Low impact to fixed cell processing aiming transmission electron microscopy.

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.

Morphological Differentiation of the Eggs of North American Container-Inhabiting Aedes Mosquitoes.

Aedes aegypti , Ae. albopictus, Ae. japonicus japonicus, and Ae. triseriatus eggs are described with the aid of color images and images from scanning electron microscopy. All eggs were broadly cigar shaped with Ae. triseriatus and Ae. j japonicus eggs being dull or matte black, while Ae. albopictus and Ae. aegypti eggs were shiny jet black. Aedes triseriatus eggs were lighter in color and had a rougher appearance when compared to those of Ae. j. japonicus. Aedes albopictus and Ae. aegypti eggs can be differentiated by the distinct presence of micropylar collars in Ae. aegypti. Our results reduce the need to rear these mosquitoes, reducing labor and time while providing an accurate identification of the vector mosquitoes.

Cytotoxicity of piperamides towards Aedes aegypti (Diptera: Culicidae).

The effectiveness of the amides piplartine and piperlonguminine isolated from Piper species for controlling L3 and L4 of Aedes aegypti (L.) was assessed through bioassays at concentrations ranging from 1 to 300 g/l ml. Piplartine reduced the mosquito development period and caused larval mortality only at concentrations > 100 microg/ml, whereas piperlonguminine resulted in an extended period of mosquito development (10 microg/ml) and caused 100% larval mortality (30 microg/ml) within 24 h. The toxicity and cytotoxic effects of piperlonguminine on epithelial cells of the digestive system of Ae. aegypti were viewed using transmission electron microscopy, which indicated vacuolization of cytoplasm, mitochondrial swelling and leaking of nuclear material. Piperlonguminine was the more effective amide, showing toxic activity with LD50 of approximately 12 microg/ml against the larvae of Ae. aegypti.

[The first finding of invasive species Aedes (Finlaya) koreicus (Edwards, 1917) in European Russia].

For the first time mosquito Aedes koreicus were identified by morphology and molecular genetics in the south of European Russia (Sochi city) in 2013.

Behavioral insensitivity to DEET in Aedes aegypti is a genetically determined trait residing in changes in sensillum function.

N,N-Diethyl-m-toluamide (DEET) is one of the most effective and commonly used mosquito repellents. However, during laboratory trials a small proportion of mosquitoes are still attracted by human odors despite the presence of DEET. In this study behavioral assays identified Aedes aegypti females that were insensitive to DEET, and the selection of either sensitive or insensitive groups of females with males of unknown sensitivity over several generations resulted in two populations with different proportions of insensitive females. Crossing experiments showed the "insensitivity" trait to be dominant. Electroantennography showed a reduced response to DEET in the selected insensitive line compared with the selected sensitive line, and single sensillum recordings identified DEET-sensitive sensilla that were nonresponders in the insensitive line. This study suggests that behavioral insensitivity to DEET in A. aegypti is a genetically determined dominant trait and resides in changes in sensillum function.

Using adult mosquitoes to transfer insecticides to Aedes aegypti larval habitats.

Vector control is a key means of combating mosquito-borne diseases and the only tool available for tackling the transmission of dengue, a disease for which no vaccine, prophylaxis, or therapeutant currently exists. The most effective mosquito control methods include a variety of insecticidal tools that target adults or juveniles. Their successful implementation depends on impacting the largest proportion of the vector population possible. We demonstrate a control strategy that dramatically improves the efficiency with which high coverage of aquatic mosquito habitats can be achieved. The method exploits adult mosquitoes as vehicles of insecticide transfer by harnessing their fundamental behaviors to disseminate a juvenile hormone analogue (JHA) between resting and oviposition sites. A series of field trials undertaken in an Amazon city (Iquitos, Peru) showed that the placement of JHA dissemination stations in just 3-5% of the available resting area resulted in almost complete coverage of sentinel aquatic habitats. More than control mortality occurred in 95-100% of the larval cohorts of Aedes aegypti developing at those sites. Overall reductions in adult emergence of 42-98% were achieved during the trials. A deterministic simulation model predicts amplifications in coverage consistent with our observations and highlights the importance of the residual activity of the insecticide for this technique.

Morphological and morphometrical assessment of spermathecae of Aedes aegypti females.

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.

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.

Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues.

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.

Larvicidal activity, route of interaction and ultrastructural changes in Aedes aegypti exposed to entomopathogenic fungi.

Blastospores or conidia (formulated or not) of entomopathogenic fungi were assessed against Aedes aegypti larvae. Larvae (L2) were exposed to 105, 106, 107, and 108 propagules mL-1 water suspension. Mineral oil at 0.1%, 0.5%, or 1.0% (v/v) was employed to observe the effect on larval survival. The 0.1% mineral oil did not affect larval survival. Accordingly, 107 propagules mL-1 and 0.1% mineral oil were used to prepare all fungal emulsions. The fungal suspension or formulation was prepared as follows: 107 propagules mL-1 on 0.03% TweenⓇ 80 (v/v) aqueous solution or 107 propagules mL-1 on 0.03% TweenⓇ 80 plus 0.1% mineral oil; larval survival rates were evaluated for 7 days, and median survival time (S50) was also determined. The presence of fungi in larvae was examined both histologically and by scanning electron microscopy 24 h or 48 h after exposure. To evaluate the larval growth, larvae were exposed to 107 propagules mL-1 for 48 hours and their length measured using a digital caliper. Here, propagules had similar results in reducing the larvae survival rate and time. The treatment with Beauveria bassiana s.l. at 108 propagules mL-1 or with Metarhizium anisopliae s.l. at 108 blastopores mL-1 reduced the larval survival time to two days. M. anisopliae s.l. at 108 conidia mL-1 reduced the survival time to three days. The survival time of larvae submitted to the other treatments ranged from 6 days to over 7 days. M. anisopliae s.l. or B. bassiana s.l. oil-in-water emulsions at 107 propagules mL-1 yielded better results than the water suspensions, the larvae survival rate was 2 days for both propagules in oil-in-water emulsion. Larvae exposed to blastospores from both isolates or M. anisopliae conidia were longer than in the other treatments. Scanning electron microscopy and histology analyzes found fungi predominantly in the gut, mouthparts, and perispiracular lobes of larvae. Formulated fungus yielded better results than the aqueous suspensions for control of mosquito larvae. Thus, for the first time, the effect of mineral oil on the fungal interaction on A. aegypti larvae was observed as well as the effect of entomopathogenic fungi in the growth of larvae, supporting the search for strategies to control this arthropod.

Scanning electron microscopy of the eggs of Aedes scapularis from southern South America.

The eggs of Aedes scapularis analyzed by scanning electron microscopy are black and elliptical in outline, measuring approximately 620.4 +/- 16.74 microm long and 163.7 +/- 16.90 microm (n = 10) wide, with an egg index (length/width ratio) of 3.79. The anterior extremity tapered abruptly from a width of 51.6 microm, while such tapering was more gradual at the posterior extremity, from a width of 61.4 microm. The ventral surface of the chorionic coating presented cells with a tubular aspect containing tubercles in rows at a density of 5 to 9 per cell with 2 different sizes, the largest measuring 7.23 +/- 0.98 microm in a longitudinal diameter and the smallest 4.15 +/- 0.53 microm (n = 30). In the dorsal region, the external chorionic reticulum had a porous appearance, and its thickness ranged from 2.5 to 4.1 microm. Isolated tubercles presented wide variation per cell. In the central region of some chorionic cells were tubercles of greater diameter, characterized as central tubercles of 8.45 +/- 0.67 microm, and around them 3 to 5 smaller tubercles measuring 2.57 +/- 0.26 microm. The micropylar apparatus presented a collar with a very evident molding and edges with defined margins for the transition area and a thickness of around 11.1 microm. The micropyle disc margins were raised, measuring around 17.8 microm in diameter and 229 microm in circumference. The micropyle orifice was very evident, with a diameter of 1.41 microm.

Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential.

The recent Zika virus (ZIKV) and chikungunya virus epidemics highlight the explosive nature of arthropod-borne viruses (arboviruses) transmitted by Aedes spp. mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious blood meal and temporally sampling mosquitoes to determine the infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behaviour of many mosquito vectors that refeed frequently (every 2-3 d)4. Here, we demonstrate that acquisition of a second non-infectious blood meal significantly shortens the EIP of ZIKV-infected Aedes aegypti by enhancing virus dissemination from the mosquito midgut. Similarly, a second blood meal increases the competence of this species for dengue virus and chikungunya virus as well as Aedes albopictus for ZIKV, suggesting that this phenomenon may be common among other virus-vector pairings and that A. albopictus might be a more important vector than once thought. Blood-meal-induced microperforations in the virus-impenetrable basal lamina that surrounds the midgut provide a mechanism for enhanced virus escape. Modelling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0, estimated from experimental data. This helps to explain how A. aegypti can sustain explosive epidemics such as ZIKV despite relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behaviour, EIP and vector competence.

Surface morphology and morphometric analysis of sensilla of Asian tiger mosquito, Aedes albopictus (Skuse): an SEM investigation.

BACKGROUND & OBJECTIVES: The sensilla and sensory mechanism play a significant role in hostseeking and oviposition behaviour of mosquitoes, which enable them to transmit various diseases to humans. Aedes albopictus (Skuse) has emerged as a major vector of Chikungunya virus in the recent epidemics in most parts of southern India. Studies on the sensory structures of dengue vector, Aedes aegypti (Linn) are comprehensive; whereas information on the sensillary systems of Asian tiger mosquito, Ae. albopictus is inadequate. Therefore, the present study has been carried out to observe various types of sensilla located on the antenna, maxillary palp, labial palp, tarsi and ovipositor of Ae. albopictus using scanning electron microscopy. METHODS: The antennae, maxillary palpi, labellum, tarsi and ovipositor of 10 different female mosquito of Ae. albopictus were fixed individually in 2.5% glutaraldehyde solution, washed twice and dehydrated with ascending grades of ethanol. Samples were cleared with xylene, air-dried, mounted on stubs, gold coated in an ion-sputtering unit and the sensilla were viewed between 5 and 10 KV using FEI-Quanta 400-EDAX scanning electron microscope. ANOVA revealed significant differences in the morphometric features of various sensilla. RESULTS: In the antenna Sensilla trichoidea are numerously distributed in all flagellar segments revealed four distinct subtypes. Two types of grooved peg sensilla were observed. Sensilla coeloconica was observed in the terminal flagellum of antenna and tarsomeres with large variation in diameter. Sensilla chaetica are distributed throughout the body and revealed greater variation in morphology and morphometric parameters. INTERPRETATION & CONCLUSION: The significant difference among various types of sensilla would possibly reveal their functions. The porous sensilla are olfactory and contact chemoreceptors while the aporous sensilla would play the role of mechanoreception. Sensilla coeloconica on the antenna, tarsus showed major differences with Ae. aegypti. The ovipositor sensilla revealed three types of chaetica arranged in rows but has not been reported earlier with other mosquito species.

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus.

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99-100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.

Structural changes in fat body of Aedes aegypti caused by aging and blood feeding.

The fat body is the intermediary metabolism organ of insects and the main source of hemolymph components. In the current study, the microanatomy of Aedes aegypti (L., 1762) fat body was studied through scanning electron microscopy to observe the effects of blood feeding and aging. Three groups of female mosquitoes were used: newly emerged females, 18-d-old sugar-fed females, and 18-d-old blood-fed females. In Ae. aegypti, the fat body is located beneath the integument, and it is subdivided into dorsal, ventral, and lateral lobes, with the latter two being larger than the dorsal lobes. The lobes projected into the body cavity, and they were covered externally by a basal lamina with rounded cells beneath it. In 18-d-old sugar-fed females, the ventral and dorsal fat bodies seemed more developed than in newly emerged mosquitoes. The fat body hypertrophy caused by aging in the sugar-fed mosquito was probably associated with lipid accumulation due to the sugar diet. The blood-fed 18-d-old mosquitoes showed flattened fat bodies in all locations. The fat body modifications after the blood ingestion may be associated with midgut expansion after blood feeding, followed by ovary hypertrophy that mechanically compresses the fat body against the body wall. The structural changes in the fat body after a bloodmeal may be important for midgut extension to maximize blood storage and subsequent ovary enlargement, leading to the organ's reorganization in the body cavity. In addition, the depletion of fat body content during vitellogenesis could be responsible for the shrinking and flattening of the fat body lobes.

Hemocytes ultrastructure of Aedes aegypti (Diptera: Culicidae).

Mosquitoes have an efficient defence system against infection. Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. We have identified by light and transmission electron microscopy six hemocytes cell types from the hemolymph of Aedes aegypti. They were: prohemocytes (20%), adipohemocytes (29%), granulocytes (16%), plasmatocytes (27%), oenocytoids (7%) and thrombocytoids (0.9%). The prohemocytes were the smallest hemocytes found in the hemolymph. Its cytoplasm occupies only a narrow area around the nucleus. The adipohemocytes were the most abundant cell type presented. These hemocytes exhibited a large lipid like vesicle and mitochondria. In electron micrographs, the granulocytes showed cytoplasm containing dilated rough endoplasmic reticulum (RER) and a round or elongated mitochondria. Electron-dense granules with a proteinaceous material were also present. The plasmatocytes were polymorphic and exhibited plasma membrane with irregular processes, philopodia and pseudopodia. Ultrastructural investigation revealed that the reticular cytoplasm showed a well-developed RER, a Golgi and vacuoles. Oenocytoids showed homogeneous cytoplasm with many mitochondria and ribosomes are scattered throughout the cytoplasm, abundant RER and a small smooth endoplasmic reticulum (SER) present at the cell poles. Thrombocytoids were very fragile and few in number. Similar characteristics were found in oenocytoids, possessing a homogeneous cytoplasm with poorly developed organelles, few mitochondria and granules.

Ultrastructure and morphology of midgut visceral muscle in early pupal Aedes aegypti mosquitoes.

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.

[Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae)]. / Morfología y citoquímica de cultivos celulares de Aedes aegypti (Diptera: Culicidae) y susceptibilidad a Leishmania panamensis (Kinetoplastida: Trypanosomatidae).

Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04 +/- 4.00 microm in length and 13.96 3.70 microm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08% of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90%) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week.

Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor.

BACKGROUND: Mosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni. METHODS: In this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin. RESULTS: Calcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa. CONCLUSIONS: Our results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.