亚致死剂量杀虫剂对意蜂工蜂嗅觉敏感性的影响

喙伸反应(PER)试验适用于评价杀虫剂对蜜蜂行为的影响。本实验利用喙伸反应研究了亚致死剂量(LD50/100～LD50/10)溴氰菊酯和吡虫啉对意蜂Apis mellifera ligustica L.工蜂嗅觉敏感性的影响。结果发现,经口饲喂溴氰菊酯5ng和10ng后,工蜂对0.1%的蔗糖溶液的敏感性显著下降(P<0.05),水应激指数降低,但对0.3%,1%,3%,10%和30%的蔗糖溶液的敏感性没有显著变化;而经口饲喂吡虫啉0.3ng和0.6ng后,工蜂对上述各浓度蔗糖溶液的敏感性变化不明显,但其水应激指数升高。     

RFID tracking of sublethal effects of two neonicotinoid insecticides on the foraging behavior of Apis mellifera

The development of insecticides requires valid risk assessment procedures to avoid causing harm to beneficial insects and especially to pollinators such as the honeybee Apis mellifera. In addition to testing according to current guidelines designed to detect bee mortality, tests are needed to determine possible sublethal effects interfering with the animal's vitality and behavioral performance. Several methods have been used to detect sublethal effects of different insecticides under laboratory conditions using olfactory conditioning. Furthermore, studies have been conducted on the influence insecticides have on foraging activity and homing ability which require time-consuming visual observation. We tested an experimental design using the radiofrequency identification (RFID) method to monitor the influence of sublethal doses of insecticides on individual honeybee foragers on an automated basis. With electronic readers positioned at the hive entrance and at an artificial food source, we obtained quantifiable data on honeybee foraging behavior. This enabled us to efficiently retrieve detailed information on flight parameters. We compared several groups of bees, fed simultaneously with different dosages of a tested substance. With this experimental approach we monitored the acute effects of sublethal doses of the neonicotinoids imidacloprid (0.15-6 ng/bee) and clothianidin (0.05-2 ng/bee) under field-like circumstances. At field-relevant doses for nectar and pollen no adverse effects were observed for either substance. Both substances led to a significant reduction of foraging activity and to longer foraging flights at doses of ≥0.5 ng/bee (clothianidin) and ≥1.5 ng/bee (imidacloprid) during the first three hours after treatment. This study demonstrates that the RFID-method is an effective way to record short-term alterations in foraging activity after insecticides have been administered once, orally, to individual bees. We contribute further information on the understanding of how honeybees are affected by sublethal doses of insecticides.     

Assessment of Chronic Sublethal Effects of Imidacloprid on Honey Bee Colony Health

Here we present results of a three-year study to determine the fate of imidacloprid residues in hive matrices and to assess chronic sublethal effects on whole honey bee colonies fed supplemental pollen diet containing imidacloprid at 5, 20 and 100 μg/kg over multiple brood cycles. Various endpoints of colony performance and foraging behavior were measured during and after exposure, including winter survival. Imidacloprid residues became diluted or non-detectable within colonies due to the processing of beebread and honey and the rapid metabolism of the chemical. Imidacloprid exposure doses up to 100 μg/kg had no significant effects on foraging activity or other colony performance indicators during and shortly after exposure. Diseases and pest species did not affect colony health but infestations of Varroa mites were significantly higher in exposed colonies. Honey stores indicated that exposed colonies may have avoided the contaminated food. Imidacloprid dose effects was delayed later in the summer, when colonies exposed to 20 and 100 μg/kg experienced higher rates of queen failure and broodless periods, which led to weaker colonies going into the winter. Pooled over two years, winter survival of colonies averaged 85.7, 72.4, 61.2 and 59.2% in the control, 5, 20 and 100 μg/kg treatment groups, respectively. Analysis of colony survival data showed a significant dose effect, and all contrast tests comparing survival between control and treatment groups were significant, except for colonies exposed to 5 μg/kg. Given the weight of evidence, chronic exposure to imidacloprid at the higher range of field doses (20 to 100 μg/kg) in pollen of certain treated crops could cause negative impacts on honey bee colony health and reduced overwintering success, but the most likely encountered high range of field doses relevant for seed-treated crops (5 μg/kg) had negligible effects on colony health and are unlikely a sole cause of colony declines.     

Effects of Sublethal Doses of Imidacloprid on Young Adult Honeybee Behaviour

Imidacloprid (IMI), a neonicotinoid used for its high selective toxicity to insects, is one of the most commonly used pesticides. However, its effect on beneficial insects such as the honeybee Apis mellifera L is still controversial. As young adult workers perform in-hive duties that are crucial for colony maintenance and survival, we aimed to assess the effect of sublethal IMI doses on honeybee behaviour during this period. Also, because this insecticide acts as a cholinergic-nicotinic agonist and these pathways take part in insect learning and memory processes; we used IMI to assess their role and the changes they suffer along early adulthood. We focused on appetitive behaviours based on the proboscis extension response. Laboratory reared adults of 2 to 10 days of age were exposed to sublethal IMI doses (0.25 or 0.50ng) administered orally or topically prior to behavioural assessment. Modification of gustatory responsiveness and impairment of learning and memory were found as a result of IMI exposure. These outcomes differed depending on age of evaluation, type of exposure and IMI dose, being the youngest bees more sensitive and the highest oral dose more toxic. Altogether, these results imply that IMI administered at levels found in agroecosystems can reduce sensitivity to reward and impair associative learning in young honeybees. Therefore, once a nectar inflow with IMI traces is distributed within the hive, it could impair in-door duties with negative consequences on colony performance.     

Acute exposure to a sublethal dose of imidacloprid and coumaphos enhances olfactory learning and memory in the honeybee Apis mellifera

The decline of honeybees and other pollinating insects is a current cause for concern. A major factor implicated in their decline is exposure to agricultural chemicals, in particular the neonicotinoid insecticides such as imidacloprid. Honeybees are also subjected to additional chemical exposure when beekeepers treat hives with acaricides to combat the mite Varroa destructor. Here, we assess the effects of acute sublethal doses of the neonicotinoid imidacloprid, and the organophosphate acaricide coumaphos, on honey bee learning and memory. Imidacloprid had little effect on performance in a six-trial olfactory conditioning assay, while coumaphos caused a modest impairment. We report a surprising lack of additive adverse effects when both compounds were administered simultaneously, which instead produced a modest improvement in learning and memory.     

亚致死剂量吡虫啉胁迫对意大利蜜蜂内勤蜂与外勤蜂免疫解毒相关基因表达及免疫解毒酶系活力的影响

摘要: 【目的】吡虫啉(imidaclorprid)是广泛使用的新烟碱类杀虫剂之一。大量研究表明亚致死剂量吡虫啉影响意大利蜜蜂Apis mellifera ligustica(简称“意蜂”)幼虫的发育和成年蜜蜂的采集、学习等行为。本实验旨在探究亚致死剂量吡虫啉对意大利蜜蜂内勤蜂(1日龄成年工蜂)与外勤蜂(21日龄成年工蜂)免疫解毒相关基因表达及免疫解毒酶系活力的影响,进而为蜜蜂健康的维护提供科学依据。【方法】测定饲喂含0.1 ng/μL吡虫啉的50%蔗糖溶液不同时间后意蜂成年工蜂的存活率;利用荧光定量PCR检测饲喂含0.1 ng/μL吡虫啉的50%蔗糖溶液6 d后其体内免疫基因多酚氧化酶基因(PPOA3, GenBank登录号： GB43738), Abaecin类抗菌肽基因(ABA, GenBank登录号： GB18323),葡萄糖脱氢酶基因(GLD, GenBank登录号： GB43007)和解毒基因细胞色素P450基因(CYP450 6a2, GenBank登录号： GB49876)的表达,并采用双抗体一步夹心法酶联免疫吸附试验测定其体内细胞色素P450酶(cytochrome P450, CYP450)含量和多酚氧化酶(polyphenol oxidase,PPO)活力。【结果】1日龄和21日龄意蜂成年工蜂连续饲喂6 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,其存活率与对照组(饲喂含0.1 ng/μL丙酮的50%蔗糖溶液)无显著差异;连续饲喂9 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,1日龄意蜂成年工蜂存活率与对照组无显著差异,而21日龄意蜂成年工蜂存活率与对照组有显著差异。1日龄意蜂成年工蜂自由取食含0.1 ng/μL吡虫啉的蔗糖溶液6 d后, PPOA3, CYP450 6a2, ABA和GLD表达水平,细胞色素P450含量以及多酚氧化酶活力与对照组相比均有显著下调趋势;而21日龄意蜂成年工蜂取食该药液6 d后,CYP450 6a2, ABA和GLD表达水平及多酚氧化酶活力与对照组相比均有显著下调趋势,PPOA3表达水平和细胞色素P450含量有显著上调趋势。【结论】亚致死剂量吡虫啉影响意大利蜜蜂内勤蜂与外勤蜂免疫解毒相关基因的表达及免疫解毒酶系活力;吡虫啉短期胁迫对意大利蜜蜂内勤蜂与外勤蜂的存活无显著影响,长期胁迫则会影响意大利蜜蜂内勤蜂与外勤蜂的存活。     

Honeybees (Apis mellifera) modulate dance communication in response to pollution by imidacloprid

Imidacloprid, one of the most commonly used insecticides, is highly toxic to honeybees and other beneficial insects. Imidacloprid is a chloronicotinyl insecticide, which has a highly specific affinity to the nicotinic acetylcholine receptors (nAChRs) in the honeybee’s nervous system. So it may interfere with dance behavior and memory formation. We found the waggle dances were modulated in honeybees fed sucrose water containing imidacloprid (pesticide group) compared to those fed normal sucrose water (control group). In our data, dancers of the pesticide group significantly increased the variance of divergence angle and the return phases in waggle dances than the control group. And the dance followers in pesticide group significantly increased the variance of crop content than the control group. Furthermore, four learning and memory related genes were significantly regulated at the gene expression levels between pesticide and control group. Our data revealed that the sub-lethal dose of imidacloprid impaired the honeybees’ learning and memory and resulted in cognitive disorder. The dancers may adjust their recruitment behavior leading to the observed reduced number of followers. We conclude that modulation of in-hive communication serves to protect the colony from foraging toxic food.     

Impaired Olfactory Associative Behavior of Honeybee Workers Due to Contamination of Imidacloprid in the Larval Stage

The residue of imidacloprid in the nectar and pollens of the plants is toxic not only to adult honeybees but also the larvae. Our understanding of the risk of imidacloprid to larvae of the honeybees is still in a very early stage. In this study, the capped-brood, pupation and eclosion rates of the honeybee larvae were recorded after treating them directly in the hive with different dosages of imidacloprid. The brood-capped rates of the larvae decreased significantly when the dosages increased from 24 to 8000 ng/larva. However, there were no significant effects of DMSO or 0.4 ng of imidacloprid per larva on the brood-capped, pupation and eclosion rates. Although the sublethal dosage of imidacloprid had no effect on the eclosion rate, we found that the olfactory associative behavior of the adult bees was impaired if they had been treated with 0.04 ng/larva imidacloprid in the larval stage. These results demonstrate that a sublethal dosage of imidacloprid given to the larvae affects the subsequent associative ability of the adult honeybee workers. Thus, a low dose of imidacloprid may affect the survival condition of the entire colony, even though the larvae survive to adulthood.     

The effect of temperature on candidate gene expression in the brain of honey bee Apis mellifera (Hymenoptera: Apidae) workers exposed to neonicotinoid imidacloprid

Neonicotinoid insecticides are potent agonists of nicotinic acetylcholine receptors and are a major factor in the decline of pollinators worldwide. Several studies show that low doses of this neurotoxin influence honey bee physiology, however, little is known about how insecticides interact with other environmental variables. We studied the effects of two neonicotinoid Imidacloprid doses (IMD, 0, 2.5, and 10 ppb), and three temperatures (20, 28, and 36°C) on gene expression in the brains of worker honey bees (Apis mellifera). Using qRT-PCR we quantified the expression of eight key genes related to the nervous system, stress response, and motor and olfactory capacities. Gene expression tended to increase with the low IMD dose, which was further intensified in individuals maintained in the cold treatment (20°C). At 20°C the octopamine receptor gene (oa1) was underexpressed in bees that were not exposed to IMD, but overexpressed in individuals exposed to 2.5 ppb IMD. Also, heat shock proteins (hsp70 and hsp90) increased their expression at high temperatures (36°C), but not with IMD doses. These results suggest that despite the low insecticide concentrations used in this study (a field-realistic dose), changes in gene expression associated with honey bee physiological responses could be induced. This study contributes to the understanding of how neonicotinoid residual doses may alter honey bee physiology.     

Proteome Changes Paralleling the Olfactory Conditioning in the Forager Honey Bee and Provision of a Brain Proteomics Dataset

The olfactory conditioning of the bee proboscis extension reflex (PER) is extensively used as a paradigm in associative learning of invertebrates but with limited molecular investigations. To investigate which protein changes are linked to olfactory conditioning, a non‐sophisticated conditioning model is applied using the PER in the honeybee (Apis mellifera). Foraging honeybees are assigned into three groups based on the reflex behavior and training: conditioned using 2‐octanone (PER‐conditioned), and sucrose and water controls. Thereafter, the brain synaptosomal proteins are isolated and analyzed by quantitative proteomics using stable isotope labeling (TMT). Additionally, the complex proteome dataset of the bee brain is generated with a total number of 5411 protein groups, including key players in neurotransmitter signaling. The most significant categories affected during olfactory conditioning are associated with “SNARE interactions in vesicular transport” (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.     

Long‐term risk assessment on noneffective and effective toxic doses of imidacloprid to honeybee workers

Imidacloprid is the most widely used insecticide in agriculture. Its impact on honeybees has received worldwide attention. Foliar sprays are commonly and frequently used for piercing insect control, particularly on cotton in southern USA. To simulate field exposures of formulated imidacloprid (Advise^® 2Fl), we used a modified spray tower to treat honeybee workers and monitored five enzyme activities and survival for up to 52 days. Results indicated that spray treatments twice a week for 52 days with 0.001 and 1 mg/L and once a week for three weeks with 4.3 mg/L Advise showed no adverse effect on bee survival, where imidacloprid‐treated bees could live as long as untreated bees. Concentration ≥80 mg/L significantly reduced bee survival, and substantial number of bees continued to die after 48‐hr of post‐treatment period which was commonly used for measuring insecticide toxicity. The body weight of imidacloprid‐treated bees (at LC₂₀ and LC₅₀) was also significantly reduced. Enzymatic data showed that activities of detoxification enzymes esterase and glutathione S‐transferase (GST), insecticide‐target enzyme acetylcholinesterase (AChE) and honey enzyme invertase in imidacloprid‐treated survivors were mostly similar to those found in untreated bees. The immunity‐related phenoloxidase (PO) activity in imidacloprid‐treated survivors was also mostly similar to that of untreated control, but higher PO activity was detected in bees treated with higher concentrations for 3 weeks. By using both bioassays and enzymatic assays, this study revealed long‐term noneffective and effective concentrations of imidacloprid that may be useful for accurate assessment of toxicity risk of neonicotinoids to bees.     

A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee’s locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD_48h, i.e. causing no mortality at 48h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD_48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.     

Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers,Apis mellifera

Pollinators, honey bees in particular, are continuously exposed to various mixtures of pesticides, which contribute to their population decline. Both amitraz and thiacloprid have been proven less toxic to honey bees and are frequently applied in- and out-hive, respectively. We examined the sub-lethal effects of amitraz, thiacloprid and their sequential exposure on learning, memory and sugar responsiveness in Apis mellifera using the Proboscis extension response (PER). Sub-lethal doses of amitraz (0.1, 0.2 and 0.4 µg/bee) and thiacloprid (0.05, 0.1 and 0.2 µg/bee) were tested. Sub-lethal effects were observed only at the highest doses of each pesticide treatment; amitraz (0.4 µg/bee) and thiacloprid (0.2 µg/bee) but not in lower doses. In sequential treatment of amitraz and thiacloprid, reduced acquisition and memory retention were significant across all tested doses. The same profile was also obtained on sugar responsiveness of foragers. Our results suggest that the sequential exposure would pose higher risk to honey bee compared to single pesticide exposure by reducing the bees’ appetitive olfactory learning, memory and sugar acuity more than individual pesticide exposures.     

Odor discrimination in classical conditioning of proboscis extension in two stingless bee species in comparison to Africanized honeybees

Learning in insects has been extensively studied using different experimental approaches. One of them, the proboscis extension response (PER) paradigm, is particularly well suited for quantitative studies of cognitive abilities of honeybees under controlled conditions. The goal of this study was to analyze the capability of three eusocial bee species to be olfactory conditioned in the PER paradigm. We worked with two Brazilian stingless bees species, Melipona quadrifasciata and Scaptotrigona aff. depilis, and with the invasive Africanized honeybee, Apis mellifera. These three species present very different recruitment strategies, which could be related with different odor-learning abilities. We evaluated their gustatory responsiveness and learning capability to discriminate floral odors. Gustatory responsiveness was similar for the three species, although S. aff. depilis workers showed fluctuations along the experimental period. Results for the learning assays revealed that M. quadrifasciata workers can be conditioned to discriminate floral odors in a classical differential conditioning protocol and that this discrimination is maintained 15 min after training. During conditioning, Africanized honeybees presented the highest discrimination, for M. quadrifasciata it was intermediate, and S. aff. depilis bees presented no discrimination. The differences found are discussed considering the putative different learning abilities and procedure effect for each species.     

Evaluation of synergistic interactions between the Colorado potato beetle (Coleoptera: Chrysomelidae) pathogen Beauveria bassiana and the insecticides, imidacloprid, and cyromazine

Laboratory studies investigated the interaction between the fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin and sublethal doses of the insecticides imidacloprid and cyromazine when applied to larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say). When second instars were fed potato leaf discs treated with sublethal doses of imidacloprid and a range of doses of B. bassiana, a synergistic action was demonstrated. Similar results were observed when larvae were sprayed directly with B. bassiana conidia and immediately fed leaf discs treated with imidacloprid. No synergistic interaction was detected when larvae were fed leaf discs treated with sublethal doses ofimidacloprid 24 h after application of B. bassiana conidia to larvae. However, a synergistic interaction was detected when larvae were fed leaf discs treated with imidacloprid and sprayed with B. bassiana conidia 24 h later. Although sublethal doses of both imidacloprid and the triazine insect growth regulator (IGR) cyromazine prolonged the duration of the second instar, only imidacloprid interacted with B. bassiana to produce a synergistic response in larval mortality. In leaf consumption studies, the highest dose of B. bassiana tested promoted feeding in inoculated second instars. Feeding was inhibited when larvae were fed foliage treated with sublethal doses of imidacloprid and significantly reduced when fed foliage treated with a sublethal dose of cyromazine. Starvation of larvae for 24 h immediately after B. bassiana treatment produced a similar result to the combined treatment of B. bassiana and imidacloprid and increased the level of mycosis when compared with B. bassiana controls. Imidacloprid treatment affected neither the rate of germination of B. bassiana conidia on the insect cuticle nor the rate at which conidia were removed from the integument after application. The statistical analysis used to detect synergism and the possible role of starvation-induced stress factors underlying the observed synergistic interactions are discussed.     

Pre‐treatment of Stegomyia aegypti mosquitoes with a sublethal dose of imidacloprid impairs behavioural avoidance induced by lemon oil and DEET

The present study was conducted to determine whether imidacloprid can impair the avoidance behaviour of the mosquito Stegomyia aegypti. Laboratory investigations using a T‐maze apparatus showed that St. aegypti mosquitoes present long term avoidance behaviour when they are exposed to repetitive trials with lemon oil and DEET. The present study tested the effect of a sublethal dose of imidacloprid on the avoidance behaviour of St. aegypti mosquitoes over a 48 h period. Data suggest that 0.5 ng of imidacloprid/mosquito reduces the avoidance behaviour of mosquitoes exposed to lemon oil, on the first day of exposure, after the second trial; whereas imidacloprid affected DEET repellency only the first day of exposure, after the second trial. Imidacloprid was toxic against St. aegypti mosquitoes, and at sublethal doses was able to impair the repellency induced by lemon oil and DEET. The present data were consistent with the finding that St. aegypti mosquitoes exhibit long term avoidance behaviour, and treatment of mosquitoes with a sublethal dose of imidacloprid under DEET application can affect the repellency of DEET against St. aegypti.     

吡虫啉对成年意大利蜜蜂脑神经细胞致凋亡作用

【目的】蜜蜂是重要的授粉昆虫, 但一直受到新烟碱类杀虫剂（如吡虫啉）的危害, 该类杀虫剂主要作用于蜜蜂脑神经细胞。本研究旨在明确吡虫啉对意大利蜜蜂Apis mellifera ligustica脑神经细胞致凋亡作用。【方法】利用亚致死剂量吡虫啉饲喂成年意大利蜜蜂, 通过原位末端标记法（TUNEL）检测脑神经细胞的凋亡情况, 利用免疫荧光法检测Caspase-1激活情况, 通过透射电镜观察脑神经细胞的超微结构。【结果】在对选取的脑部7个部位进行总体观测后, 摄入亚致死剂量的吡虫啉（9.90 ng/蜜蜂）诱导成年意蜂工蜂脑神经细胞凋亡率随时间递增而增加, 在处理9 d和12 d时, 处理组与空白对照组差异极显著（P<0.01）; Caspase-1阳性细胞率也随时间递增而增加, 在处理3, 6, 9和12 d时, 处理组与空白对照组差异极显著（P<0.01）。凋亡率和Caspase-1阳性细胞率均表现出时间效应, 且两者成正相关性。超微结构表明凋亡的神经细胞呈现凋亡和自噬双重特征, 包括： 细胞固缩、 染色质浓缩、 凋亡小体和自噬体出现; 线粒体肿胀, 有的被自噬泡包裹后进行线粒体自噬。【结论】本研究从细胞水平证实了亚致死剂量吡虫啉对成年意蜂工蜂脑神经细胞具有致凋亡作用, 并且其凋亡途径与Caspase-1和细胞自噬有关, 这为利用细胞凋亡法评价杀虫剂对非靶标生物的神经毒性提供一定的理论基础。     

吡虫啉对意大利蜜蜂脑乙酰胆碱受体分布的影响

蜜蜂是自然界主要的授粉昆虫; 新烟碱类杀虫剂(neonicotinoid insecticide)通过结合害虫体内乙酰胆碱受体（nAChR）使害虫致死, 是目前广泛用于田间害虫防控的杀虫剂。本研究以意大利蜜蜂Apis mellifera ligustica和新烟碱类杀虫剂的代表品种吡虫啉为材料, 应用免疫组织化学的方法, 研究了正常成年蜜蜂脑内蘑菇体及视叶nAChR-α7的表达和分布; 分析了亚致死剂量新烟碱类杀虫剂吡虫啉对nAChR-α7表达和分布的影响。结果表明, nAChR-α7在正常蜜蜂脑蘑菇体和视叶中均可检测到, 在蘑菇体中分布相对较少, 但在视叶分布丰富。吡虫啉对nAChR-α7在视叶的表达和分布有显著抑制作用, 但对蘑菇体nAChR-α7的表达没有显著影响。结果提示, 新烟碱类杀虫剂吡虫啉除了文献报道的抑制nAChR的表达外, 还能抑制nAChR-α7的表达量, 这是新烟碱类杀虫剂作用机制的新发现。     

Activity of protein kinase A and gustatory responsiveness in the honey bee (Apis mellifera L.)

The cAMP-dependent protein kinase A is involved in the induction of long-term memory and habituation in the bee. Gustatory responsiveness correlates strongly with associative and non-associative learning in bees. We tested whether protein kinase A activity in the antennal lobes correlates with gustatory responsiveness. Thirty minutes after feeding, bees with high gustatory responsiveness had a significantly higher protein kinase A activity than bees with low responsiveness. Ninety minutues after feeding, when gustatory responsiveness had increased in initially unresponsive bees, no changes in protein kinase A activity were found. We also tested time-dependent effects of protein kinase A activator and protein kinase A inhibitor on gustatory responsiveness. Injection of the protein kinase A activator adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt or of the protein kinase A inhibitor KT 5720 did not affect gustatory responsiveness within the first 4 h after treatment. Feeding of adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt over 4 days increased gustatory responsiveness in newly emerged bees and adult foragers. These results enable us to distinguish between two different forms of gustatory responsiveness: basal and transient gustatory responsiveness. Basal gustatory responsiveness correlates with protein kinase A activity and can only be modulated in the range of several days. Transient gustatory responsiveness appears to be independent of protein kinase A activity and can be modulated in the range of minutes to hours.     

Effects of acute sublethal exposure to coumaphos or diazinon on acquisition and discrimination of odor stimuli in the honey bee (Hymenoptera: Apidae)

Two organophosphate compounds, coumaphos and diazinon, were examined for effects of sublethal exposure on odor learning and generalization in honey bees, Apis mellifera L. Using proboscis extension response training as a measure of odor learning and discrimination, a series of two experiments tested whether these compounds would inhibit bees from learning a new odor or discriminating between different odors. Bees were exposed to coumaphos or diazinon in acetone applied to the thorax, or to coumaphos or diazinon in hexane injected intracranially. At no dose tested or exposure method used was coumaphos shown to inhibit acquisition of a novel odor stimulus, although it was shown to slightly reduce discriminatory ability when given by intracranial injection. Diazinon had effects on odor learning at several small doses, and a small injected dose was shown to significantly inhibit learning of an odor stimulus paired with a sucrose reward. When bee head acetylcholineasterase activity was measured after dermal applications of both pesticides, only the higher doses of diazinon showed reduced activity, indicating that externally-applied coumaphos shows no significant effect on bee brain acetylcholinesterase activity. These data suggest that acute application of coumaphos has only slight nonlethal effects upon the behavior of honey bees and should have little effect upon bee tasks that involve odor learning.