Extract from Opuntia ficus-indica cladode delays the Aedes aegypti larval development by inducing an axenic midgut environment.

This study evaluated the effects of acute exposure of Aedes aegypti third instar (L3 ) larvae to the saline extract of Opuntia ficus-indica cladodes on the biological cycle and fertility of the emerging adults. For this, larvae were treated for 24 h with the extract at » LC50 (lethal concentration to kill 50% of larvae), ½ LC50 or LC50 ; the development and reproduction of the emerged adults were evaluated after a recovery period of 9 days. The resistance of proteins in the extract to hydrolysis by L3 digestive enzymes and histomorphological alterations in the larval midgut were also investigated. The extract contained lectin, flavonoids, cinnamic derivatives, terpenes, steroids, and reducing sugars. It showed a LC50 of 3.71% for 48 h. The data indicated mean survival times similar in control and extract treatments. It was observed development delay in extract-treated groups, with a lower number of adults than in control. However, the females that emerged laid similar number of eggs in control and treatments. Histological evaluation revealed absence of bacterial and fungal microorganisms in the food content in midguts from larvae treated with cladode extract. Electrophoresis revealed that three polypeptides in the extract resisted to hydrolysis by L3 digestive proteases for 90 min. The lectin activity was not altered even after 24-h incubation with the enzymes. In conclusion, the extract from O. ficus-indica can delay the development of Ae. aegypti larvae, which may be linked to induction of an axenic environment at larval midgut and permanence of lectin activity even after proteolysis.

FMRF-related peptides in Aedes aegypti midgut: neuromuscular connections and enteric nervous system.

FMRFamide-related peptides (FaRPs) are a class of neuropeptides that participate in a variety of physiological processes in invertebrates. They occur in nerves of stomatogastric ganglia and enteroendocrine cells of the insect digestive tract, where they may control muscle functions. However, their direct involvement in muscle function has never been shown in situ. We studied the relationship between FaRPs and midgut muscle during larval-pupal transition of the mosquito Aedes aegypti. In late L4, FaRP-positive neuronal extensions attach to the bundles of the external circular muscle layer, and muscle stem cells start to undergo mitosis in the internal circular layer. Thereafter, the external muscle layer degenerates, disappearing during early pupal development, and is completely absent in the adult mosquito. Our results indicate that FaRP-based neural signals are involved in the reorganization of the muscle fibers of the mosquito midgut during the larval-pupal transition. In addition to confirming FaRP involvement in muscle function, we show that the mosquito midgut muscles are largely innervated, and that circular and longitudinal muscle have specific neuron bodies associated with them.

Detrimental effects of pyriproxyfen on the detoxification and abilities of Belostoma anurum to prey upon Aedes aegypti larvae.

Despite being effective in controlling mosquito larvae and a few other target organisms, the application of insecticides into aquatic systems may cause unintended alterations to the physiology or behavioral responses of several aquatic non-target organisms, which can ultimately lead to their death. Here, we firstly evaluated whether the susceptibility of the giant water bug, Belostoma anurum (Hemiptera: Belostomatidae), a predator of mosquito larvae, to pyriproxyfen would be similar to that of its potential prey, larvae of Aedes aegypti (Diptera: Culicidae). Secondly, we recorded the nominal concentrations of pyriproxyfen in water and evaluated whether sublethal exposures would lead to physiological or behavioral alterations on the B. anurum nymphs. We characterized the activities of three major families of detoxification enzymes (i.e., cytochrome P450 monooxygenases, glutathione-S-transferase, and general esterases) and further evaluated the abilities of pyriproxyfen sublethally-exposed B. anurum to prey upon A. aegypti larvae at different prey densities. Our findings revealed that nominal pyriproxyfen concentration significantly decreased (approximately 50%) over the first 24 h. Furthermore, when applied at the concentration of 10 µg a.i./L, pyriproxyfen was approximately four times more toxic to A. aegypti larvae (LT50 = 48 h) than to B. anurum nymphs (LT50 = 192 h). Interestingly, the pyriproxyfen sublethally-exposed (2.5 µg a.i./L) B. anurum nymphs exhibited reduced enzyme activities (cytochrome P450 monooxygenases) involved in detoxication processes and preyed significantly less on A. aegypti larvae when compared to unexposed predators. Collectively, our findings demonstrate that mortality-based pyriproxyfen risk assessments are not always protective of aquatic non-target organisms.

Toxicological assessments of agrochemical effects on stingless bees (Apidae, Meliponini).

Bee pollination is crucial for ecosystem maintenance and crop production. The ubiquity of bee pollinators in agricultural landscapes frequently results in their exposure to agrochemicals, which has been associated with their decline. Stingless bees are wild pollinators restricted to the Pantropical region, and like honey bees, are suffering colony losses. However, stingless bees and honey bees do not show the same behaviors, therefore, methods used for risk assessment of honey bees cannot be utilized on stingless bees. Herein, we describe protocols to standardize methods that allow for the exploration of lethal and sublethal effects of agrochemicals via acute and chronic exposure of stingless bees. The in vitro rearing used for chronic exposure from the egg to the adult stage proved to be effective in obtaining relevant screenings. In addition, we performed a meta-analysis and summarized the results of toxicological studies conducted with the protocols described. The meta-analyses indicated a reduction in survival under acute and chronic exposures to agrochemicals, and revealed that our protocols for toxicological assessments did not have publication bias for either acute or chronic exposure. These findings proved that these standardized protocols are reliable for toxicological research on stingless bee.

Frequently encountered pesticides can cause multiple disorders in developing worker honey bees.

Pesticide exposure is regarded as a contributing factor to the high gross loss rates of managed colonies of Apis mellifera. Pesticides enter the hive through contaminated nectar and pollen carried by returning forager honey bees or placed in the hive by beekeepers when managing hive pests. We used an in vitro rearing method to characterize the effects of seven pesticides on developing brood subjected dietary exposure at worse-case environmental concentrations detected in wax and pollen. The pesticides tested included acaricides (amitraz, coumaphos, fluvalinate), insecticides (chlorpyrifos, imidacloprid), one fungicide (chlorothalonil), and one herbicide (glyphosate). The larvae were exposed chronically for six days of mimicking exposure during the entire larval feeding period, which is the worst possible scenario of larval exposure. Survival, duration of immature development, the weight of newly emerged adult, morphologies of the antenna and the hypopharyngeal gland, and gene expression were recorded. Survival of bees exposed to amitraz, coumaphos, fluvalinate, chlorpyrifos, and chlorothalonil was the most sensitive endpoint despite observed changes in many developmental and physiological parameters across the seven pesticides. Our findings suggest that pesticide exposure during larvae development may affect the survival and health of immature honey bees, thus contributing to overall colony stress or loss. Additionally, pesticide exposure altered gene expression of detoxification enzymes. However, the tested exposure scenario is unlikely to be representative of real-world conditions but emphasizes the importance of proper hive management to minimize pesticide contamination of the hive environment or simulates a future scenario of increased contamination.

"Armed to the teeth": The multiple ways to survive insecticidal and predatory challenges in Aedes aegypti larvae.

Environmental pollutants, such as insecticides, can alter the equilibrium of aquatic ecosystems, particularly those closely located to human occupations. The use of such anthropogenic compounds frequently results in the selection of resistant individuals. However, how the underlying insecticide resistance mechanisms interplay with the abilities of the resistant individuals to cope with other environmental challenges (e.g., predators) has not received adequate attention. Here, we evaluated whether resistance to pyrethroid insecticides in larvae of the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), would affect their abilities to survive other environmental challenges. We assessed the susceptibilities of the pyrethroid-resistant larvae to other insecticides (i.e., the oxadiazine indoxacarb and juvenile hormone mimic pyriproxyfen) and determined the activities of potential detoxification enzymes. Finally, we also recorded potential alterations in larva swimming behavior in the presence of predators, such as the water bug Belostoma anurum (Hemiptera: Belostomatidae). Our results revealed that high pyrethroid resistance was associated with moderate resistance to the other two insecticides. Furthermore, this multiple resistance was associated with higher detoxification activity by glutathione-S-transferases and general esterases. Interestingly, in comparison with insecticide-susceptible larvae, the pyrethroid-resistant larvae not only swam for longer periods and distances, but also took longer to be captured by B. anurum nymphs. Collectively, our findings revealed increased abilities to survive natural environmental challenges (e.g., predatory attacks) in mosquito larvae that express physiological and behavioral changes associated with multiple resistance to insecticides.

Morphology and Morphometry of the Midgut in the Stingless Bee Friesella schrottkyi (Hymenoptera: Apidae).

Friesella schrottkyi is a small stingless bee (3-mm long) important for agricultural and native forest pollination. This study describes the morphology and morphometry of the midgut in F. schrottkyi forager workers. The F. schrottkyi midgut presents a single-layered epithelium with digestive, regenerative and endocrine cells. The digestive cells are similar along the entire midgut length with a spherical nucleus, apex with long striated border, cytoplasmic granules in the apical region and well-developed basal labyrinth associated with mitochondria, suggesting they are multifunctional, synthesizing digestive enzymes and peritrophic matrix compounds and absorbing nutrients. Regenerative cells are located around the basal region organized in nests with some cells with a spherical nucleus. Phe-Met-Arg-Phe-NH2-amide (FMRFamide) positive endocrine cells are restricted to the posterior midgut region, suggesting a paracrine function in the midgut. This is the first morphological description of the F. schrottkyi midgut contributing to the comprehension of the digestive process of this bee.

Hydrophobic Nanoprecipitates of ß-Cyclodextrin/Avermectins Inclusion Compounds Reveal Insecticide Activity against Aedes aegypti Larvae and Low Toxicity against Fibroblasts.

In the present work, hydrophobic nanoprecipitates (HNPs) of inclusion complexes formed between ß-cyclodextrin (ßCD) and the avermectins (AVMs) named eprinomectin (EPRI) and ivermectin (IVER) were synthesized and characterized, and their larvicidal activity against Aedes aegypti and human safety against fibroblasts were evaluated. Initially, thermogravimetric analysis/differential thermal analysis data revealed that inclusion increased the thermal stability of AVMs in the presence of ßCD. Nuclear magnetic resonance experiments and density functional theory calculations pointed out the inclusion of the benzofuran ring of the two AVMs in the ßCD cavity. Isothermal titration calorimetry experiments allowed identification of different binding constants for EPRI/ßCD ( Kb = 1060) and ßCD/IVER ( Kb = 1700) systems, despite the structural similarity. Dynamic light scattering titrations of AVMs' dimethyl sulfoxide solution in ßCD aqueous solution demonstrated that the formed HNPs have lower sizes in the presence of ßCD. Finally, the inclusion of EPRI in ßCD increased its larval toxicity and reduced its human cytotoxicity, while for IVER/ßCD no beneficial effect was observed upon inclusion. These results were rationalized in terms of structural differences between the two molecules. Finally, the EPRI/ßCD complex has great potential as an insecticide against A. aegypti larvae with high human safety.

Alternative control of Aedes aegypti resistant to pyrethroids: lethal and sublethal effects of monoterpene bioinsecticides.

BACKGROUND: The mosquito Aedes aegypti is intensively controlled because it is a vector of viruses that cause numerous diseases, especially in tropical regions. As a consequence of the indiscriminate use of insecticides, populations from different regions have become resistant to pyrethroids. Here, we analyzed the lethal and sublethal effects of essential oil of Aristolochia trilobata and its major compounds on A. aegypti from susceptible and pyrethroid-resistant populations. RESULTS: Our results showed that the toxicity of the different compounds and behavioral changes in response to them are dependent on the stage of the insect life cycle. The monoterpene ρ-cymene caused high mortality in both larvae and adult females of A. aegypti, including those from the pyrethroid-resistant population. The monoterpenes limonene and linalool caused a sublethal effect in the larvae, triggering changes in the swimming pattern. CONCLUSION: This study highlights the potential of the essential oil of A. trilobata and its major compounds ρ-cymene and limonene for the control of A. aegypti and reveals the importance of analyzing sublethal effects on the population dynamics of the A. aegypti mosquito. © 2017 Society of Chemical Industry.

Detection of a new pyrethroid resistance mutation (V410L) in the sodium channel of Aedes aegypti: a potential challenge for mosquito control.

The yellow fever mosquito, Aedes aegypti, particularly in Neotropical regions, is the principal vector of dengue, yellow fever, Zika and Chikungunya viruses. Pyrethroids remain one of the most used insecticides to control Aedes mosquitoes, despite the development of pyrethroid resistance in many mosquito populations worldwide. Here, we report a Brazilian strain of A. aegypti with high levels (approximately 100-60,000 fold) of resistance to both type I and type II pyrethroids. We detected two mutations (V410L and F1534C) in the sodium channel from this resistant strain. This study is the first report of the V410L mutation in mosquitoes. Alone or in combination with the F1534C mutation, the V410L mutation drastically reduced the sensitivity of mosquito sodium channels expressed in Xenopus oocytes to both type I and type II pyrethroids. The V410L mutation presents a serious challenge for the control of A. aegypti and will compromise the use of pyrethroids for the control of A. aegypti in Brazil; therefore, early monitoring of the frequency of the V410L mutation will be a key resistance management strategy to preserve the effectiveness of pyrethroid insecticides.

The insect spermatheca: an overview.

In the female insect, the spermatheca is an ectodermal organ responsible for receiving, maintaining, and releasing sperm to fertilize eggs. The number and morphology of spermathecae vary according to species. Within the spermathecal lumen, substances in the semen and secretions from the spermathecal gland nourish the sperm. Thus, the spermatheca provides an appropriate environment that ensures the long-term viability of sperm. Maintaining sperm viability for long periods within the spermatheca is crucial for insect reproductive success; however, the details of this process remain poorly understood. This review examines several aspects of and gaps in the current understanding of spermatheca biology, including morphology, function, reservoir filling, development, and biochemistry. Despite the importance of the spermatheca in insects, there is little information on the gland secretions and their role in the maintenance and protection of male gametes. Furthermore, in this review, we highlight the current information on spermathecal gland secretions and the likely roles they play in the maintenance and protection of sperm.

Termiticidal lectins from Myracrodruon urundeuva (Anacardiaceae) cause midgut damage when ingested by Nasutitermes corniger (Isoptera: Termitidae) workers.

BACKGROUND: Myracrodruon urundeuva is a hardwood tree, and its bark, heartwood and leaf contain lectins (MuBL, MuHL and MuLL respectively) with termiticidal activity against Nasutitermes corniger. In this work, the effects of these lectins on the midgut of N. corniger workers were evaluated. RESULTS: The insects were supplied with an artificial diet containing the lectins at their respective LC50 (previously determined). At 48 h after treatment, the midguts were dissected and fixed for histopathology analyses. Toluidine-blue-stained midguts from lectin-treated workers showed disorganisation, with the presence of debris in the lumen and the absence of brush border. Fluorescence microscopy revealed that the numbers of digestive and proliferating cells were lower in lectin-treated individuals than in the control, and caspase-3 staining confirmed the occurrence of cell apoptosis. Enteroendocrine cells were not seen in the treated individuals. The midguts from treated insects showed greater staining for peroxidase than the control, suggesting that the lectins caused oxidative stress. Staining with wheat germ agglutinin conjugated to FITC revealed that the lectins interfered with the integrity of the peritrophic matrix. CONCLUSION: This study showed that termiticidal lectins from M. urundeuva cause severe injuries, oxidative stress and cell death in the midgut of N. corniger workers. © 2016 Society of Chemical Industry.

Midgut of the non-hematophagous mosquito Toxorhynchites theobaldi (Diptera, Culicidae).

In most mosquito species, the females require a blood-feeding for complete egg development. However, in Toxorhynchites mosquitoes, the eggs develop without blood-feeding, and both females and males exclusively feed on sugary diets. The midgut is a well-understood organ in blood-feeding mosquitoes, but little is known about it in non-blood-feeding ones. In the present study, the detailed morphology of the midgut of Toxorhynchites theobaldi were investigated using histochemical and ultrastructural methods. The midgut of female and male T. theobaldi adults consists of a long, slender anterior midgut (AMG), and a short, dilated posterior midgut (PMG). The AMG is subdivided into AMG1 (short, with folds) and AMG2 (long, without folds). Nerve branches and enteroendocrine cells are present in AMG and PMG, respectively. Compared with the PMG of blood-feeding female mosquitoes, the PMG of T. theobaldi is smaller; however, in both mosquitoes, PMG seems be the main region of food digestion and absorption, and protein secretion. The epithelial folds present in the AMG of T. theobaldi have not been reported in other mosquitoes; however, the midgut muscle organization and endocrine control of the digestion process are conserved in both T. theobaldi and blood-feeding mosquitoes.

Spinosad in the native stingless bee Melipona quadrifasciata: regrettable non-target toxicity of a bioinsecticide.

The risks imposed by novel insecticides, mainly bioinsecticides, are largely unknown despite their increased use and their perceived environmental safety, which is based on their natural origin. Furthermore, unlike honeybees, native pollinator species have received little attention. In the present study, the lethal and sublethal effects of the neonicotinoid imidacloprid and the bioinsecticide spinosad were assessed in the stingless bee species Meliponaquadrifasciata, an important native pollinator in the Neotropical region. The adult stingless bee workers exhibited high oral insecticide susceptibility, with LD50s of 23.54 and 12.07 ng a.i./bee for imidacloprid and spinosad, respectively. Imidacloprid also impaired worker respiration and overall group activity and flight, while spinosad significantly impaired only worker flight despite exhibiting higher oral toxicity to adult workers than imidacloprid. These findings indicate the hazardous nature not only of imidacloprid but also the bioinsecticide spinosad to adult workers of the native pollinator M. quadrifasciata. Therefore, bioinsecticides should not be exempted from risk assessment analysis due to their lethal and sublethal components.

Survival and swimming behavior of insecticide-exposed larvae and pupae of the yellow fever mosquito Aedes aegypti.

BACKGROUND: The yellow fever mosquito Aedes aegypti is essentially a container-inhabiting species that is closely associated with urban areas. This species is a vector of human pathogens, including dengue and yellow fever viruses, and its control is of paramount importance for disease prevention. Insecticide use against mosquito juvenile stages (i.e. larvae and pupae) is growing in importance, particularly due to the ever-growing problems of resistance to adult-targeted insecticides and human safety concerns regarding such use in human dwellings. However, insecticide effects on insects in general and mosquitoes in particular primarily focus on their lethal effects. Thus, sublethal effects of such compounds in mosquito juveniles may have important effects on their environmental prevalence. In this study, we assessed the survival and swimming behavior of A. aegypti 4th instar larvae (L4) and pupae exposed to increasing concentrations of insecticides. We also assessed cell death in the neuromuscular system of juveniles. METHODS: Third instar larvae of A. aegypti were exposed to different concentrations of azadirachtin, deltamethrin, imidacloprid and spinosad. Insect survival was assessed for 10 days. The distance swam, the resting time and the time spent in slow swimming were assessed in 4th instar larvae (L4) and pupae. Muscular and nervous cells of L4 and pupae exposed to insecticides were marked with the TUNEL reaction. The results from the survival bioassays were subjected to survival analysis while the swimming behavioral data were subjected to analyses of covariance, complemented with a regression analysis. RESULTS: All insecticides exhibited concentration-dependent effects on survival of larvae and pupae of the yellow fever mosquito. The pyrethroid deltamethrin was the most toxic insecticide followed by spinosad, imidacloprid, and azadirachtin, which exhibited low potency against the juveniles. All insecticides except azadirachtin reduced L4 swimming speed and wriggling movements. A similar trend was also observed for swimming pupa, except for imidacloprid, which increased the swimming activity of pupa. Curiously, the insecticides did not affect cell damage in the neuromuscular system of larvae and pupae. CONCLUSIONS: Deltamethrin and spinosad were the main compounds to exhibit lethal effects, which allowed the control of A. aegypti larvae and pupae, and impair their swimming potentially compromising foraging and predation likelihood.

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.

Histochemistry and ultrastructure of urocytes in the pupae of the stingless bee Melipona quadrifasciata (Hymenoptera: Meliponini).

The main cell types of the adult bee fat body are trophocytes and oenocytes; however, in pupae of some newly emerged bees, trophocytes are modified into cells called urocytes, which possibly function as a substitute for Malpighian tubules during metamorphosis when larval tubules are not functional and/or storage of urate salts is required. This study evaluated the morphology of urocytes in the stingless bee Melipona quadrifasciata and the possibility of maintaining these cells in primary culture. The urocytes M. quadrifasciata are white spherical cells with an irregular surface as observed by stereomicroscopy. They may be found individually or in groups associated with tracheae. Urocytes have a single, small, and spherical nucleus and cytoplasm rich in neutral polysaccharides, lipid droplets, protein, and granules containing calcium and urate salts. Our findings suggest that urocytes play a role in storage of neutral polysaccharides and calcium in M. quadrifasciata pupae and that these cells can be cultured for 72 h.

Imidacloprid-induced impairment of mushroom bodies and behavior of the native stingless bee Melipona quadrifasciata anthidioides.

Declines in pollinator colonies represent a worldwide concern. The widespread use of agricultural pesticides is recognized as a potential cause of these declines. Previous studies have examined the effects of neonicotinoid insecticides such as imidacloprid on pollinator colonies, but these investigations have mainly focused on adult honey bees. Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, we assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival. The ecological and economic importance of neotropical stingless bees as pollinators, their susceptibility to insecticides and the vulnerability of their larvae to insecticide exposure emphasize the importance of studying these species.