The larval midgut of Anopheles, Aedes, and Toxorhynchites mosquitoes (Diptera, Culicidae): a comparative approach in morphophysiology and evolution.

The mosquito larval midgut is responsible for acquiring and storing most of the nutrients that will sustain the events of metamorphosis and the insect's adult life. Despite its importance, the basic biology of this larval organ is poorly understood. To help fill this gap, we carried out a comparative morphophysiological investigation of three larval midgut regions (gastric caeca, anterior midgut, and posterior midgut) of phylogenetically distant mosquitoes: Anopheles gambiae (Anopheles albimanus was occasionally used as an alternate), Aedes aegypti, and Toxorhynchites theobaldi. Larvae of Toxorhynchites mosquitoes are predacious, in contrast to the other two species, that are detritivorous. In this work, we show that the larval gut of the three species shares basic histological characteristics, but differ in other aspects. The lipid and carbohydrate metabolism of the An. gambiae larval midgut is different compared with that of Ae. aegypti and Tx. theobaldi. The gastric caecum is the most variable region, with differences probably related to the chemical composition of the diet. The peritrophic matrix is morphologically similar in the three species, and processes involved in the post-embryonic development of the organ, such as cell differentiation and proliferation, were also similar. FMRF-positive enteroendocrine cells are grouped in the posterior midgut of Tx. theobaldi, but individualized in An. gambiae and Ae. aegypti. We hypothesize that Tx. theobaldi larval predation is an ancestral condition in mosquito evolution.

A Genetically Modified Anti-Plasmodium Bacterium Is Harmless to the Foragers of the Stingless Bee Partamona helleri.

Paratransgenesis consists of genetically engineering an insect symbiont to control vector-borne diseases. Biosafety assessments are a prerequisite for the use of genetically modified organisms (GMOs). Assessments rely on the measurement of the possible impacts of GMOs on different organisms, including beneficial organisms, such as pollinators. The bacterium Serratia AS1 has been genetically modified to express anti-Plasmodium effector proteins and does not impose a fitness cost on mosquitoes that carry it. In the present study, we assessed the impact of this bacterium on the native bee Partamona helleri (Meliponini), an ecologically important species in Brazil. Serratia eGFP AS1 (recombinant strain) or a wild strain of Serratia marcescens were suspended in a sucrose solution and fed to foragers, followed by measurements of survival, feeding rate, and behavior (walking and flying). These bacteria did not change any of the variables measured at 24, 72, and 144 h after the onset of the experiment. Recombinant and wild bacteria were detected in the homogenates of digestive tract during the 144 h period analyzed, but their numbers decreased with time. The recombinant strain was detected in the midgut at 24 h and in the hindgut at 72 h and 144 h after the onset of the experiment under the fluorescent microscope. As reported for mosquitoes, Serratia eGFP AS1 did not compromise the foragers of P. helleri, an ecologically relevant bee.

The virulence of Salmonella Enteritidis in Galleria mellonella is improved by N-dodecanoyl-homoserine lactone.

Salmonella is a food and waterborne pathogen responsible for outbreaks worldwide, and it can survive during passage through the stomach and inside host phagocytic cells. Virulence genes are required for infection and survival in macrophages, and some are under the regulation of the quorum sensing (QS) system. This study investigated the influence of the autoinducer 1 (AI-1), N-dodecanoyl-homoserine lactone (C12-HSL), on the virulence of Salmonella PT4 using Galleria mellonella as an infection model. Salmonella PT4 was grown in the presence and absence of C12-HSL under anaerobic conditions for 7 h, and the expression of rpoS, arcA, arcB, and invA genes was evaluated. After the inoculation of G. mellonella with the median lethal dose (LD50) of Salmonella PT4, the survival of bacteria inside the larvae and their health status (health index scoring) were monitored, as well as the pigment, nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT) production. Also, the hemocyte viability, the induction of caspase-3, and microtubule-associated light chain 3 (LC3) protein in hemocytes were evaluated. Salmonella PT4 growing in the presence of C12-HSL showed increased rpoS, arcA, arcB, and invA expression and promoted higher larvae mortality and worse state of health after 24 h of infection. The C12-HSL also increased the persistence of Salmonella PT4 in the hemolymph and in the hemocytes. The highest pigmentation, NO production, and antioxidant enzymes were verified in the larva hemolymph infected with Salmonella PT4 grown with C12-HSL. Hemocytes from larvae infected with Salmonella PT4 grown with C12-HSL showed lower viability and higher production of caspase-3 and LC3. Taken together, these findings suggest that C12-HSL could be involved in the virulence of Salmonella PT4.

Ethoflow: Computer Vision and Artificial Intelligence-Based Software for Automatic Behavior Analysis.

Manual monitoring of animal behavior is time-consuming and prone to bias. An alternative to such limitations is using computational resources in behavioral assessments, such as tracking systems, to facilitate accurate and long-term evaluations. There is a demand for robust software that addresses analysis in heterogeneous environments (such as in field conditions) and evaluates multiple individuals in groups while maintaining their identities. The Ethoflow software was developed using computer vision and artificial intelligence (AI) tools to monitor various behavioral parameters automatically. An object detection algorithm based on instance segmentation was implemented, allowing behavior monitoring in the field under heterogeneous environments. Moreover, a convolutional neural network was implemented to assess complex behaviors expanding behavior analyses' possibilities. The heuristics used to generate training data for the AI models automatically are described, and the models trained with these datasets exhibited high accuracy in detecting individuals in heterogeneous environments and assessing complex behavior. Ethoflow was employed for kinematic assessments and to detect trophallaxis in social bees. The software was developed in desktop applications and had a graphical user interface. In the Ethoflow algorithm, the processing with AI is separate from the other modules, facilitating measurements on an ordinary computer and complex behavior assessing on machines with graphics processing units. Ethoflow is a useful support tool for applications in biology and related fields.

Transcriptional profiling and physiological roles of Aedes aegypti spermathecal-related genes.

BACKGROUND: Successful mating of female mosquitoes typically occurs once, with the male sperm being stored in the female spermatheca for every subsequent oviposition event. The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. Aedes aegypti is a major vector of arboviruses, including Yellow Fever, Dengue, Chikungunya, and Zika. Vector control is difficult due to this mosquito high reproductive capacity. RESULTS: Following comparative RNA-seq analyses of spermathecae obtained from virgin and inseminated females, eight transcripts were selected based on their putative roles in sperm maintenance and survival, including energy metabolism, chitin components, transcriptional regulation, hormonal signaling, enzymatic activity, antimicrobial activity, and ionic homeostasis. In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts. Following RNA interference (RNAi), observed outcomes varied between targeted transcripts, affecting mosquito survival, egg morphology, fecundity, and sperm motility within the spermathecae. CONCLUSIONS: This study identified spermatheca-specific transcripts associated with sperm storage in Ae. aegypti. Using RNAi we characterized the role of eight spermathecal transcripts on various aspects of female fecundity and offspring survival. RNAi-induced knockdown of transcript AeSigP-66,427, coding for a Na+/Ca2+ protein exchanger, specifically interfered with egg production and reduced sperm motility. Our results bring new insights into the molecular basis of sperm storage and identify potential targets for Ae. aegypti control.

Spinosad-mediated effects on survival, overall group activity and the midgut of workers of Partamona helleri (Hymenoptera: Apidae).

Populations of stingless bees have declined around the world and pesticides have been indicated as one of the possible causes of this decrease. Spinosad, which is synthesized from the fermentation process produced by the soil actinomycete Saccharopolyspora spinosa, is one of the most used bioinsecticides today. This study aimed to evaluate the possible effects of spinosad (formulation) on survival, general group activity and the processes of autophagy, apoptosis and oxidative stress in two organs (midgut and brain) of workers of Partamona helleri, after 24 h of oral exposure. Workers were orally exposed to different concentrations of spinosad. The concentration (8.16 × 10-3 mg a.i./mL) that led to the mortality of approximately half the number of treated bees was considered LC50 and was used in behavior, histology and immunofluorescence bioassays. The results revealed that bee survival was substantially reduced with increasing spinosad concentrations. The LC50 of the bioinsecticide compromised general group activity, caused morphological alterations in the midgut and intensified the processes of autophagy, apoptosis and oxidative stress in this organ. The brain, on the other hand, did not present significant alterations under the tested conditions. The data obtained demonstrate, therefore, that spinosad negatively affects individual survival, general group activity and the midgut epithelium of P. helleri.

Exposure of mosquito (Aedes aegypti) larvae to the water extract and lectin-rich fraction of Moringa oleifera seeds impairs their development and future fecundity.

Aedes aegypti control is a key component of the prophylaxis of dengue fever and other diseases. Moringa oleifera seeds contain a water-soluble lectin (WSMoL) with larvicidal and ovicidal activities against this insect. In this study, A. aegypti individuals were exposed at the third larval instar for 24 h to the water extract (0.1-1.0 mg/mL of protein) or lectin-rich fraction (0.05-0.6 mg/mL of protein) containing WSMoL, and then their survival and development were followed for 9 days post-exposure. The feeding capacity of adult females that developed from the treated larvae and the hatching success of eggs laid by them were also evaluated. Further, any alterations to the midgut histology of treated larvae, pupae, and adults were investigated. The extract and fraction induced the death of A. aegypti larvae along the post-exposure period. Both preparations also delayed the developmental cycle. The midguts of treated larvae and pupae showed disorganization and epithelial vacuolization, while in treated adults, the epithelium was underdeveloped compared to control. Unlike in control mosquitos, proliferating cells were not detected in treated larvae, and appeared in lower numbers in treated pupae than in control pupae. Adult females that developed from larvae treated with the fraction gained less weight after a blood meal compared with control. The amount of eggs laid by females that developed from larvae treated with both the extract and fraction was significantly lower than in control. In addition, the eggs showed lower hatching rates. In conclusion, females that developed from larvae treated with both the water extract and lectin-rich fraction showed reduced engorgement after a blood meal, with the consequent impairment of their fertility and fecundity. These results were probably due to the damage to midgut organization and impairment of the remodeling process during metamorphosis.

Post-embryonic changes in the hindgut of honeybee Apis mellifera workers: Morphology, cuticle deposition, apoptosis, and cell proliferation.

In insects, the hindgut is a homeostatic region of the digestive tract, divided into pylorus, ileum, and rectum, that reabsorbs water, ions, and small molecules produced during hemolymph filtration. The hindgut anatomy in bee larvae is different from that of adult workers. This study reports the morphological changes and cellular events that occur in the hindgut during the metamorphosis of the honeybee Apis mellifera. We describe the occurrence of autophagosomes and the ultrastructure of the epithelial cells and cuticle, suggesting that cuticular degradation begins in prepupae, with the cuticle being reabsorbed and recycled by autophagosomes in white- and pink-eyed pupae, followed by the deposition of new cuticle in light-brown-eyed pupae. In L5S larvae and prepupae, the hindgut undergoes cell proliferation in the anterior and posterior ends. In the pupae, the pylorus, ileum, and rectum regions are differentiated, and cell proliferation ceases in dark-brown-eyed pupae. Apoptosis occurs in the hindgut from the L5S larval to the pink-eyed pupal stage. In light-brown- and dark-brown-eyed pupae, the ileum epithelium changes from pseudostratified to simple only after the production of the basal lamina, whereas the rectal epithelium is always flattened. In black-eyed pupae, ileum epithelial cells have large vacuoles and subcuticular spaces, while in adult forager workers these cells have long invaginations in the cell apex and many mitochondria, indicating a role in the transport of compounds. Our findings show that hindgut morphogenesis is a dynamic process, with tissue remodeling and cellular events taking place for the formation of different regions of the organ, the reconstruction of a new cuticle, and the remodeling of visceral muscles.

Variations in circulating hemocytes are affected by age and caste in the stingless bee Melipona quadrifasciata.

The insect immune system faces various challenges; particularly in social bees, caste system and age polyethism expose individuals to numerous environmental and working conditions. However, little is known about how cellular defenses in social bees may be organized to respond to a variety of immune challenges. Here, we describe the morphological features and the total and differential counts of hemocytes in different female classes (newly emerged workers, nurses, foragers, and virgin queens) of the eusocial stingless bee Melipona quadrifasciata. Granulocytes and prohemocytes were, respectively, the most and the least abundant cells among all classes of females. Furthermore, there were more prohemocytes in virgin queens than in foragers. The total number of hemocytes was smaller in foragers, whereas the largest number was observed in nurse workers. This reduced amount of hemocytes in foragers might allow energy savings to perform colony activities such as foraging and defense. Foragers also had the biggest hemocytes (either prohemocytes, granulocytes, or plasmatocytes) in comparison to the other classes of females, which might have arisen as a compensation for the reduction in number of these cells during aging. These results suggest that profiles of hemocytes of M. quadrifasciata vary according to the caste and age of this eusocial bee.

Acute and oral exposure to imidacloprid does not affect the number of circulating hemocytes in the stingless bee Melipona quadrifasciata post immune challenge.

In the present work, the effects of the neonicotinoid imidacloprid formulation on the total hemocyte counts (THC) and differential hemocyte counts (DHC) were investigated in foraging workers of the stingless bee Melipona quadrifasciata under (or not) the challenge with the bacteria Escherichia coli. The THC was not altered with the insecticide exposure and/or bacterial infection. However, the DHC of the bees changed with the imidacloprid exposure and/or bacterial infection. The number of prohemocytes (stem cells) increased in bees exposed to imidacloprid, but it did not change after the bacterial infection. The number of plasmatocytes (phagocytic cells) increased in imidacloprid-exposed and uninfected bees and decreased in infected bees regardless of the exposure to imidacloprid. On the other hand, there was a reduction of granulocytes, the most active immune cells, after imidacloprid exposure and an increase of granulocytes after the infection. Previous studies have shown that the neonicotinoids exposure can impair the immune system of bees. Our findings showed that the relative number of granulocytes declined with imidacloprid exposure, but the overall capacity of hemocyte responses in terms of total numbers after bacterial infection persisted even after the insecticide exposure.

Midgut morphological changes and autophagy during metamorphosis in sand flies.

During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.

The antennal sensilla of Melipona quadrifasciata (Hymenoptera: Apidae: Meliponini): a study of different sexes and castes.

The sensilla of insects are integumental units that play a role as sensory structures and are crucial for the perception of stimuli and for communication. In this study, we compared the antennal sensilla of females (workers and queens), males (haploid (n) and diploid (2n)), and queen-like males (QLMs, resulting from 2n males after juvenile hormone (JH) treatment) in the stingless bee Melipona quadrifasciata. Images of the dorsal antenna surfaces were acquired using a scanning electron microscope. As reported for other hymenopterans, this species exhibits a heterogeneous sensillar distribution along the antennae. Thirteen different types of sensilla were found in the antennae of M. quadrifasciata: trichodea (subtypes I to VI), chaetica (subtypes I and II), placodea, basiconica, ampullacea, coeloconica, and coelocapitula. Sensilla trichodea I were the most abundant, followed by sensilla placodea, which might function in olfactory perception. Sensilla basiconica, sensilla chaetica I, sensilla coeloconica, and sensilla ampullacea were found exclusively in females. In terms of the composition and size of the sensilla, the antennae of QLMs most closely resemble those of the 2n male, although QLMs exhibit a queen phenotype. This study represents the first comparative analysis of the antennal sensilla of M. quadrifasciata. The differences found in the type and amount of sensilla between the castes and sexes are discussed based on the presumed sensillary functions.

Midgut morphology of the predator mosquito Lutzia bigoti (Diptera: Culicidae) and its implications for feeding behavior.

Lutzia mosquitoes (Theobald, 1903) are predaceous during their larval stages, but the adult feeding is not clearly understood, especially in relation to blood feeding. In case these mosquitoes are harmless to humans and related animals, they can be useful in biological control of mosquito vectors of pathogens. Investigating the midgut morphology is a good strategy to understand the feeding behavior of this species. The midgut in Lutzia bigoti Bellardi, 1862 displays two distinct portions, a thin anterior midgut and a more dilated posterior midgut. Digestive cells form a single epithelium in the midgut. These cells have long and packed microvilli at their apex and membrane infoldings at their basal portion, the basal labyrinth. The epithelium is supported by a basal lamina. Regarding their cytoplasm, it is noteworthy the abundance of mitochondria, distributed in an apical-basal fashion, and also a whirl-shaped endoplasmic reticulum in the posterior midgut. Basal cells are also found in the midgut of L. bigoti, resembling regenerative cells. The general organization of L. bigoti's midgut closely resembles that of numerous hematophagous mosquitoes previously documented. However, it diverges due to the presence of a peritrophic matrix even when exclusively fed on sugary solutions. Peculiar aspects of L. bigoti's midgut are discussed and compared to those of other mosquito species.

Toxicological Assessments of Agrochemicals in Stingless Bees in Brazil: a Systematic Review.

The growing concern with the decline of pollinators worldwide is centered on honey bees, due to their wide distribution, economic, and ecological importance. This type of concern remained less evident for stingless bees, which are widely distributed in the Neotropics, until recently. Since exposure to agrochemicals has been identified as one of the potential threats to bees, the present systematic review compiled information from toxicological evaluations in stingless bees in Brazil, home to a considerable portion of the existing species. This systematic review was performed considering species, research institutions, scientific journals, metrics, experimental set ups, and agrochemicals. The first article in this topic was published in 2010. Since then, 93 scientific papers were published, which showed that there are few species of stingless bees used for toxicological evaluations and Brazilian institutions lead these evaluations. Only 1.5% of the stingless bees' species that occur in Brazil were assessed through chronic exposure in the larval stage. The Universidade Federal de Viçosa (UFV) is responsible for 37% of the total publications. The main route of exposure was acute, using adults in laboratory conditions. The main group of agrochemicals studied were insecticides, in particular the neonicotinoids. The current results reveal the advances achieved and point out the gaps that still need to be filled considering toxicological evaluations in stingless bees.

Morphological characterization of antennal sensilla in Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti adults: a comparative study using scanning electron microscopy.

Some mosquitoes, including species of the genus Toxorhynchites, are known for actively preying on other mosquito larvae, making these predators valuable allies in the fight against vector-borne diseases. A comprehensive understanding of the anatomy and physiology of these potential biological control agents is helpful for the development of effective strategies for controlling vector populations. This includes the antennae, a crucial component in the search for hosts, mating, and selection of oviposition sites. This study utilized scanning electron microscopy to characterize the sensilla on the antennae of adult mosquitoes from two species that are exclusively phytophagous, including Toxorhynchites theobaldi and Toxorhynchites violaceus, as well as Lutzia bigoti, which females are allegedly hematophagous. The types of sensilla in each species were compared, and five basic types of antennal sensilla were identified: trichoid, chaetic, coeloconic, basiconic, and ampullacea. The analysis also found that they were morphologically similar across the three species, regardless of feeding habits or sex. The identification and characterization of basic types of antennal sensilla in T. theobaldi, T. violaceus, and L. bigoti suggest that these structures, which play a crucial role in the behavior and ecology, have common functions across different mosquito species, despite differences in feeding habits or sex.

A mixture of mesotrione and atrazine harms adults and larvae of the predatory wasp Polistes satan.

Herbicides have been intensively used for weed control, raising concerns about their potentially adverse effects on non-target organisms. Research on the effects of these common agrochemicals on beneficial insects and the ecosystem services they provide (e.g., predation and pollination) is scarce. Therefore, we tested whether a commercial formulation comprising a mixture of mesotrione and atrazine was detrimental to adult females and larvae of the Neotropical predatory social wasp Polistes satan, which is an effective natural enemy of crop pests. Wasps were individually fed syrups contaminated with different concentrations of the herbicide above and below the maximum label rate (MLR = 12 mL/L). Survival was assessed. The locomotor activity, immune response, and midgut morphology of adults as well as the immune response of the larvae were also studied. Herbicide concentrations far above the MLR (12, 40, and 100 times) caused adult mortality, whereas lower concentrations (0.5, 1, and 6 times) did not. Herbicide exposure at 0.5 to 12 times the MLR increased adult activity. Adult exposure at 0.1 or 0.5 times the MLR did not affect melanotic encapsulation of foreign bodies but led to changes in the morphology of the midgut epithelium and peritrophic matrix. In larvae, the ingestion of herbicide at 0.1 or 0.2 times the MLR (corresponding to 9.6 and 19.2 ng of herbicide per individual) did not cause mortality but decreased their melanization-encapsulation response. Increased locomotor activity in herbicide-exposed adults can affect their foraging activity. The altered midgut morphology of adults coupled with the decreased immune response in larvae caused by herbicide exposure at realistic concentrations can increase the susceptibility of wasps to infections. Therefore, herbicides are toxic to predatory wasps.

Exploring honey bee toxicological data as a proxy for assessing dimethoate sensitivity in stingless bees.

The high diversity and distinctive characteristics of stingless bees pose challenges in utilizing toxicity test results for agrochemical registrations. Toxicity assessments were performed on 15 stingless bee species, along with the honey bee, using the insecticide dimethoate, following adapted OECD protocols. Median lethal doses over 24 h (24 h-LD50) were determined for exposure routes (acute oral or contact) and species. Species sensitivity distribution (SSD) curves were constructed and the 5% hazard doses (HD5) were estimated based on 24 h-LD50 values. The SSD curve was adjusted as the body weight and dimethoate response were correlated. Lighter bees (<10 mg) had lower 24 h-LD50 values. Contact exposure for adjusted HD5 suggested insufficient protection for Melipona mondury, whereas the oral exposure HD5 indicated no risks for the other 14 species. Comprehensive risk assessments are crucial for understanding the agrochemical impact on stingless bees, emphasizing the need for a broader species range in formulating conservation strategies.

Exposure to low-concentration fipronil impairs survival, behavior, midgut morphology and physiology of Aedes aegypti larvae.

The Aedes aegypti mosquito is a vector for various arboviruses, including dengue and yellow fever. Insecticides, such as pyrethroids and organophosphates, are widely used to manage and control these insects. However, mosquitoes have developed resistance to these chemicals. Therefore, this study aimed to investigate the effects of the commercial formulation of fipronil (Tuit® Florestal; 80% purity) on the survival, behavior, morphology, and proteins related to signaling pathways of the midgut in A. aegypti larvae under controlled laboratory conditions. Significant reductions in immature survival were observed in all concentrations of fipronil tested. Low insecticide concentration (0.5 ppb) led to decreased locomotor activity in the larvae and caused disorganization of the epithelial tissue in the midgut. Moreover, exposure to the insecticide decreased the activity of detoxifying enzymes such as catalase, superoxide dismutase, and glutathione-S-transferase. On the other hand, the insecticide increased protein oxidation and nitric oxide levels. The detection of LC3, caspase-3, and JNK proteins, related to autophagy and apoptosis, increased after exposure. However, there was a decrease in the positive cells for ERK 1/2. Furthermore, the treatment with fipronil decreased the number of positive cells for the proteins FMRF, Prospero, PH3, Wg, Armadillo, Notch, and Delta, which are related to cell proliferation and differentiation. These findings demonstrate that even at low concentrations, fipronil exerts larvicidal effects on A. aegypti by affecting behavior and enzymatic detoxification, inducing protein oxidation, free radical generation, midgut damage and cell death, and inhibiting cell proliferation and differentiation. Thus, this insecticide may represent a viable alternative for controlling the spread of this vector.