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Morphological and histochemical analysis of trophocytes, oenocytes and pericardial cells of the Bombus morio exposure group. (A-C) stained with haematoxylin and eosin; (D-F) stained with Acridine Orange; (G-I) HSP70. Key: tr = trophocytes; en = oenocytes; n = nucleus; arrow = in (c) pericardial cells with chromatin fragmented and peripheral localisation; arrow = in (d), (e) and (f) fragmentation of the chromatin in the nuclei of the trophocytes, oenocytes and pericardial cells, respectively; pc = pericardial cells
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... 3A and 4A). In the exposed group, trophocytes and oenocytes were identified as collapsed structures; an effect that was especially pronounced in B. atratus (Figs. 3A and 3B). The oenocytes of the exposed groups lost their characteristic shape (Figs. 3B and 4B) and these cells of B. morio exhibited irregular contours and central pyknotic nuclei (Fig. 4B). In B. atratus, the oenocytes formed an indistinguishable cellular mass (Fig. ...
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... cytoplasm and an absence of pinocytosis (Figs. 1C and 2C). The pericardial cells of B. atratus, following exposure to mercury, presented pyknotic nuclei (Fig. 3C). In mercury-exposed B. morio workers, the pericardial cells were turgescent with obvious chromatin fragmentation and condensation, as well as peripherally- located lumps of chromatin (Fig. ...
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... species showed weak HSP70 fluorescence (Figs. 1I and 2I). When the exposed group of B. atratus was examined, HSP70 was not detected in the nuclei of trophocytes, oenocytes or pericardial cells (Figs. 3G-I). In the exposed group of B. morio, overexpression of HSP70 was observed in the cytoplasm of the trophocytes, oenocytes and pericardial cells (Figs. 4G-I). ...
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... 3A and 4A). In the exposed group, trophocytes and oenocytes were identified as collapsed structures; an effect that was especially pronounced in B. atratus (Figs. 3A and 3B). The oenocytes of the exposed groups lost their characteristic shape (Figs. 3B and 4B) and these cells of B. morio exhibited irregular contours and central pyknotic nuclei (Fig. 4B). In B. atratus, the oenocytes formed an indistinguishable cellular mass (Fig. ...
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... cytoplasm and an absence of pinocytosis (Figs. 1C and 2C). The pericardial cells of B. atratus, following exposure to mercury, presented pyknotic nuclei (Fig. 3C). In mercury-exposed B. morio workers, the pericardial cells were turgescent with obvious chromatin fragmentation and condensation, as well as peripherally- located lumps of chromatin (Fig. ...
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... species showed weak HSP70 fluorescence (Figs. 1I and 2I). When the exposed group of B. atratus was examined, HSP70 was not detected in the nuclei of trophocytes, oenocytes or pericardial cells (Figs. 3G-I). In the exposed group of B. morio, overexpression of HSP70 was observed in the cytoplasm of the trophocytes, oenocytes and pericardial cells (Figs. 4G-I). ...
Citations
... These cells have a mesodermal origin and are responsible for capturing, filtering, and eliminating toxic components in the hemolymph [48,54]. According to Mills and King [36], and confirmed in bees by other studies [37,44,51,55,56], the pericardial cells have four stages of activation related to stressful conditions. The presence of altered pericardial cells (stages II, III, and IV) identified in the FG1 and FG2 groups, with high cytoplasmic vacuolization and resulting peripheral displacement of the nucleus, suggests a great activity in the uptake of substances from the hemolymph [37]. ...
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.
... Toxicants such as Hg may be neutralized by such a system, being taken up by the pericardial cells, metabolized by the trophocytes and/or modified by oenocytes, or disrupt part of the entire system Domingues et al., 2017). Therefore, morphological and molecular responses of HNS when exposed by Hg estimated field concentration can be considered a precise biomarker of risk assessment for bees in response to Hg environmental contamination (Abdalla & Domingues, 2015;Ceschi-Bertoli et al., 2020;Nogueira et al., 2019;Skaldina & Sorvari, 2017). ...
... B. atratus worker bees exposed to 110 ppb of mercury had both trophocyte and oenocyte cells that were negatively impacted. In bees exposed to environmentally safe concentrations of Hg (0.2 ppb), the collapsing of the HNS cells was more drastic with the trophocytes and oenocytes becoming a mass of indistinguishable cells (Nogueira et al., 2019), something that did not happen at Hg levels of 110 ppb. That was not the first time that trace and sublethal concentrations of toxins have been observed to cause more damage to HNS cells than higher concentrations of the same trace metal Domingues et al., 2017). ...
... In a driving experiment, we found that bees normally consume 2 mL of water or a solution of 2 ppb cadmium per day but decrease this consumption at 3 ppb. We also noticed that the total ingestion of mercury 0.2 ppb is 2 mL per day (Nogueira et al., 2019). In the present study, bees consumed all 2 mL of 110 ppb Hg solution offered per day, having no consumption differences regarding the mercury concentration. ...
We analyzed the effect of mercury (Hg) on the hepato-nephrocitic system (HNS) of Bombus atratus workers exposed to an estimated concentration similar to that found in honey stores of neotropical bees (110 ppb). The bees were divided into control and experimental groups. A solid mixture of honey, pollen and organic soy was offered to both experimental groups, ad libitum. The control group received distilled water and the exposed group received a 110 µg.L⁻¹ (110 ppb) Hg solution. After 48 h of exposure, the bees were cryo-anesthetized, and the dorsal vessel was dissected directly in 4% paraformaldehyde. The samples were prepared for routine morphological analysis (HE), fluorescent histochemical staining (Acridine Orange and F-actin + DAPI), and in situ immunohistochemical labeling (Hsp70 and Hsp90). Our results showed that both the trophocytes and oenocytes of bees exposed to mercury exhibited chromatin damages. The Hg exposure also induced trophocytes deactivation of the nucleus-cytoplasm exchange as a result of branched contour loss of the nuclei. The pericardial cells were predominantly found at stage IV with pyknotic nuclei. Although the fluorescence intensity of both Hsp70 and Hsp90 was reduced in the exposed group compared to the control group, there was an indication of misfolded proteins. In conclusion, our results showed that the concentration of 110 µg.L⁻¹Hg, which can be found in the honey stores of B. atratus colonies, has severely damaged the HNS of B. atratus workers. These effects can trigger major damage to their populations, contributing to bee declines in natural environments worldwide.
The pervasiveness of human imprint on Earth is alarming and most animal species, including bees (Hymenoptera: Apoidea: Anthophila), must cope with several stressors. Recently, exposure to trace metals and metalloids (TMM) has drawn attention and has been suggested as a threat for bee populations. In this review, we aimed at bringing together all the studies (n = 59), both in laboratories and in natura, that assessed the effects of TMM on bees. After a brief comment on semantics, we listed the potential routes of exposure to soluble and insoluble (i.e. nanoparticle) TMM, and the threat posed by metallophyte plants. Then, we reviewed the studies that addressed whether bees could detect and avoid TMM in their environment, as well as the ways bee detoxify these xenobiotics. Afterwards, we listed the impacts TMM have on bees at the community, individual, physiological, histological and microbial levels. We discussed around the interspecific variations among bees, as well as around the simultaneous exposure to TMM. Finally, we highlighted that bees are likely exposed to TMM in combination or with other stressors, such as pesticides and parasites. Overall, we showed that most studies focussed on the domesticated western honey bee and mainly addressed lethal effects. Because TMM are widespread in the environment and have been shown to result in detrimental consequences, evaluating their lethal and sublethal effects on bees, including non-Apis species, warrants further investigations.
Although mercury neurotoxic effects are well known in several species, it is poorly studied in bees. Mercury contamination is increasing in several regions of the Brazilian Amazon Rainforest due to illegal and indiscriminate gold mining. Therefore, this study aimed to evaluate the effects of mercury (Hg) in brain Kenyon cells of foraging workers of Bombus atratus exposed to an average concentration (110 ppb) found in pots of honey from native bees of South America and Australia. Twenty forager workers were collected in the field (23° 34' S 47° 31' W), divided into control (n=10) and exposed (n=10) groups, and individually kept in special boxes for 48 hours. For the exposed group, we offered Hg solution (at 110 ppb) ad libitum, while for the control group we offered water, and for both sucrose syrup at 70%. After the exposure time, the bees were crio-anesthezied at 4°C. Brains were dissected and processed for morphological, morphometric, and histochemical analyses. Morphological results showed that the Kenyon cells of the Hg-exposed group presented both cytoplasmic vacuolization and nuclear pyknosis, which indicate cell death. These findings were corroborated by the acridine orange staining. Hg exposure also induced significant nuclear chromatin compaction in Kenyon cells. The calyces and peduncles of the mushroom bodies showed disorganization and vacuolization. In summary, these changes may imply in a severe impairment of the cognitive abilities of the bees, which could lead them to the loss of many tasks, such as foraging or even nest founding by the queen.
The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics.
