Sublethal exposure to thiamethoxam and pyraclostrobin affects the midgut and Malpighian tubules of the stingless bee Frieseomelitta varia (Hymenoptera: Apidae: Meliponini).

The stingless bee Frieseomelitta varia Lepeletier 1836 (Hymenoptera: Apidae) is an essential pollinator in natural and agricultural ecosystems in the Neotropical region. However, these bees may be exposed to pesticides during foraging, which can affect both individuals and their colonies. One example comes from the use of pyraclostrobin (a fungicide) and thiamethoxam (an insecticide) for pest control in pepper crops, which F. varia visits. This study aimed to evaluate the isolated and combined sublethal effects of thiamethoxam (TMX) (0.000543 ng a.i./µL) and pyraclostrobin (PYR) (1.5 ng i.a./µL) on the morphology of the midgut and Malpighian tubules of F. varia workers. Results showed that both pesticides, regardless of the exposure time (through feeding during 48 h or 96 h), disturbed the morphology of the analyzed organs. Specifically, F. varia exposed orally to sublethal concentrations of thiamethoxam and pyraclostrobin, either alone or in combination, exhibited a higher rate of damage to the midgut (e.g., vacuolization, apocrine secretion, and cellular elimination) compared to the bees in the control groups, both after 48 h and 96 h of exposure. In Malpighian tubules, vacuolation is the only damage present. As the observed morphological alterations likely compromise the excretion and absorption functions, exposure to pyraclostrobin and thiamethoxam may lead to disturbances at both the individual and colony levels. These results highlight the urgent need for a future reassessment of the safety of fungicides and insecticides regarding their potential effects on bee populations.

Exposure to the herbicide tebuthiuron affects behavior, enzymatic activity, morphology and physiology of the midgut of the stingless bee Partamona helleri.

Partamona helleri is an important pollinator in the Neotropics. However, this bee faces an increased risk of pesticide exposure, potentially affecting both individual bees and entire colonies. Thus, this study aimed to evaluate the effects of the herbicide tebuthiuron on behavior, antioxidant activity, midgut morphology, and signaling pathways related to cell death, cell proliferation and differentiation in P. helleri workers. tebuthiuron significantly reduced locomotor activity and induced morphological changes in the midgut. The activity of the detoxification enzymes superoxide dismutase and glutathione S-transferase increased after exposure, indicating a detoxification mechanism. Furthermore, the herbicide led to alterations in the number of positive cells for signaling-pathway proteins in the midgut of bees, suggesting induction of apoptotic cell death and disruption of midgut epithelial regeneration. Therefore, tebuthiuron may negatively impact the behavior, antioxidant activity, morphology, and physiology of P. helleri workers, potentially posing a threat to the survival of this non-target organism.

Acute exposure to fungicide fluazinam induces cell death in the midgut, oxidative stress and alters behavior of the stingless bee Partamona helleri (Hymenoptera: Apidae).

Stingless bees (Hymenoptera: Meliponini) are pollinators of both cultivated and wild crop plants in the Neotropical region. However, they are susceptible to pesticide exposure during foraging activities. The fungicide fluazinam is commonly applied in bean and sunflower cultivation during the flowering period, posing a potential risk to the stingless bee Partamona helleri, which serves as a pollinator for these crops. In this study, we investigated the impact of acute oral exposure (24 h) fluazinam on the survival, morphology and cell death signaling pathways in the midgut, oxidative stress and behavior of P. helleri worker bees. Worker bees were exposed for 24 h to fluazinam (field concentrations 0.5, 1.5 and 2.5 mg a.i. mL-1), diluted in 50 % honey aqueous solution. After oral exposure, fluazinam did not harm the survival of worker bees. However, sublethal effects were revealed using the highest concentration of fluazinam (2.5 mg a.i. mL-1), particularly a reduction in food consumption, damage in the midgut epithelium, characterized by degeneration of the brush border, an increase in the number and size of cytoplasm vacuoles, condensation of nuclear chromatin, and an increase in the release of cell fragments into the gut lumen. Bees exposed to fluazinam exhibited an increase in cells undergoing autophagy and apoptosis, indicating cell death in the midgut epithelium. Furthermore, the fungicide induced oxidative stress as evidenced by an increase in total antioxidant and catalase enzyme activities, along with a decrease in glutathione S-transferase activity. And finally, fluazinam altered the walking behavior of bees, which could potentially impede their foraging activities. In conclusion, our findings indicate that fluazinam at field concentrations is not lethal for workers P. helleri. Nevertheless, it has side effects on midgut integrity, oxidative stress and worker bee behavior, pointing to potential risks for this pollinator.

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.

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology.

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

Exposure to sublethal concentrations of imidacloprid, pyraclostrobin, and glyphosate harm the behavior and fat body cells of the stingless bee Scaptotrigona postica.

Pesticide use in agriculture threatens non-target insects such as bees. Considering the ecological and economic relevance of native bees, such as Scaptotrigona postica, and the insufficient studies on the effects of pesticides on their behavior and physiology, improving the current knowledge on this issue is essential. Therefore, this study investigated the sublethal effects of imidacloprid, pyraclostrobin, and glyphosate on the behavior and fat body cells of S. postica. Pesticide ingestion decreased the walking distance and mean velocity of bees compared to the control and solvent control groups. The oenocytes of the control groups were spherical, with central nuclei containing decondensed chromatin, and the trophocytes presented irregular morphology, with cells varying in shape and the cytoplasm filled with vacuoles and granules. However, bees exposed to pesticides showed extensive cytoarchitectural disruption in the fat body, such as vacuolization and shape changes in oenocytes and altered nuclei morphology in trophocytes. Moreover, pesticide exposure increased the number of atypical oenocytes and altered trophocytes, except for the PYR group, which showed a lower number of atypical oenocytes. Caspase-positive labeling significantly increased in all exposed bee groups. Alternatively, TLR4 labeling was significantly decreased in the exposed groups compared to the control groups. There was a significant increase in HSP90 immunolabeling in all exposed groups compared to the control. These findings reinforce the importance of research on the sublethal effects of low pesticide concentrations on key neotropical pollinators and prove that these toxic substances can impair their detoxification and immune defense.

Effects of chronic exposure to sublethal doses of neonicotinoids in the social wasp Polybia paulista: Survival, mobility, and histopathology.

Several studies have investigated the consequences of exposure to neonicotinoids in honeybees. Given the lack of studies concerning the consequences of exposure of social wasps to neonicotinoids, as well as the ecological importance of these insects, the objective of this study was to test the hypothesis that chronic exposure to sublethal concentrations of thiamethoxam decreases survival and mobility by causing damage to the brain and midgut of the social wasp Polybia paulista. The wasps were exposed to different concentrations of thiamethoxam, in order to obtain the mean lethal concentration (LC50), which was used as a reference for calculation of two sublethal concentrations (LC50/100 and LC50/10) employed in subsequent experiments. To calculate survival, groups of exposed (EW) and unexposed (UW) wasps were monitored until death, allowing calculation of the average lethal time. The EW and UW groups were evaluated after 12, 24, 48, and 72 h of exposure, considering their mobility and histopathological parameters of the midgut and brain. A lesion index based on semiquantitative analyses was used for comparison of histopathological damage. The results demonstrated that exposure to the LC50/10 led to a significantly shorter survival time of the P. paulista workers, compared to unexposed wasps. In addition, both sublethal concentrations decreased mobility and caused damage to the intestine (loss of brush border, presence of spherocrystals, loss of cytoplasmic material, and pyknosis) and the brain (loss of cell contact and pyknosis), regardless of the exposure time. The findings showed that, like bees, social wasps are nontarget insects susceptible to the detrimental consequences of neonicotinoid use, with exposure leading to impaired survival, locomotion, and physiology.

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.

Lethal and sublethal effects of an emulsion based on Pogostemon cablin (Lamiaceae) essential oil on the coffee berry borer, Hypothenemus hampei.

The global search for eco-friendly and human-safe pesticides has intensified, and research on essential oils (EOs) has expanded due to their remarkable insecticidal activities and apparent human-safe. Despite this, most of the literature focuses on short-term and simplified efforts to understand lethal effects, with only a few comprehensive studies addressing sublethal exposures. To fill this shortcoming, we explore the lethal and sublethal effects of Pogostemon cablin (Lamiaceae) EO and an EO-based emulsion (18%) using the coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae) as a model. First, we determine the toxicity of EO and EO-based emulsion using dose-mortality curves and lethal times. Second, we subjected adult females of H. hampei to sublethal doses to assess whether they affected their behavior, reproductive output, and histological features. Our findings reveal that patchoulol (43.05%), α-Guaiene (16.06%), and α-Bulnesene (13.69%) were the main components of the EO. Furthermore, the EO and its emulsion had similar toxicity, with dose-mortality curves and lethal times overlapping 95% confidence intervals. We also observed that sublethal exposure of females of H. hampei reduces reproduction and feeding, increases walking activity, and causes histopathological changes in the midgut. This study advances the knowledge of the lethal and sublethal effects of an eco-friendly substance on insects.Responsible Editor: Giovanni Benelli.

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.

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.

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.

Pest potencial of Sitophilus zeamais on Brazil nut under storage conditions: Sitophilus zeamais on Brazil nut / Potencial de Sitophilus zeamais como praga da castanha-do-Brasil em condições de armazenamento

The Brazil nut, fruit of Bertholletia excelsa H.B.K., a native plant of the Amazon region and may be considered one of the main extractivist products of countries like Bolivia, Colombia, Guyana, Venezuela, Peru and Brazil. In Brazil this plant can be found in the states of Acre, Amapá, Amazonas, Mato Grosso, Pará, Rondônia, Roraima and Tocantins. Species of the Coleoptera and Lepidoptera, have been classified as pest potentials of Brazil nut under storage conditions. Sitophilus zeamais (Coleoptera: Curculionidae) is one of the main insect pests attacking grain stores worldwide. This specie may be observed attacking grains in the field and in storage facilities (cross-infestation), presenting a wide variety of hosts. The purpose of this study was to assess the potential of adults of this species feed on Brazil nuts, and also to identify the type of injury as well as the life span of this coleopteran exposed to the meat of the Brazil nut. No feeding injury was observed on the surface of Brazil nuts and the life span of the insects was similar in treatments both with and without this food source in all populations evaluated. Due to the fact that S. zeamais adults do not injure, or feed on the Brazil nut, this beetle is not able to colonize this product and therefore may be considered a non-pest insect for stored Bertholletia excelsa almonds stored.

A castanha-do-brasil, fruto da Bertholletia excelsa H.B.K., que é uma planta nativa da região Amazônica e esse fruto pode ser considerado um dos principais produtos extrativistas de países como Bolívia, Colômbia, Guiana, Venezuela, Peru e Brasil. No Brasil, essa planta pode ser encontrada nos estados do Acre, Amapá, Amazonas, Mato Grosso, Pará, Rondônia, Roraima e Tocantins. Espécies de Coleoptera e Lepidoptera foram classificadas como potenciais pragas da castanha-do-brasil em condições de armazenamento. Sitophilus zeamais (Coleoptera: Curculionidae) é uma das principais pragas de insetos que atacam os armazéns de grãos em todo o mundo. Esta espécie pode ser observada atacando grãos no campo e em unidades armazenadoras (infestação cruzada), apresentando uma grande variedade de hospedeiros. O objetivo deste estudo foi avaliar o potencial de adultos dessa espécie em se alimentarem de castanha-do-brasil, e também, identificar o tipo de lesão e o tempo de vida deste coleóptero exposto castanha-do-brasil. Não foi observada lesão na superfície da castanha-do-brasil e o tempo de vida dos insetos foram semelhantes nos tratamentos com e sem alimento em todas as populações avaliadas. Devido ao fato de que adultos de S. zeamaisnão lesionaram ou se alimentaram da castanha-do-brasil, este besouro não é capaz de colonizar este produto e, portanto, pode ser considerado um inseto não-praga para as amêndoas de Bertholletia excelsa armazenadas.

Morphology of ovary and spermathecae of the parasitoid Eibesfeldtphora tonhascai Brown (Diptera: Phoridae).

Eibesfeldtphora tonhascai (Diptera: Phoridae) is a parasitoid of leaf-cutting ants and a potential biological control agent against these insect pests. This study describes the morphology of the ovary and spermatheca of E. tonhascai. The female reproductive tract of this parasitoid has a pair of meroistic polytrophic ovaries, two lateral oviducts that open into a common oviduct, an elongated accessory gland, and two spermathecae. Young oocytes are small and spherical, and their size increases as yolk is stored in the cytoplasm. This process is followed by chorion production by follicular cells. Mature oocytes are elliptical or torpedo-shaped. The reservoir wall of the spermatheca has type III glandular cells with cytoplasm rich in free ribosomes, rough endoplasmic reticulum, and secretory vesicles. The apical surface of these cells has microvilli associated with mitochondria. The reservoir lumen is lined by a cuticle and filled with spermatozoa. This is the first report of the ovary and spermatheca morphology of E. tonhascai and contributes to the comprehension of the reproductive biology of this parasitoid of leaf-cutting ants.

Parasitism, sexual dimorphism and effect of host size on Apocephalus attophilus offspring, a parasitoid of the leaf-cutting ant Atta bisphaerica.

Atta bisphaerica (Forel) is a leaf-cutting ant that specializes on grass and causes productivity losses in sugar cane fields and pastures. Three phorid species, Apocephalus attophilus (Borgmeier), Myrmosicarius grandicornis (Borgmeier) and Eibesfeldtphora bragancai (Brown), have been found parasitizing A. bisphaerica workers. These parasitoids can reduce plant material transported into the nests and ant traffic on the trails. Therefore, phorid flies have been considered potential biological control agents for leaf-cutting ants. Here, we evaluated which parasitoid species attack the leaf-cutting ant A. bisphaerica in pasture areas of a Brazilian Savannah-Atlantic Forest ecotone, parasitism rate, effect of host size, sexual dimorphism and sex ratio of the emerged parasitoids. Four nests of A. bisphaerica were selected in pasture areas from August 2016 to August 2017, with 400 workers collected from each colony monthly. A total of 23,714 A. bisphaerica workers were collected during the study, of which 236 (0.99%) were parasitized by phorid parasitoids. Apocephalus attophilus, E. bragancai and M. grandicornis parasitized 217, 17 and 2 ants, respectively. The higher parasitism rate was found in the hottest/rainy season of the year. Non-parasitized ants survived longer than those parasitized by A. attophilus. The larval and pupal periods of this parasitoid were 2.2 ± 0.8 and 16 ± 1.4 days, respectively, and the number of pupae per parasitized ant ranged from 1 to 7. The number of A. attophilus pupae per host increased with the host head size. Likewise, the size of the adult parasitoids also increased according to the host ant. Apocephalus attophilus females were larger than males and the sex ratio (male: female) did not differ from 1: 1. Our results showed that A. attophilus would be a potential biocontrol agent of leaf-cutting ants because it produces multiple larvae per host, allowing a great production of parasitoids with short developmental time and kills the host ant faster than other phorids.