Sensitivities of three bumblebee species to four pesticides applied commonly in greenhouses in China

Abstract  This study was conducted to evaluate the effects of four pesticides (Mosplian, Kingbo, Score and Lvrtong) applied commonly in greenhouses in China, on three bumblebee species ( Bombus hypocrita , Bombus ignitus and Bombus patagiatus ). The study used a contact experiment and oral toxicity LD₅₀ values. The results showed that the mortality for B. hypocrita after contacting the four pesticides was significantly lower than B. patagiatus and B. ignitus , but there was no significant difference between B. patagiatus and B. ignitus . The oral toxicity median lethal dose (LD₅₀) value of Mosplian to B. hypocrita (0.0028 μg active ingredient/bee) was significantly higher than that to B. ignitus (0.0023 μg active ingredient/bee) and B. patagiatus (0.0021 μg active ingredient/bee). Of the bee species, it can be concluded that B. hypocrita was the least susceptible to the four pesticides. The mortality rates of each bumblebee species after contact with Mosplian were significantly higher than for the other three pesticides and the control group. For Kingbo, the rates were significantly higher than the control group, but Score and Lvrtong exposed groups showed no significant increase in mortality relative to the control group. It can therefore be concluded that the pesticides differ in their negative influences on bumblebees, and that Mosplian is the most harmful.     

河北地区熊蜂物种多样性与蜂群繁育特性

基于2005-2009年从河北地区（包括河北省、北京市和天津市）所采集的1893号熊蜂标本资料,结合中国科学院动物研究所馆藏记录,分析了河北地区熊蜂物种多样性和蜂群繁育特性. 结果表明：河北地区共有熊蜂属昆虫8亚属32种,其中河北省32种,北京市18种,天津市5种;西部太行山区、北部燕山山区和坝上高原地区熊蜂种类丰富度和多度较高;河北地区熊蜂的访花植物涉及到21科80种,其中,菊科、豆科和唇形科植物是大多数熊蜂种类访问的主要对象;小峰熊蜂、红光熊蜂、密林熊蜂、火红熊蜂和重黄熊蜂5种熊蜂群势强大,平均单群蜂的工蜂数量在110只以上,雄蜂数量在160只以上,子代蜂王数在30只以上;这5种熊蜂的繁育成群率均在50%以上,易于人工驯养,具有重要的传粉利用价值.     

Displacement of Japanese native bumblebees by the recently introduced <Emphasis Type="Italic">Bombus terrestris</Emphasis> (L.) (Hymenoptera: Apidae)

The introduced Bombus terrestris has recently been naturalized in Japan and become dominant in some local communities. We investigated potential niche overlaps between introduced and native bumblebees in terms of morphological characteristics, seasonal flight activity, foraging and nesting habitat use, and plant species visited. There were considerable niche overlaps in flower resource use between B. terrestris and B. hypocrita sapporoensis/B. pseudobaicalensis. Bombus terrestris also potentially competes for nest sites with B. hypocrita sapporoensis. During 3-year monitoring, B. pseudobaicalensis showed no noticeable change, but B. hypocrita sapporoensis decreased while B. terrestris increased. Abundant flower resources provided by exotic plants may buffer native bumblebees from competition for food with introduced species. By contrast, the number of nest usurpers found in B. terrestris nests increased between 2003 and 2005, indicating that availability of nest sites was limiting and queens strongly competed for nest sites. Our findings suggest that competition for nest sites rather than flower resources is the major ecological mechanism for displacement of native bees. The large reduction of B. hypocrita sapporoensis queen indicates that B. terrestris may cause local extinction of native bumblebees. Control of established B. terrestris populations and prevention of further range expansion are urgently needed.     

意大利蜜蜂和小峰熊蜂在温室桃园的传粉行为及其影响因素

意大利蜜蜂Apis mellifera ligustica和小峰熊蜂Bombus hypocrita是我国设施农业中的两种主要授粉昆虫, 二者的传粉方式和传粉效率因不同作物而各有不同。为了选用最理想的蜂种为温室作物授粉, 提高作物的授粉效率, 我们于2008-2010年连续3年在北京的温室桃园进行了意大利蜜蜂和小峰熊蜂的传粉行为及其影响因素的研究。结果表明, 两种蜂的日活动规律不同, 和意大利蜜蜂相比, 小峰熊蜂的活动起点温度低、 日工作时间长、 单花访问时间长, 采集蜂在温室内距离蜂巢不同距离之间扩散比较均匀。而意大利蜜蜂采集蜂的数量随授粉半径的增大而明显减少。温室内环境因子对两种蜂传粉活动的影响基本一致, 温度对两种蜂的传粉活动影响最大, 其次是湿度、 单花花蜜浓度和光照强度, 而单花花蜜体积对蜂活动无明显影响。本研究认为对于温室桃授粉应优先选用授粉性能稳定的小峰熊蜂, 并且适当调节温室内环境条件, 以提高其授粉效率。     

华北地区六种熊蜂的地理分布及生态习性

2005～2007年连续3年对华北地区小峰熊蜂Bombus hypocrita、密林熊蜂B.patagiatus、红光熊蜂B.ignitus、明亮熊蜂B.lucorum、火红熊蜂B.pyrosoma和重黄熊蜂B.picipes等6种熊蜂的地理分布和生态习性进行研究。结果表明,这6种熊蜂在华北地区的地理分布差异很大,火红熊蜂分布最广,重黄熊蜂分布最窄。6种熊蜂在垂直分布上也存在差异,红光熊蜂分布于较低海拔高度地区,明亮熊蜂分布于较高海拔高度地区,但在海拔1300～1800m范围内6种熊蜂都有分布。6种熊蜂中,火红熊蜂的年活动周期较长,为180天左右。小峰熊蜂、明亮熊蜂、密林熊蜂和重黄熊蜂的年活动周期居中。红光熊蜂的年活动周期最短,为140天左右。6种熊蜂采访植物涉及到17科63种,采访植物存在一定的偏好性,主要采访豆科、菊科、蔷薇科和唇形科等植物;而且,不同种熊蜂采集植物的种类也存在较大差异。     

小峰熊蜂头部乙酰胆碱酯酶测定条件的优化及其对六种常用杀虫剂的敏感性

小峰熊蜂Bombus hypocrita是我国优势熊蜂种群之一, 因其易于饲养、 群势较强且授粉性能优良而成为我国设施农业常用优良授粉蜂种, 但常受到以乙酰胆碱酯酶(AChE)为靶标酶的有机磷和氨基甲酸酯类杀虫剂的危害。 为合理规避这两类杀虫剂对熊蜂的危害, 同时也为完善熊蜂授粉配套技术和保护野生熊蜂资源提供理论基础, 本研究利用正交试验对小峰熊蜂头部乙酰胆碱酯酶活性的测定条件进行了优化, 并明确了6种常用有机磷和氨基甲酸酯类杀虫剂对乙酰胆碱酯酶活性的影响。结果表明： 各测定因素对小峰熊蜂乙酰胆碱酯酶活性测定影响的大小顺序依次为： 酶浓度＞pH＞温度＞底物浓度＞反应时间; 小峰熊蜂头部乙酰胆碱酯酶活性的最适反应条件为： 酶浓度0.25 g 蛋白质/L, 底物浓度0.8 mmol/L, pH值7.5, 温度40℃, 反应时间5 min。毒死蜱、 三唑磷、 丙溴磷、 异丙威、 仲丁威和残杀威6种杀虫剂对小峰熊蜂头部乙酰胆碱酯酶离体抑制作用均呈现明显的剂量-效应关系, 其抑制中浓度IC₅₀分别为0.39, 1.79, 0.42, 0.04, 0.43和0.63 mmol/L。这6种杀虫剂对小峰熊蜂AChE抑制作用的强弱依次为： 异丙威＞毒死蜱＞三唑磷＞仲丁威＞残杀威＞丙溴磷, 即小峰熊蜂对异丙威最敏感, 而对丙溴磷的敏感性最弱。     

马先蒿属花冠形态的多样性与传粉式样的关系

马先蒿属(Pediculais)是有花植物中花冠形态多样化最为集中的属。该属主要的传粉者是熊蜂属(Bormbus)昆虫;在北美,熊蜂和蜂鸟是马先蒿植物一些种类有效的传粉者;也发现壁蜂(Osmia)为其传粉。不同的传粉机制要求某一特定的取食式样储藏和释放花粉。本文讨论了花冠类型的进化趋势与传粉式样和花粉形态的关系。传粉者的选择压力是决定花冠多样化的重要因素之一;花冠类型与传粉者和传粉行为紧密相关。马先蒿植物和传粉者的相互依赖与其花冠类型、功能和物候互相适应,但花冠类型与花粉形态两者之间似乎没有明显的一一对应关系。通过北美、日本和喜马拉雅不同地理分布马先蒿种类的比较研究表明,具有相同花冠类型的种类有着相同的传粉方式,花冠形态与传粉式样存在紧密的协同进化关系。     

Ecological Variation in Response to Mass-Flowering Oilseed Rape and Surrounding Landscape Composition by Members of a Cryptic Bumblebee Complex

The Bombus sensu stricto species complex is a widespread group of cryptic bumblebee species which are important pollinators of many crops and wild plants. These cryptic species have, until now, largely been grouped together in ecological studies, and so little is known about their individual colony densities, foraging ranges or habitat requirements, which can be influenced by land use at a landscape scale. We used mass-flowering oilseed rape fields as locations to sample bees of this complex, as well as the second most common visitor to oilseed rape B. lapidarius, and molecular RFLP methods to distinguish between the cryptic species. We then used microsatellite genotyping to identify sisters and estimate colony densities, and related both proportions of cryptic species and their colony densities to the composition of the landscape surrounding the fields. We found B. lucorum was the most common member of the complex present in oilseed rape followed by B. terrestris. B. cryptarum was also present in all but one site, with higher proportions found in the east of the study area. High numbers of bumblebee colonies were estimated to be using oilseed rape fields as a forage resource, with B. terrestris colony numbers higher than previous estimates from non-mass-flowering fields. We also found that the cryptic species responded differently to surrounding landscape composition: both relative proportions of B. cryptarum in samples and colony densities of B. lucorum were negatively associated with the amount of arable land in the landscape, while proportions and colony densities of other species did not respond to landscape variables at the scale measured. This suggests that the cryptic species have different ecological requirements (which may be scale-dependent) and that oilseed rape can be an important forage resource for many colonies of bumblebees. Given this, we recommend sustainable management of this crop to benefit bumblebees.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.     

The utility of wing morphometrics for assigning type specimens to cryptic bumblebee species

Since the beginning of taxonomy, species have been described based on morphology, but the advent of using semio-chemicals and genetics has led to the discovery of cryptic species (i.e. morphologically similar species). When a new cryptic species is described, earlier type specimens have to be re-evaluated, although this process can be challenging as only nondestructive methods ought to be used in order to preserve the integrity of the type specimens. Methods should allow comparison with recently collected specimens clustered based on chemical, ethological and/or genetic traits with old specimens (i.e. type specimens) where only morphological traits are available. Here we develop a method based on geometric morphometric analyses of wing shape for a taxonomically challenging group of bumblebees, the subgenus Alpinobombus Skorikov. We consider nine monophyletic taxa (including several cryptic species) to assess the accuracy of this method to discriminate the taxa based on their wing shape and then to attribute type specimens using a leave-one-out cross-validation procedure. We show that, for these bees, wing shape is taxon-specific, except for two sister taxa for which the species status is still debated. Moreover, for most of the taxa, type specimens were correctly attributed with high posterior probabilities of attribution, except for a few type specimens corresponding to the same two sister taxa where taxa delimitation based on wing shape was previously the subject of discussion. Our study highlights the potential of geometric morphometric analyses to help in the re-attribution of type specimens when the existence of cryptic species is revealed.     

Bombus terrestris in a mass‐flowering pollinator‐dependent crop: A mutualistic relationship?

Bumblebees (Bombus spp.) rely on an abundant and diverse selection of floral resources to meet their nutritional requirements. In farmed landscapes, mass‐flowering crops can provide an important forage resource for bumblebees, with increased visitation from bumblebees into mass‐flowering crops having an additional benefit to growers who require pollination services. This study explores the mutualistic relationship between Bombus terrestris L. (buff‐tailed bumblebee), a common species in European farmland, and the mass‐flowering crop courgette (Cucurbita pepo L.) to see how effective B. terrestris is at pollinating courgette and in return how courgette may affect B. terrestris colony dynamics. By combining empirical data on nectar and pollen availability with model simulations using the novel bumblebee model Bumble‐BEEHAVE, we were able to quantify and simulate for the first time, the importance of courgette as a mass‐flowering forage resource for bumblebees. Courgette provides vast quantities of nectar to ensure a high visitation rate, which combined with abundant pollen grains, enables B. terrestris to have a high pollination potential. While B. terrestris showed a strong fidelity to courgette flowers for nectar, courgette pollen was not found in any pollen loads from returning foragers. Nonetheless, model simulations showed that early season courgette (nectar) increased the number of hibernating queens, colonies, and adult workers in the modeled landscapes. Synthesis and applications. Courgette has the potential to improve bumblebee population dynamics; however, the lack of evidence of the bees collecting courgette pollen in this study suggests that bees can only benefit from this transient nectar source if alternative floral resources, particularly pollen, are also available to fulfill bees’ nutritional requirements in space and time. Therefore, providing additional forage resources could simultaneously improve pollination services and bumblebee populations.     

Dynamics of gut microbiome upon pollination in bumblebee (Bombus terrestris)

Bumblebees are crucial buzz pollinators of poricidal plants and commercial crops. The population of these crucial pollinators is globally declining. The need to focus on factors contributing to bee health such as gut microbiota is imperative. We evaluated the effect of source on gut microbiota composition in adult worker Bombus terrestris from two environments using multiplexed 16S rRNA amplicon sequencing on the Illumina MiSeq platform. Indoor reared adult worker bees were segregated into non-pollination (NP) and pollination (P) groups. The NP group bumblebees were raised and kept in the insectary for the entire experiment. P group bumblebees worked in a tomato-planted greenhouse for 15 days as pollinators. Our results show that members of Proteobacteria and Firmicutes were significantly different between the two groups. Bifidobacterium bombi (Actinobacteriota) and Candidatus_Schmidhempelia (Proteobacteria) were found to be important gut colonizer exclusive to NP and P group bees, respectively. DESeq2 analysis showed differentially abundant OTUs belonging to Lactobacillus spp. in each group. But for the differentially abundant detected OTUs, resolution till genus level was obtained which impairs our knowledge of the species associated with each group. Our data show that the gut microbiota between the groups differed when indoor- reared adult worker bees were exposed to different environments. The distinct gut microbiota between the two groups may also have been influenced by the different diets fed to bees upon segregation. Further studies can give important insights into the role of gut microbiota on bee health when indoor reared bees are employed for pollination.     

Predator crypsis enhances behaviourally mediated indirect effects on plants by altering bumblebee foraging preferences

Predators of pollinators can influence pollination services and plant fitness via both consumptive (reducing pollinator density) and non-consumptive (altering pollinator behaviour) effects. However, a better knowledge of the mechanisms underlying behaviourally mediated indirect effects of predators is necessary to properly understand their role in community dynamics. We used the tripartite relationship between bumblebees, predatory crab spiders and flowers to ask whether behaviourally mediated effects are localized to flowers harbouring predators, or whether bees extend their avoidance to entire plant species. In a tightly controlled laboratory environment, bumblebees (Bombus terrestris) were exposed to a random mixture of equally rewarding yellow and white artificial flowers, but foraging on yellow flowers was very risky: bees had a 25 per cent chance of receiving a simulated predation attempt by ‘robotic’ crab spiders. As bees learnt to avoid ‘dangerous’ flowers, their foraging preferences changed and they began to visit fewer yellow flowers than expected by chance. Bees avoided spider-free yellow flowers as well as dangerous yellow flowers when spiders were more difficult to detect (the colour of yellow spiders was indistinguishable from that of yellow flowers). Therefore, this interaction between bee learning and predator crypsis could lead flower species harbouring cryptic predators to suffer from reduced reproductive success.     

Speed-accuracy tradeoffs and false alarms in bee responses to cryptic predators

Learning plays a crucial role in predator avoidance [1-3], but little is known about how the type of experience with predators molds future prey behavior. Specifically, is predator-avoidance learning and memory retention disrupted by cryptic coloration of predators, such as crab spiders [4, 5]? How does experience with different predators affect foraging decisions? We evaluated these questions by exposing foraging bumblebees to controlled predation risk from predators (robotic crab spiders) that were either cryptic or highly contrasting, as assessed by a quantitative model of bee color perception [6]. Our results from 3D tracking software reveal a speed-accuracy tradeoff [7]: Bees slow their inspection flights after learning that there is a risk from cryptic spiders. The adjustment of inspection effort results in accurate predator detection, leveling out predation risk at the expense of foraging time. Overnight-retention tests reveal no decline in performance, but bees that had experienced cryptic predators are more prone to "false alarms" (rejection of foraging opportunities on safe flowers) than those that had experienced conspicuous predators. Therefore, bees in the cryptic-spider treatment made a functional decision to trade off reduced foraging efficiency via increased inspection times and false-alarm rates against higher potential fitness loss from being injured or eaten.     

小峰熊蜂蜂毒磷脂酶A2基因的克隆及表达分析

磷脂酶A2 （phospholipase A2, PLA2）是蜂毒主要成分, 也是蜂毒的主要过敏原, 在熊蜂个体和群体防御方面具有重要功能。为了探究熊蜂A2基因的生物学功能, 本研究以小峰熊蜂Bombus hypocrita为材料进行了蜂毒PLA2基因的克隆、 鉴定与表达特性分析。结果表明： 该基因全长为2 272 bp, GenBank登录号为KF214771, 由4个外显子和3个内含子组成, 编码区（CDS）长为543 bp, 共编码180个氨基酸残基。氨基酸序列相似性分析显示, 成熟的小峰熊蜂PLA2（含有136个氨基酸）与其他蜂类PLA2的氨基酸序列相似性较高, 均包含10个保守的半胱氨酸残基、 1个保守的Ca2+结合位点和1个酶活性中心。基于PLA2氨基酸序列的系统进化树分析表明, 熊蜂属Bombus与蜜蜂属Apis在不同分支上, 属单系群, 且蜜蜂属分化较早。荧光定量PCR结果表明, PLA2基因在小峰熊蜂各日龄均有表达, 且随日龄增长, 表达量呈先上升后下降的趋势, 10日龄时出现峰值, 其表达量显著高于其他日龄（P<0.05）。半定量PCR结果表明, PLA2基因在毒腺、 卵巢、 中肠中表达量较高, 在足、 触角、 食道腺中表达量较低, 在脂肪体、 肌肉、 神经、 气管、 复眼、 脑中未表达。本研究探明了小峰熊蜂PLA2的基因结构及其表达特性, 丰富了熊蜂PLA2的生物学基础, 为进一步深入研究熊蜂PLA2生物学功能和作用机制以及开发蜂毒生物制剂等鉴定了基础。     

Comparative analysis of thermoregulation in the nests of certain species of social insects

Studies have been made on thermal regulation in the nests of families of the honey bee Apis mellifera, wasp Dolihovespula silvestris and bumblebees Bombus terrestris, B. agrorum and B. lapidaris during their maximum development. It was shown that thermoregulation significantly stimulated the brood in the nest. Among the species investigated, the highest thermoregulatory capacities are exhibited by honey bees, this fact being associated with the large number of individuals in their colonies. During cooling, bees group around the brood and their bodies make a cover of thermal insulation. Sheltering of the nests plays the main role in heat preservation of wasps and bumblebees. The latter, using contact method of heating of cells with the brood, provide rather constant temperature for its development. Honey bees, wasps and bumblebees react to overheating in the nests essentially in a similar way, i. e. by active aeration of their nests by vigorous wing beatings. The frequency of beatings increases with the increase in temperature.     

A Preliminary Study of the Pollination Ecology of Pedicularis (Scrophulariaceae) in Japan

Abstract An introductory study of the pollination ecology of six Japanese species of Pedicularis conducted in 1982 and 1983 indicated a close parallel between characteristics of floral mechanisms in Japan and those previously investigated in North America. Floral color, floral form, total dissolved solids in floral nectar, and polinator behavior on flowers of Japanese species are similar to those of North American species. Blooming phenology of Japanese species also corresponds to the North American sequence of long-tongued, nectar-foraging queen bumblebees (Bombus Latr.) pollinating nectariferous , earlier-blooming species nototribically and short-tongued, pollen-foraging workers pollinating nectarless, rostrate flowers of later-blooming ones sternotribically. Electrofocus analysis of peroxidase systems in two Japanese species indicated considerable intrapopulational heterozygosity. Nectar component analysis of two transboreal species indicated intraspecific differences in kinds of sugars between widely disjunct Japanese and North American populations. A tentative conclusion is that the Japanese and North American Pedicularis floras have evolved in a parallel manner from Himalayan migrants, while their transboreal elements are in process of vicariant diversification.     

Observations on Aulacoseira nipponica from Lake Biwa, Japan, and Aulacoseira solida from North America (Bacillariophyceae)

A recent diatom, Aulacoseira nipponica (Skvortzow) Tuji comb. et stat. nov., is described from Lake Biwa, Japan, where they had been identified previously as Aulacoseira solida (Eulenstein) Krammer. These forms are compared with populations of A. solida from North America. The Japanese species differs from the North American specimens in characteristics related to the density of striae and form of the rimoportulae.     

Diversity and functional analysis of Chinese bumblebee gut microbiota reveal the metabolic niche and antibiotic resistance variation of Gilliamella

Bumblebees play an important role in maintaining the balance of natural and agricultural ecosystems,and the characteristic gut microbiota of bumblebees exhibit significant mutualistic functions.China has the highest diversity of bumblebees;however,gut microbiota of Chinese bumblebees have mostly been investigated through cultureindependent studies.Here,we analyzed the gut communities of bumblebees from Sichuan,Yunnan,and Shaanxi provinces in China through 16S ribosomal RNA amplicon sequencing and bacterial isolation.It revealed that the bumblebees examined in this study harbored two gut enterotypes as previously reported:one is dominated by Gilliamella and Snodgrassella,and the other is distinguished by prevalent environmental species.The gut compositions obviously varied among different individual bees.We then isolated 325 bacterial strains and the comparative genomic analysis of Gillianiella strains revealed that galactose and pectin digestion pathways were conserved in strains from bumblebees,while genes for the utilization of arabinose,mannose,xylose,and rhamnose were mostly lost.Only two strains from the Chinese bumblebees possess the multidrug-resistant gene emrB,which is phylogenetically closely related to that from the symbionts of soil entomopathogenic nematode.In contrast,tetracycline-resistant genes were uniquely present in three strains from the USA.Our results illustrate the prevalence of strain-level variations in the metabolic potentials and the distributions of antibiotic-resistant genes in Chinese bumblebee gut bacteria.