Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Ultrastructural Changes in the Midgut of Brazilian Native Stingless Bee Melipona scutellaris Exposed to Fungicide Pyraclostrobin.

Melipona scutellaris is a Brazilian stingless bee that is important for pollinating wild flora and agriculture crops. Fungicides have been widely used in agriculture, and floral residues can affect forager bees. The goal of our study was to evaluate the effects of sublethal concentrations of pyraclostrobin on the midgut ultrastructure of M. scutellaris forager workers. The bees were collected from three non-parental colonies and kept under laboratory conditions. The bees were orally exposed continuously for five days to pyraclostrobin in syrup at concentrations of 0.125 ng a.i./µL (FG1) and 0.005 ng a.i./µL (FG2). The control bees (CTL) were fed a no-fungicide sucrose solution, and the acetone solvent control bees (CAC) received a sucrose solution containing acetone. At the end of the exposure, the midguts were sampled, fixed in Karnovsky solution, and routinely processed for transmission electron microscopy. Ultrastructural analysis demonstrated that both the fungicide concentrations altered the midgut, such as cytoplasmic vacuolization (more intense in FG1), the presence of an atypical nuclear morphology, and slightly dilated mitochondrial cristae in the bees from the FG1 and FG2 groups (both more intense in FG1). Additionally, there was an alteration in the ultrastructure of the spherocrystals (FG1), which could be the result of cellular metabolism impairment and the excretion of toxic metabolites in the digestive cells as a response to fungicide exposure. The results indicate that ingested pyraclostrobin induced cytotoxic effects in the midgut of native stingless bees. These cellular ultrastructural responses of the midgut are a prelude to a reduced survival rate, as observed in previous studies.

Harmful Effects of Pyraclostrobin on the Fat Body and Pericardial Cells of Foragers of Africanized Honey Bee.

Managed honey bees are daily exposed in agricultural settings or wild environments to multiple stressors. Currently, fungicide residues are increasingly present in bees' pollen and nectar and can harm colonies' production and survival. Therefore, our study aimed to evaluate the effects of the fungicide pyraclostrobin on the fat body and pericardial cells of Africanized honey bees. The foragers were divided into three experimental treatment groups and two controls: pyraclostrobin 0.125 ng/µL (FG1), 0.025 ng/µL (FG2), 0.005 ng/µL (FG3), untreated control (CTL), and acetone control (CAC). After five days of oral exposure (ad libitum), the bees were dissected and prepared for histopathological and morphometric analysis. The FG1-treated bees showed extensive cytoarchitecture changes in the fat body and pericardial cells, inducing cell death. Bees from the FG2 group showed disarranged oenocytes, peripheral vacuolization, and pyknotic nuclei of pericardial cells, but the cytoarchitecture was not compromised as observed in FG1. Additionally, immune system cells were observed through the fat body in the FG1 group. Bees exposed to FG3 demonstrated only oenocytes vacuolization. A significant decrease in the oenocyte's surface area for bees exposed to all pyraclostrobin concentrations was observed compared to the CTL and CAC groups. The bees from the FG1 and FG2 treatment groups presented a reduced surface area of pericardial cells compared to the controls and the FG3 group. This study highlighted the harmful effects of fungicide pyraclostrobin concentrations at the individual bee cellular level, potentially harming the colony level on continuous exposure.

A high quality method for hemolymph collection from honeybee larvae.

The drastic decline of bees is associated with several factors, including the immune system suppression due to the increased exposure to pesticides. A widely used method to evaluate these effects on these insects' immune systems is the counting of circulating hemocytes in the hemolymph. However, the extraction of hemolymph from larvae is quite difficult, and the collected material is frequently contaminated with other tissues and gastrointestinal fluids, which complicates counting. Therefore, the present work established a high quality and easily reproducible method of extracting hemolymph from honeybee larvae (Apis mellifera), the extraction with ophthalmic scissors. Extraction methods with the following tools also were tested: 30G needle, fine-tipped forceps, hypodermic syringe, and capillaries tubes. The hemolymph was obtained via an incision on the larvae's right side for all methods, except for the extraction with ophthalmic scissors, in which the hemolymph was extracted from the head region. To assess the purity of the collected material, turbidity analyses of the samples using a turbidimeter were proposed, tested, and evaluated. The results showed that the use of ophthalmic scissors provided the clearest samples and was free from contamination. A reference range between 22,432.35 and 24,504.87 NTU (nephelometric turbidity units) was established, in which the collected samples may be considered of high quality and free from contamination.

Nanopesticide based on botanical insecticide pyrethrum and its potential effects on honeybees.

Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and nanopesticides can control agricultural pests and increase farm productivity with little environmental impact. However, it is important to evaluate their toxicity on non-target organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term toxicity on a non-target organism (honeybee). SLN + PYR was physicochemically stable after 120 days. SLN + PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a polydispersion index of 0.39 ± 0.12 nm. SLN + PYR had an encapsulation efficiency of 99%. The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological alterations in the bees' midguts, whereas pyrethrum-loaded nanoparticles had no significant effect on digestive cells, so are considered safer, at least in the short term, for honeybees. These results are important in understanding the effects of nanopesticides on beneficial insects and may decrease the environmental impacts of pesticides.

Late effect of larval co-exposure to the insecticide clothianidin and fungicide pyraclostrobin in Africanized Apis mellifera.

Among the factors that contribute to the reduction of honeybee populations are the pesticides. These chemical compounds reach the hive through forager bees, and once there, they can be ingested by the larvae. We evaluated the effects of repeated larval exposure to neonicotinoid insecticide, both in isolation and in combination with strobilurin fungicide, at environmentally relevant doses. The total consumption of the contaminated diet was 23.63 ng fungicide/larvae (pyraclostrobin) and 0.2364 ng insecticide/larvae (clothianidin). The effects on post-embryonic development were evaluated over time. Additionally, we assessed the survival pattern of worker bees after emergence, and the pesticides' effects on the behavior of newly emerged workers and young workers. Young bees that were exposed to the fungicide and those subjected to co-exposure to both pesticides during larval phase showed behavioral changes. The insecticide, both in isolation and in combination with fungicide reduced the bees' longevity; this effect of larval exposure to pesticides was stronger in bees that were exposed only to the insecticide. Although the larvae did not have sensitivity to exposure to pesticides, they showed later effects after emergence, which may compromise the dynamics of the colony, contributing to the reduction of the populations of bees in agroecosystems.

Vitamin E supplementation and caloric restriction promotes regulation of insulin secretion and glycemic homeostasis by different mechanisms in rats.

Vitamin E and caloric restriction have antioxidant effects in mammals. The aim of this study was to evaluate effects of vitamin E supplementation and caloric restriction upon insulin secretion and glucose homeostasis in rats. Male Wistar rats were distributed among the following groups: C, control group fed ad libitum; R, food quantity reduction of 40%; CV, control group supplemented with vitamin E [30 mg·kg-1·day-1]; and RV, food-restricted group supplemented with vitamin E. The experiments ran for 21 days. Glucose tolerance and insulin sensitivity was higher in the CV, R, and RV groups. Insulin secretion stimulated with different glucose concentrations was lower in the R and RV groups, compared with C and CV. In the presence of glucose and secretagogues, insulin secretion was higher in the CV group and was lower in the R and RV groups. An increase in insulin receptor occurred in the fat pad and muscle tissue of groups CV, R, and RV. Levels of hepatic insulin receptor and phospho-Akt protein were higher in groups R and RV, compared with C and CV, while muscle phospho-Akt was increased in the CV group. There was a reduction in hepatic RNA levels of the hepatocyte growth factor gene and insulin degrading enzyme in the R group, and increased levels of insulin degrading enzyme in the CV and RV groups. Thus, vitamin E supplementation and caloric restriction modulate insulin secretion by different mechanisms to maintain glucose homeostasis.

Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee.

Apis mellifera perform important pollination roles in agroecosystems. However, there is often intensive use of systemic pesticides in crops, which can be carried to the colony by forage bees through the collection of contaminated pollen and nectar. Inside the colony, pollen loads are stored by bees that add honey and several enzymes to this pollen. Nevertheless, intra-colonial chronic exposure could induce sublethal effects in young bees exposed to a wide range of pesticides present in these pollen loads. This study was aimed to both determine the survival rate and evaluate the sublethal effects on the hepato-nephrocitic system in response to continuous oral exposure to lower concentrations of neonicotinoid thiamethoxam (TXT) and picoxystrobin fungicide (PXT). Exposure to a single chemical and co-exposure to both pesticides were performed in newly emerged honeybee workers. A significant decrease in the bee survival rates was observed following exposure to TXT (0.001 ng a.i./µL) and PXT (0.018 ng a.i./µL), as well as following co-exposure to TXT+PXT/2. After five days of continuous exposure, TXT induced sub-lethal effects in the organs involved in the detoxification of xenobiotics, such as the fat body and pericardial cells, and it also induced a significant increase in the hemocyte number. Thus, the hepato-nephrocitic system (HNS) reached the greatest level of activity of pericardial cells as an attempt to eliminate this toxic compound from hemolymph. The HNS was activated at low levels by PXT without an increase in the hemocyte number; however, the mobilization of neutral glycoconjugates from the trophocytes of the fat body was prominent only in this group. TXT and PXT co-exposure induced intermediary morphological effects in trophocytes and pericardial cells, but oenocytes from the fat body presented with atypical cytoplasm granulation only in this group. These data showed that the realistic concentrations of these pesticides are harmful to newly emerged Africanized honeybees, indicating that intra-colonial chronic exposure drastically reduces the longevity of bees exposed to neonicotinoid insecticide (TXT) and the fungicide strobilurin (PXT) as in single and co-exposure. Additionally, the sublethal effects observed in the organs constituting the HNS suggest that the activation of this system, even during exposure to low concentrations of theses pesticides, is an attempt to maintain homeostasis of the bees. These data together are alarming because these pesticides can affect the performance of the entire colony.