A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Differences in venom and cuticular peptides in individuals of Apis mellifera (Hymenoptera: Apidae) determined by MALDI-TOF MS

The fraction between 950 and 4000 Da of the venom of Apis mellifera has been analyzed with MALDI-TOF mass spectrometry and statistical facilities of the ClinProTools™ software. Consistent differences in the composition of this venom fraction were observed between queens and workers while younger and older workers (nurses and guards as well as foragers) differ for the relative percentages of two well known cytolytic peptides, namely Melittin and Apamin. Total in situ body methanol extracts and methanol micro-extractions on the cuticle of various parts of the body of drones and females confirmed that venom peptides are smeared on the body surface of females in a not yet clarified way. The observation that venom peptides have been found also on comb wax rises the hypothesis that the use of venom as antimicrobial agent makes part of the social immunity system of A. mellifera.     

低温导致中华蜜蜂后翅翅脉的新变异

在封盖发育期,将中华蜜蜂Apis cerana cerana封盖子放入24℃低温环境中分别处理24、48、72 h,再恢复到正常的发育温度35℃至羽化,或将封盖子放在30℃低温中培养至羽化,发现羽化后的蜜蜂后翅翅脉发生明显变异。发现翅脉变异有3种类型,翅脉增加、翅脉突出和翅脉缺失。后翅翅脉增加的变异只发现一种,基室、缘室共有的径分脉与基室、盘室共有的中脉之间,新增径中横脉。新增的径中横脉发生的程度不同,有的在径分脉和中脉相对的位置相向伸出,有的形成完整的新脉,新增的径中横脉与原径中横脉相似,在末端均有"弱化点"。由于原有径中横脉较短,一直忽略其存在,这次径中横脉的确定,可以判定原来公认的"中脉分叉"应分别是径分脉和中脉延伸出来的两条翅脉。后翅突出的变异有2种,中脉向基室突出,径分脉向基室突出。翅脉突出的程度不同,有的略突出,有的明显突出,有的伸出翅脉较长。中脉突出均发生在中脉的转折点上,且此类翅脉变异,仅发生在雄蜂样本中,在工蜂样本中还未见。后翅翅脉缺失的变异有2种,发生在肘臀横脉和中脉。肘臀横脉"弱化点"处缺失。将中华蜜蜂雄蜂封盖子放入低温30℃恒温培养箱中发育,发现2个羽化的雄蜂样本发生肘臀横脉在"弱化点"处缺失。中脉向盘室伸出的部分发生缺失。翅脉缺失的程度不同,有的明显缩短,有的仅存痕迹,有的缺失大部分或完全缺失。中脉向盘室伸出的部分是东方蜜蜂Apis cerana区别于西方蜜蜂Apis mellifera的主要形态特征之一,如果东方蜜蜂这段翅脉全部缺失,此部位的形态与西方蜜蜂完全相同。这意味着这个翅脉作为区分东方蜜蜂和西方蜜蜂的翅脉特征将面临挑战。对蜜蜂种质资源鉴定,蜜蜂的系统发育和进化生物学等领域的研究将具有重要影响。温度影响翅脉发育的深入研究,为研究蜜蜂翅脉发育过程,揭示翅脉发育调控机制,以及蜜蜂发育的温度适应生理机制提供一个新思路。     

Morphological and functional aspects of volatile-producing glands in bees (Hymenoptera: Apidae)

Abstract In this paper we focus on the occurrence and morphological aspects of exocrine glands in several bee species. Morphology of head labial, mandibular, Dufour, and abdominal tegumentar glands was investigated under light microscopy, scanning electron microscopy and transmission electron microscopy. Most of such glands present cells with cytoplasm homogeneous and acidophilic, or contain small apparently empty vacuoles. The cytoplasm cells' ultrastructure showed a well developed smooth endoplasmic reticulum, many polymorphic mitochondria, rare Golgi, lipid droplets, myelin figures, and many basal and apical plasma membrane infoldings. All these results are discussed in the text.     

Bombus folsomi and the origin of Philippine bumble bees (Hymenoptera: Apidae)

Abstract The type female of Bombus folsomi (Frison) is redescribed and synomymized with B.ephippiatus Say. It is concluded that the putative type locality of B.folsomi , Mt Kinabalu in Borneo, is in error and that the genus appears to be absent from Borneo. This lends support to the hypothesis that bumble bees immigrated to the Philippines by an unusual northern route through Taiwan. More rigorous testing of this idea will require further cladistic analysis of the genus Bombus .     

Comparative morphology of the postmentum of bees (Hymenoptera: Apoidea) with special remarks on the evolution of the lorum

A comparative morphological study of the basal sclerites of the bee labium was undertaken. The term postmentum was applied to the basal sclerite of the bee labium. In contrast to recent interpretations, the undivided postmentum was found to be ancestral for bees and homologous with the single postmental sclerite of other Hymenoptera. This sclerite has previously been incorrectly indentified as two separate sclerites, usually termed mentum and lorum (= submentum), for many short-tongued bees (Colletidae, Halictidae, Andrenidae, Melittidae) and for the long-tongued families of bees (Anthophoridae, Fideliidae Megachilidae) excepting most members of the Apidae. The postmentum, divided into a true mentum and lorum, was found only in certain members of the Apidae. A phylogenetic implication resulting from this study shows that the Andrenidae may be the sister group to the MCL-T group (Melittidae, Ctenoplectridae and long-tongued families of bees). In addition, it is proposed that the Euglossinae should be the sister group to the Apinae (Meliponini, Apini and Bombini). This means that eusociality may be considered a synapomorphy for the Apinae.     

Nonrandom visitation of brood cells by worker honey bees (Hymenoptera: Apidae)

The visitation pattern by worker honey bees to cells in the brood nest was monitored on an artificially created brood pattern consisting of about one-fourth brood cells evenly distributed among empty cells. The majority (63 %) of the observed workers selectively entered larval cells. In contrast, some workers avoided egg cells when presented a choice of egg vs empty cells. The results suggest that larvae produce a general signal indicating their presence to worker bees. Eggs also seem to produce a signal, which is perceived to be different from the one from larvae.     

Nest preference and ecology of cavity-nesting bees (Hymenoptera: Apoidea) in Punjab,Pakistan

Among bees, 85 % are solitary species, most of them are ground-nester and some are cavity-nesting and construct their nests in pre-existing cavities. This work was conducted to evaluate the substrate preference and nest architecture (acceptance, occupation percentage, seasonality and parasitism) of cavity-nesting bees in different substrates. Trap nests offered comprised five different materials (drilled cavities in wood, bamboo, cardboard tubes, plastic soda straws, and mud blocks). These were installed in four districts for two years. The nesting cavities of five different diameters (6, 8, 10, 12 and 14 mm) were provided in each nesting material with an average length of 180 ± 9.92 mm. In all the materials, 5400 nesting cavities were offered, out of which 628 were colonized by bees from two families (Megachilidae and Apidae) and six species (Megachile cephalotes, M. lanata, M. bicolor, Xylocopa basalis, X. fenestrata and Ceratina smaragdula), including one parasitic bee (Euaspis carbonaria). The bee species differed significantly in occupying five nesting materials. The most preferred diameters were 8 mm and 10 mm, with 52.20% and 29.45% of colonization, respectively. Nesting was done throughout the year except in winter. This study will serve as a baseline for future studies and conservation programs of cavity-nesting bees in Pakistan.     

木蜂的访花行为和传粉作用

木蜂作为蜜蜂科的重要类群,是热带和亚热带地区显花植物常见的传粉者.木蜂具有访花的季节时间长、访花植物种类多、能耐高温或低光照、能嗡声传粉等特点.这些访花特点是木蜂作为热带植物、尤其是温室作物、夜间开花植物和部分茄属植物的重要传粉者.近年来国外已经证明木蜂能有效为蓝莓、西番莲、荷包豆、温室西红柿和温室甜瓜授粉,但我国有关木蜂为多数农作物传粉的重要性和有效性的证据较少.本文综述了近年来木蜂的访花行为和传粉作用的研究进展,为进一步保护、管理和利用木蜂资源提供理论依据.     

Lipid distribution in salivary glands of larvae and adult bees (Hymenoptera: Apidae)

Cytochemical studies were carried out to establish lipid distribution in the salivary glands of larvae and adult bees, using the imidazole buffer technique. In the duct cells of the larval salivary gland, the reaction was positive in the epicuticle and negative in the glandular lumen. The absence of smooth endoplasmic reticulum and the presence of lipids in the intercellular space suggest that lipids absorbed from the haemolymph could be used in the constitution of the epicuticle, after having been conveyed through the epithelium. In adult workers (new-emerged, nurse and forager workers), the head salivary glands presented a positive reaction in the secretion in glandular lumen, identifying its lipidic nature.     

Diversity and distribution of orchid bees (Hymenoptera: Apidae) with a revised checklist of species

The aim of this study was to investigate the diversity and distribution patterns of orchid bees (Euglossina). Cluster and correlation analyses were applied to data extracted from 28 orchid-bee surveys throughout the Neotropical Region. The 28 sampling sites were grouped in three main biogeographic areas that roughly correspond to the Amazonian Basin, the Atlantic Forest and Central America. These three regions, as well as subregions within each of them, correspond approximately to biogeographic components identified through phylogeny-based analyses for other bees and organisms. The Amazonian Forest as a whole has the richest fauna and the highest levels of endemism. The Atlantic Forest, on the other hand, showed the poorest fauna and the lowest levels of endemism. However, a major neotropical biome, in which orchid bees are known to occur, has not been sampled yet, the savanna-like cerrado. At least 30% of the species are endemic to each biome. An updated checklist of the species of Euglossina is provided.     

Transfers ofVarroa mites from newly emerged bees: Preferences for age- and function-specific adult bees (Hymenoptera: Apidae)

Movements of the parasitic honey bee mite,Varroa jacobsoni (Oud.) were monitored in several assays as they moved among adult host honey bees,Apis mellifera. We examined the propensity of mites to leave their hosts and to move onto new bee hosts. We also examined their preference for bees of different age and hive function. Mites were standardized by selecting mites from newly emerged worker bees (NEWs). In closed jars, 50% ofVarroa left NEWs irreversibly when no physical path was present for the mites to return to the NEWs; about 90% of mites left newly emerged drones in identical assays. In petri dish arenas, mites were rarely seen off NEW hosts when monitored at 15-min intervals for 4 h; this was the case for single NEWs with one mite (NEWs+) and when a NEW+ and a NEW− (no mites) were placed together in a petri dish. When a NEW+ was held with either a nurse beeor a pollen forager, 25% of the mites moved to the older bees. When both a nurseand a pollen forager were placed in a petri dish with a NEW+, about 50% of the mites transferred to older bees; nurse bees received about 80% of these mites, whereas pollen foragers received significantly fewer mites (about 20%,P < 0.05). Most mite transfers occurred during the first 30 min after combining NEWs+ and test bees. When NEWs+ were combined with bees of known ages, rather than function, mites transferred more often to young bees than to older bees (1- and 5-day-old bees vs. 25-day-old bees,P < 0.05; 1-day-old vs. 13- and 25-day-old bees;P < 0.05). No differences in proportions of transferring mites were seen when the range of bee ages was ≤ 8 days (P > 0.05), implying that the factors mediating the mites’ adult-host preference change gradually with bee age. A possible chemical basis for host choice byVarroa is indicated by their greater propensity to move onto freezer-killed nurse bees than onto freezer-killed pollen foragers (P < 0.05) and by their lower movement onto heat-treated bees than onto control bees (P < 0.05). Bee age, hive function, and directional changes in cuticular chemistry are all correlated. Movements of newly emerged mites in relation to these variables may provide insights into their reproductive success inApis mellifera colonies.     

Pheromonal activity and fine structure of the mandibular glands of honeybee drones (Apis mellifera L.) (Insecta,Hymenoptera, Apidae)

Experiments were conducted to determine the role of drone mandibular gland secretions in attracting flying drones and the effect of age on the secretory activity. Extracts of mandibular glands and of cephalic tissues were applied to cotton lures which were attached below balloons tethered at 8–12 m above the ground. Most flying drones were attracted to extracts of mandibular glands but a few were drawn to other cephalic tissues or to solvent controls. Histological and electron microscope studies showed that the structure of the tiny (0.12 mm long) mandibular gland varied according to age. Its secretory activity in 0–3-day old drones was evident from the abundant rough endoplasmic reticulum. At 7-days the glands were fully developed. After 9 days the glands were no longer active and showed an autolytic process; the product was stored in the gland lumen for further emission during drone mating flights.     

Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae)

The behaviour of the endoparasitic tracheal mite, Acarapis woodi (Rennie) on honey bees (Apis mellifera L.) is a challenge to observe because of its small size. Through a microscope, we videotaped this mite's movement on young bees, dead bees and bees exposed to vegetable oil. Previous studies have shown that solid vegetable oil decreases mite infestations in a bee colony. We hypothesized that the oil alters mite behaviour to the detriment of the parasite, thus helping to safeguard the host. Habitat-seeking behaviour, identified as necessary for mites to locate a new host environment, was disrupted on both dead and oil-treated bees. Questing behaviour, which is associated with transfer between hosts, increased significantly on the dead and oily bees. The behaviours of mites were significantly different between all three treatments (x ²=494.96, p<0.001 on dead bees and x ²=851.11, p<0.001 on oily bees). Both questing and seeking behaviours were significantly different on each of the thoracic treatments (F _2,66=7.88, p<0.001 and F _2,66=21.28, p<0.001) and mite questing behaviour was not altered between males and females on live or oily bees (F _1,22=0.25, p<0.62), but habitat seeking was (F _1,22=7.42, p<0.012). The male questing and habitat-seeking behaviours were observed. We conclude that oil-treated bees gained protection from habitat-seeking mites because the normal behaviour of the mites seeking an oviposition site is interrupted.     

Phylogeny,diversification patterns and historical biogeography of euglossine orchid bees (Hymenoptera: Apidae)

The orchid bees constitute a clade of prominent insect pollinators distributed throughout the Neotropical region. Males of all species collect fragrances from natural sources, including flowers, decaying vegetation and fungi, and store them in specialized leg pockets to later expose during courtship display. In addition, orchid bees provide pollination services to a diverse array of Neotropical angiosperms when foraging for food and nesting materials. However, despite their ecological importance, little is known about the evolutionary history of orchid bees. Here, we present a comprehensive molecular phylogenetic analysis based on ～4.0 kb of DNA from four loci [cytochrome oxidase (CO1), elongation factor 1‐α (EF1‐α), arginine kinase (ArgK) and RNA polymerase II (Pol‐II)] across the entire tribe Euglossini, including all five genera, eight subgenera and 126 of the approximately 200 known species. We investigated lineage diversification using fossil‐calibrated molecular clocks and the evolution of morphological traits using disparity‐through‐time plots. In addition, we inferred past biogeographical events by implementing model‐based likelihood methods. Our dataset supports a new view on generic relationships and indicates that the cleptoparasitic genus Exaerete is sister to the remaining orchid bee genera. Our divergence time estimates indicate that extant orchid bee lineages shared a most recent common ancestor at 27–42 Mya. In addition, our analysis of morphology shows that tongue length and body size experienced rapid disparity bursts that coincide with the origin of diverse genera (Euglossa and Eufriesea). Finally, our analysis of historical biogeography indicates that early diversification episodes shared a history on both sides of Mesoamerica, where orchid bees dispersed across the Caribbean, and through a Panamanian connection, thus reinforcing the hypothesis that recent geological events (e.g. the formation of the isthmus of Panama) contributed to the diversification of the rich Neotropical biota. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 552–572.     

Thermal tolerance varies with dim-light foraging and elevation in large carpenter bees (Hymenoptera: Apidae: Xylocopini)

1. Thermal tolerance has a strong predictive power for understanding the ecology and distribution of organisms, as well as their responses to changes in land use and global warming. However, relatively few studies have assessed thermal tolerances for bees. 2. The present study aimed to determine whether the critical thermal maximum (CT_max) of carpenter bees (Apidae: genus Xylocopa Latreille) varies with different patterns of foraging activity and elevation. In addition, the influence of body size, body water content and relative age was examined with respect to their CT_max and differences in thoracic temperature (T_th) among species were evaluated. 3. The CT_max of one crepuscular (Xylocopa olivieri) and two diurnal species (Xylocopa violacea and Xylocopa iris) of carpenter bees was assessed at sea level on the Greek island of Lesvos. To detect variation as a result of elevation, the CT_max of a population of X. violacea at 625 m.a.s l. was assessed and compared with that from sea level. 4. Xylocopa olivieri displayed a similar CT_max to that of X. violacea but lower than that of X. iris. Body size, body water content, and relative age did not affect CT_max. In X. violacea, CT_max decreased with elevation and all three species have high T_th independent of ambient temperatures. 5. The results of the present study are consistent with variations in CT_max predicted by broad spatial and temporal patterns reported for other insects, including honey and bumble bees. The implications of the results are discussed aiming to understand the differences in the foraging pattern of these bees.