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.

Fipronil exposure compromises respiration and damages the Malpighian tubules of the stingless bee Partamona helleri Friese (Hymenoptera: Apidae).

Fipronil has been widely used in agriculture and forestry in Brazil to control several pests. However, this insecticide may be hazardous to non-target organisms, including stingless bees, which are essential pollinators of crops and natural environments. Here, we investigated the effect of 24-h acute oral exposure to LC50 of fipronil on the Malpighian tubules of the stingless bee Partamona helleri (Friese). Insecticide exposure decreases the respiration rate of forager bees, and the Malpighian tubules are severely affected, as shown by the epithelial architecture disorganization, loss of cytoplasmic content, degradation of the brush border, and nuclear pyknosis. In addition, fipronil ingestion increases the number of Malpighian cells positive for peroxidase, LC3, cleaved caspase-3, and JNK. However, Notch and ERK1/2-positive cells decrease in the exposed bees. These changes in the signaling proteins indicate an increase in oxidative stress, autophagy and apoptosis, and impairment of cell recovery. Overall, our results demonstrate the toxicological effects of fipronil on a stingless bee, which compromises the physiology of this important pollinator.

Impact of copper sulfate on survival, behavior, midgut morphology, and antioxidant activity of Partamona helleri (Apidae: Meliponini).

Copper sulfate (CuSO4) is widely used in agriculture as a pesticide and foliar fertilizer. However, the possible environmental risks associated with CuSO4 use, particularly related to pollinating insects, have been poorly studied. In this study, we evaluated both lethal and sublethal effects of CuSO4 on the stingless bee Partamona helleri. Foragers were orally exposed to five concentrations of CuSO4 (5000, 1666.7, 554.2, 183.4, 58.4 µg mL-1), and the concentration killing 50% (LC50) was estimated. This concentration (142.95 µg mL-1) was subsequently used in behavioral, midgut morphology, and antioxidant activity analyses. Bee mortality increased with the ingestion of increasing concentrations of CuSO4. Ingestion at the estimated LC50 resulted in altered walking behavior and damage to the midgut epithelium and peritrophic matrix of bees. Furthermore, the LC50 increased the catalase or superoxide dismutase activities and levels of the lipid peroxidation biomarker malondialdehyde. Furthermore, the in situ detection of caspase-3 and LC3, proteins related to apoptosis and autophagy, respectively, revealed that these processes are intensified in the midgut of treated bees. These data show that the ingestion of CuSO4 can have considerable sublethal effects on the walking behavior and midgut of stingless bees, and therefore could pose potential risks to pollinators including native bees. Graphical abstract.

Exposure to copper sulfate impairs survival, post-embryonic midgut development and reproduction in Aedes aegypti.

Aedes aegypti is a vector of several global human viruses responsible for high human morbidity and mortality. The method to prevent the transmission of vector-borne viruses is mainly based on the control of the insect vector using insecticides. Among these chemicals, copper sulfate is a compound widely used in agriculture with the potential to be used as an alternative to control these insects. This study evaluated the effects of the exposure of A. aegypti larvae to copper sulfate on survival, midgut morphology, blood-feeding and fecundity. The exposure to CuSO4 decreased the survival of A. aegypti during the immature phase. Adults obtained from exposed larvae had their lifespan decreased at all tested concentrations. The exposure to CuSO4 impaired the development in the transition from larvae to pupae and from pupae to adult. The number of eggs laid by females developed from larvae treated with CuSO4 was significantly lower than in control. In addition, the egg hatching rates were also negatively affected. The midguts of treated larvae and pupae showed epithelial disorganization. The number of cleaved caspase-3 cells increased in the midgut of exposed pupae compared to control. Moreover, there was a reduction in proliferating cells in treated larvae and pupae compared to the control. In conclusion, the results reveal that CuSO4 exposure has insecticidal activity against A. aegypti, which may be related to the impairment of the midgut metamorphosis and reduced proliferation of stem cells, with the consequent impairment of female mosquito fertility and fecundity.

Toxicological assessment of agrochemicals on bees using machine learning tools.

Machine learning (ML) is a branch of artificial intelligence (AI) that enables the analysis of complex multivariate data. ML has significant potential in risk assessments of non-target insects for modeling the multiple factors affecting insect health, including the adverse effects of agrochemicals. Here, the potential of ML for risk assessments of glyphosate (herbicide; formulation) and imidacloprid (insecticide, neonicotinoid; formulation) on the stingless bee Melipona quadrifasciata was explored. The collective behavior of forager bees was analyzed after in vitro exposure to agrochemicals. ML algorithms were applied to identify the agrochemicals that the bees have been exposed to based on multivariate behavioral features. Changes in the in situ detection of different proteins in the midgut were also studied. Imidacloprid exposure leads to the greatest changes in behavior. The ML algorithms achieved a higher accuracy (up to 91%) in identifying agrochemical contamination. The two agrochemicals altered the detection of cells positive for different proteins, which can be detrimental to midgut physiology. This study provides a holistic assessment of the sublethal effects of glyphosate and imidacloprid on a key pollinator. The procedures used here can be applied in future studies to monitor and predict multiple environmental factors affecting insect health in the field.

Harmful effects of fipronil exposure on the behavior and brain of the stingless bee Partamona helleri Friese (Hymenoptera: Meliponini).

Fipronil is a pesticide widely used to control agricultural and household insect pests. However, fipronil is highly toxic to non-target insects, including pollinators. In this study, we investigated the acute effects of fipronil on the behavior, brain morphology, antioxidant activity, and proteins related to signaling pathways on the brain of workers of the stingless bee Partamona helleri. The ingestion of fipronil increases both the walking distance and velocity and causes enlarged intercellular spaces in the Kenyon cells and intense vacuolization in the neuropils of the brain. Moreover, fipronil decreases the activity of catalase (CAT) and increases the activity of glutathione S-transferase (GST). However, there is no difference in superoxide dismutase (SOD) activity between the control and fipronil. Regarding immunofluorescence analysis, bees exposed to fipronil showed an increase in the number of cells positive for cleaved caspase-3 and peroxidase, but a reduction in the number of cells positive for ERK 1/2, JNK and Notch, suggesting neuron death and impaired brain function. Our results demonstrate that fipronil has harmful effects on the behavior and brain of a stingless bee, which may threaten the individuals and colonies of this pollinator.

Effects of sub-lethal doses of nisin on the virulence of Salmonella enterica in Galleria mellonella larvae.

Salmonella enterica is a pathogen that induces self-limiting gastroenteritis and is of worldwide concern. Nisin, an antimicrobial peptide, has emerged as an alternative for the control of microbial growth but its effect on the virulence of pathogenic bacteria is not yet well-explored. This work aimed to evaluate the virulence of S. enterica in the presence of sub-inhibitory nisin using the experimental model Galleria mellonella. Sub-inhibitory concentrations of nisin of 11.72 and 46.88 µM did not affect the cellular viability of S. enterica but promoted changes in gene expression within 1 h of treatment, with increases of up to 3-fold of pagC, 1.8-fold of invA and 2.3-fold of invF. Larvae of G. mellonella inoculated with S. enterica combined with nisin at 46.88 µM presented mortality, and TL50 noticeably increased to 50% and 80% at 24 and 48 h post-infection, respectively. Defence responses, such as melanisation, nodulation, pseudopodia, immune response, and expression of defence proteins of the larvae G. mellonella were enhanced when the treatments with S. enterica were combined with 11.72 or 46.88 µM nisin. These results show an increase in virulence of S. enterica by sub-MIC concentration of nisin that needs to be explored.

Acute exposure to fipronil induces oxidative stress, apoptosis and impairs epithelial homeostasis in the midgut of the stingless bee Partamona helleri Friese (Hymenoptera: Apidae).

Partamona helleri is an important pollinator in natural and agricultural ecosystems in the neotropics. However, the foraging activity of this bee increases its risk of exposure to pesticides, which may affect both the individuals and the colony. Thus, this study aims to evaluate the side effects of LC50 of fipronil (0.28 ng a.i. µL-1) on the midgut morphology, antioxidant activity and some pathways of cell death, proliferation and differentiation in workers of P. helleri, after 24 h of oral exposure. Fipronil caused morphological alterations in the midgut of the bees. The activities of the detoxification enzymes superoxide dismutase, catalase and glutathione S-transferase increased after exposure, which suggests the occurrence of a detoxification mechanism. Furthermore, exposure to fipronil changed the number of positive cells for signaling-pathway proteins in the midgut of bees, which indicates the induction of cell death by the apoptotic pathway and impairment of the midgut epithelial regeneration. These results demonstrate that fipronil may negatively affect the morphology and physiology of the midgut of the stingless bee P. helleri and impose a threat to the survival of non-target organisms.

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.

Biological, histological and immunohistochemical studies on the toxicity of spent coffee grounds and caffeine on the larvae of Aedes aegypti (Diptera: Culicidae).

The mosquito Aedes aegypti is a primary vector for major arboviruses, and its control is mainly based on the use of insecticides. Caffeine and spent coffee grounds (CG) are potential agents in controlling Ae. aegypti by reducing survival and blocking larval development. In this study, we analyzed the effects of treatment with common CG (CCG: with caffeine), decaffeinated CG (DCG: with low caffeine), and pure caffeine on the survival, behavior, and morphology of the midgut of Ae. aegypti under laboratory conditions. Third instar larvae (L3) were exposed to different concentrations of CCG, DCG, and caffeine. All compounds significantly affected larval survival, and sublethal concentrations reduced larval locomotor activity, delayed development, and reduced adult life span. Damage to the midgut of treated larvae included changes in epithelial morphology, increased number of peroxidase-positive cells (more abundant in DCG-treated larvae), and caspase 3-positive cells (more abundant in CCG-treated larvae), suggesting that the treatments triggered cell damage, leading to activation of cell death. In addition, the treatments reduced the FMRFamide-positive enteroendocrine cells and dividing cells compared to the control. CG and caffeine have larvicidal effects on Ae. aegypti that warrant field testing for their potential to control mosquitoes.

A peritrophin mediates the peritrophic matrix permeability in the workers of the bees Melipona quadrifasciata and Apis mellifera.

The permeability of the peritrophic matrix, essential for its function, depends on its chemical composition. The objective was to determine if the permeability of the peritrophic matrix varies along the midgut and in the presence of anti-peritrophin-55 antibody in Melipona quadrifasciata and Apis mellifera bees. The thickness of the peritrophic matrix in both species varies between the anterior and posterior midgut regions in workers. In A. mellifera dextran molecules with 40 kDa cross the peritrophic matrix, whereas those ≥70 kDa are retained in the endoperitrophic space. In M. quadrifasciata the peritrophic matrix permeability was for molecules <40 kDa. Bees fed on anti-peritrophin-55 antibody showed an increase in peritrophic matrix permeability, but survival was not affected. In the bees studied, the peritrophic matrices have morphological differences between midgut regions, but there is no difference in their permeability along the midgut, which is affected by peritrophin 55.

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.

Spinosad-mediated effects in the post-embryonic development of Partamona helleri (Hymenoptera: Apidae: Meliponini).

The use of insecticides based on metabolites found in live organisms, such as the insecticide spinosad, has been an option for the control of agricultural pests because of the allegedly low toxicological risk for nontarget arthropods, such as stingless bees. In the current study, we evaluate the effects of chronic oral exposure to spinosad during the larval phase on survival, developmental time, body mass, midgut epithelial remodeling, and the peritrophic matrix (PM) of Partamona helleri stingless bee workers. Worker larvae that were raised in the laboratory were orally exposed to different concentrations (0, 6.53, 13.06, 32.64, and 3,264 ng. a.i. bee-1) of spinosad (formulation), and the resulting survival, developmental time, and body mass were studied. The concentration of spinosad recommended for use in the field (3,264 ng. a.i. bee-1) reduced the survival of workers during development. Also, sublethal concentrations of spinosad delayed the development and caused morphological changes in the midgut epithelium. Finally, the chronic exposure of larvae to 32.64 ng. a.i. bee-1 spinosad also altered the remodeling of the midgut during metamorphosis and affected the organization of the PM of larvae, pupae, and adults. Our data suggest possible environmental risks for using spinosad in cultures that are naturally pollinated by stingless bees.

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.

Arsenic exposure intensifies glycogen nephrosis in diabetic rats.

It is known that either arsenic exposure or diabetes can impact renal function. However, it is unclear how these combined factors may influence kidney functions. Therefore, we evaluated morphological, functional, and oxidative parameters in the kidney of diabetic rats exposed to arsenic. Healthy male Wistar rats and streptozotocin-induced diabetic rats were exposed to 0 and 10 mg/L arsenate through drinking water for 40 days. Renal tissue was assessed using morphometry, mitosis and apoptosis markers, mineral proportion, oxidative stress markers, as well as the activity of antioxidant enzymes and membrane-bound adenosine triphosphatases. Arsenate intake altered glucose levels in healthy animals, but it did not reach hyperglycemic conditions. In diabetic animals, arsenate led to a remarkable increase of glycogen nephrosis in distal tubules. In these animals, additionally, the activity of catalase and glutathione S-transferase, besides the proportion of Fe, Cu, and K in renal tissue, was altered. Nevertheless, arsenate did not accumulate in the kidney and did not impact on other parameters previously altered by diabetes, including levels of malondialdehyde, Na, urea, creatinine, and apoptosis and mitosis markers. In conclusion, besides the intensification of glycogen nephrosis, the kidney was able to handle arsenate toxicity at this point, preventing arsenic deposition in the exposed groups and the impairment of renal function.

Aedes aegypti larvae treated with spinosad produce adults with damaged midgut and reduced fecundity.

The mosquito Aedes aegypti is the main vector of Dengue, Chikungunya, Zika, and yellow fever viruses, which are responsible for high human morbidity and mortality. The fight against these pathogens is mainly based on the control of the insect vector with the use of insecticides. Among insecticides, spinosad bioinsecticide is efficient against A. aegypti larvae and may be an alternative for vector control. Here, we investigate the sublethal effects of spinosad during midgut metamorphosis of A. aegypti females and its cumulative effects on blood acquisition capacity and fecundity in adults. We studied the midgut because it is an important model organ directly related to blood acquisition and digestion. Treatment of larvae with spinosad induced oxidative stress, apoptosis, and damage to the midgut cells at all stages of development and in adults. There was a reduction in the number of proliferating cells and the number of enteroendocrine cells in treated individuals. In addition, damage caused by spinosad led to a reduction in oviposition and egg viability of A. aegypti females. Finally, the exposure of mosquito larvae to sublethal concentrations of spinosad interfered with the development of the midgut, arresting the blood digestion and reproduction of adult females with blood digestion and reproduction difficulties.

Reproductive functions in Desmodus rotundus: A comparison between seasons in a morphological context.

Reproductive seasonality in Neotropical bats has been assessed to the better understand their reproductive behavior. This knowledge is especially important for the control of Desmodus rotundus population as it is a transmitter of rabies virus. Therefore, we aimed to evaluate the functional activity of testis and epididymis of D. rotundus in dry and rainy seasons under a morphological approach. We observed an increase in tubular diameter and epithelial height of the seminiferous tubules during the rainy season. In the latter, additionally, stereological analysis of the testis showed increased proportion of seminiferous epithelium and reduced percentage of lumen. The sperm number in caput/corpus epididymis increased in rainy season, whereas sperm count and transit time were reduced in cauda region. These alterations were probably related to the recovery of epithelium activities after mating season in dry season. Despite altered nuclear and cytoplasm parameters of Leydig cells between seasons, the volume and number of these cells were constant. Moreover, no change in serum testosterone levels, daily sperm production, and apoptotic index were observed, which indicates that the reproductive pattern in D. rotundus does not change between seasons. Our study offers a baseline for the management of vampire bat population as an attempt to control rabies disease.

Histochemistry, immunohistochemistry and cytochemistry of the anterior midgut region of the stingless bee Melipona quadrifasciata and honey bee Apis mellifera (Hymenoptera: Apidae).

The anterior midgut region of stingless bees is anatomically differentiated with tall and narrow cells, whereas in other social and solitary bees this anatomical gut region is lacking. The objective of the present study was to describe the histochemistry, immunohistochemistry and cytochemistry of the anterior midgut region of the stingless bee Melipona quadrifasciata in comparison with the honey bee Apis mellifera. The anterior midgut region of both species was evaluated for identification of the enzymes ß- galactosidase, glucose-6-phosphatase, acid phosphatase, and alkaline phosphatase, the membrane transporter aquaporin, the hormone FMRF-amide, and lysosomes. Histology of the anterior midgut region showed that this region in M. quadrifasciata workers did not present external folds of the wall, whereas the following midgut wall presented many. In A. mellifera, folds in the midgut wall occur starting from the fore- midgut transition region. Despite these morphological differences, the tests evaluated were similar in both species. ß-galactosidase was not found in the anterior midgut cells. Glucose-6-phosphatase and acid phosphatase occurred in the apical region of the gut epithelium. Alkaline phosphatase occurred in vesicles in apical cytoplasm and in the basal plasma membrane infoldings of the epithelial cells. Aquaporin was found in the basal region of the midgut epithelium and in the associated visceral muscles. FMRF-amide was found only in nerve endings in the anterior midgut region. All cells in the anterior midgut region were rich in lysosomes. These results suggest that in both bee species, although they have anatomically different anterior midgut regions, these regions present high metabolic activity and function in cellular homeostasis, lipid absorption and are under neurohormone control.

Post-embryonic development of the Malpighian tubules in Apis mellifera (Hymenoptera) workers: morphology, remodeling, apoptosis, and cell proliferation.

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.

Spinosad-mediated effects on the walking ability, midgut, and Malpighian tubules of Africanized honey bee workers.

BACKGROUND: The global decline in Apis mellifera colonies is attributed to multiple factors, including pesticides. The bioinsecticide spinosad was initially recognized as safe for non-target organisms; however, its toxicity has been changing this view. Here, we investigated the survival, behavioral changes, and structural changes in the midgut and Malpighian tubules of A. mellifera treated orally with a spinosad formulation. RESULTS: The field-recommended concentration of spinosad killed 100% of the bees. The 5% and 50% lethal concentrations (LC5 and LC50 , respectively) of spinosad altered the behavioral activity, reducing the walking distance and velocity, and increased the resting time in comparison to the control. The LC50 caused disorganization of the epithelia of tested organs and induced oxidative stress and cell death. CONCLUSIONS: The present work provides new insights into the debate about the role of bioinsecticides in the mortality of Africanized honey bees. Even at very low concentrations, the spinosad formulation was toxic to the vital organs midgut and Malpighian tubules and adversely affected walking behavior. This detailed evaluation of the impact of the bioinsecticide on A. mellifera will contribute to the clarification of disturbances probably caused by spinosad formulations, which can be used to develop more sustainable protocols in agriculture. © 2017 Society of Chemical Industry.