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.

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.

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.

Superb feeding behavior of Aedes albopictus transmitting Zika virus.

Disease-mediated mosquitoes have been receiving much attention, as the World Health Organization recently declared the Zika virus a global public health emergency. Mosquitoes transmit pathogens that cause various tropical diseases including malaria, dengue fever and yellow fever as well as Zika virus. The vector efficiency of mosquitoes depends on their blood-feeding characteristics and the mechanics of their blood-sucking pump system, but only a few studies have attempted to investigate these key issues. In this study, we demonstrate the rapid and gluttonous liquid-feeding characteristics of Ae. albopictus which transmits Zika virus can be explained by similar proportion of two blood-sucking pumps and accelerated liquid intake driven by fast expanding of pumps. Our results provide insight into the vector efficiency of Ae. albopictus in terms of feeding velocity, pumping frequency, liquid-intake rate, and wall shear stress.

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

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

Damage-Induced Cell Regeneration in the Midgut of Aedes albopictus Mosquitoes.

Mosquito-transmitted diseases cause over one million deaths every year. A better characterization of the vector's physiology and immunity should provide valuable knowledge for the elaboration of control strategies. Mosquitoes depend on their innate immunity to defend themselves against pathogens. These pathogens are acquired mainly through the oral route, which places the insects' gut at the front line of the battle. Indeed, the epithelium of the mosquito gut plays important roles against invading pathogens acting as a physical barrier, activating local defenses and triggering the systemic immune response. Therefore, the gut is constantly confronted to stress and often suffers cellular damage. In this study, we show that dividing cells exist in the digestive tract of adult A. albopictus and that these cells proliferate in the midgut after bacterial or chemical damage. An increased transcription of signaling molecules that regulate the EGFR and JAK/STAT pathways was also observed, suggesting a possible involvement of these pathways in the regeneration of damaged guts. This work provides evidence for the presence of regenerative cells in the mosquito guts, and paves the way towards a molecular and cellular characterization of the processes required to maintain mosquito's midgut homeostasis in both normal and infectious conditions.

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.

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.

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.

Differentiation of Aedes aegypti and Aedes notoscriptus (Diptera: Culicidae) eggs using scanning electron microscopy.

Aedes notoscriptus and Aedes aegypti are both peri-domestic, invasive container-breeding mosquitoes. While the two potential arboviral vectors are bionomically similar, their sympatric distribution in Australia is limited. In this study, analyses of Ae. aegypti and Ae. notoscriptus eggs were enabled using scanning electron microscopy. Significant variations in egg length to width ratio and outer chorionic cell field morphology between Ae. aegypti and Ae. notoscriptus enabled distinction of the two species. Intraspecific variations in cell field morphology also enabled differentiation of the separate populations of both species, highlighting regional and global variation. Our study provides a comprehensive comparative analysis of inter- and intraspecific egg morphological and morphometric variation between two invasive container-breeding mosquitoes. The results indicate a high degree of intraspecific variation in Ae. notoscriptus egg morphology when compared to the eggs of Ae. aegypti. Comparative morphological analyses of Ae. aegypti and Ae. notoscriptus egg attributes using SEM allows differentiation of the species and may be helpful in understanding egg biology in relation to biotope of origin.

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Insect-specific viruses detected in laboratory mosquito colonies and their potential implications for experiments evaluating arbovirus vector competence.

Recently, there has been a dramatic increase in the detection and characterization of insect-specific viruses in field-collected mosquitoes. Evidence suggests that these viruses are ubiquitous in nature and that many are maintained by vertical transmission in mosquito populations. Some studies suggest that the presence of insect-specific viruses may inhibit replication of a super-infecting arbovirus, thus altering vector competence of the mosquito host. Accordingly, we screened our laboratory mosquito colonies for insect-specific viruses. Pools of colony mosquitoes were homogenized and inoculated into cultures of Aedes albopictus (C6/36) cells. The infected cells were examined by electron microscopy and deep sequencing was performed on RNA extracts. Electron micrograph images indicated the presence of three different viruses in three of our laboratory mosquito colonies. Potential implications of these findings for vector competence studies are discussed.

Distinguishing the two forms of Egyptian Aedes (Ochlerotatus) caspius Palias species (Diptera: Culicidae) by ultra structure micrographs of eggs.

Ultrastructure of the two forms autogenous and anautogenous eggs of Aedes (Ochlerotatus) caspius of Egypt are described using Scanning Electron Microscope (SEM). The eggs of the two forms are slightly boat shape with quite difference in width. Chorionic cells of the ventral surface are ultimately different in both forms in shape, width of reticulum, number and size of tubercles. The chorionic cells of the autogenous form's egg are elongate, narrow and almost curved with unusually wide, outer reticulum contain 2 - 13 large tubercles along with a few number in small size. However, the anautogenous form's egg, the chorionic cells of the ventral surface fairly distinct, very regular in outline with thin reticulum and usually hexagonal, each cell contain one or two large tubercles with many small scattered peripheral tubercles. Fine structure micrographic work of eggs of the Egyptian Ae. caspius provides new morphological evidence that both autogenous and unautogenous forms are certainly different and suggests that those forms are two distinct species.

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.

Aedes aegypti midgut remodeling during metamorphosis.

The Aedes aegypti midgut is restructured during metamorphosis; its epithelium is renewed by replacing the digestive and endocrine cells through stem or regenerative cell differentiation. Shortly after pupation (white pupae) begins, the larval digestive cells are histolized and show signs of degeneration, such as autophagic vacuoles and disintegrating microvilli. Simultaneously, differentiating cells derived from larval stem cells form an electron-dense layer that is visible 24h after pupation begins. Forty-eight hours after pupation onset, the differentiating cells yield an electron-lucent cytoplasm rich in microvilli and organelles. Dividing stem cells were observed in the fourth instar larvae and during the first 24h of pupation, which suggests that stem cells proliferate at the end of the larval period and during pupation. This study discusses various aspects of the changes during midgut remodeling for pupating A. aegypti.

The ultrastructure of the Aedes aegypti heart.

Comparative structural analyses of the heart and associated tissues in 4th instar larvae (L4), pupae and adults of Aedes aegypti were undertaken using a combination of microscopy techniques. The Ae. aegypti heart consists of cardiomyocytes arranged in a helical fashion, and it is physically associated with intersegmental groups of pericardial cells (PCs) and the alary muscles (AMs). Ramifications commonly present in AMs are more developed in adults than in the immature stages. Pericardial cells absorb and store extracellular components as shown by the uptake of carmine dye fed in larval diet. We also observed that carmine stained inclusions corresponding to electron-dense structures resembling lysosomes that were more abundant and prominent in pupae, suggestive of increase of waste accumulation during pupation. The results presented here expand on previously known aspects of the mosquito heart and describe for the first time comparative aspects of the morphology of the heart in different developmental stages.

[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.