.     

Calibration and evaluation of field cage for oviposition study with Aedes (Stegomyia) aegypti female (L.) (Diptera: Culicidae)

Differences among results gathered from insect behavior studies conducted in laboratory and field situations are due to ambient variables that differ greatly between both environments. In laboratory studies the environmental conditions can be controlled whereas in field temperature, humidity and air velocity vary uncontrollably. The objective of this study was to calibrate and evaluate an experimental area (field cage) (14 x 7 x 3.5 m) subdivided into eight test cages (2.5 x 2.5 x 2 m) for use in behavioral oviposition tests of Aedes aegypti (L.) mosquitoes for developing a new methodology to assess attractants and oviposition traps. Test cage calibration involved: (1) minimal experiment duration tests; (2) optimal female release number per traps test and (3) trap placement tests. All tests used gravid A. aegypti females; 3-4 days post blood meal and the sticky trap MosquiTRAP to catch adults. Ninety percent of the females released were recaptured 2h after the beginning of the experiment, and this allowed up to 32 test repetitions/day to be conducted in the field cage. The minimum number of females necessary to conduct statistical analyses was 20 females/trap/test per cage. No significant difference was found in the behavioral response of gravid females to four different trap positions within test cages. Field trapping results with attractant were similar to those in the field cage. Therefore, the field cage could replace field trapping for evaluating at least mosquito traps and oviposition attractants for A. aegypti.     

Dynamics and Characterization of Aedes aegypti (L.) (Diptera: Culicidae) Key Breeding Sites

The present study aimed to analyze the dynamics of containers used as breeding sites by Aedes aegypti (L.) in the city of Aracaju, SE, one of the Northeast Brazilian states. A total of three entomological surveys were performed during different precipitation levels. Breeding sites were categorized according to their function into storage, disposable containers, and reusable containers. “Mean number of pupae” and “frequency of each type of breeding site” were the criteria considered to identify key breeding sites. House index and Breteau index were calculated in each survey. A total of 3,647 water reservoirs were found, of which 220 were breeding sites, where 22,880 immature forms were identified. There were no differences in the mean number of larvae of several types of breeding sites and in the number of larvae among surveys. Larval indices showed a reduction in the second visit, but with no effect on adult occurrence when the number of pupae was considered. Key breeding sites resulted from containers used for water storage. The area studied showed conditions favorable to a new epidemic of dengue fever.     

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae).

Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P. sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage, while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides used for mosquito control.