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.     

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 .     

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.     

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.     

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.     

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.     

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.     

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.     

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at −80 °C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Ultrastructure and Immunofluorescence of the midgut of Bombus morio (Hymenoptera: Apidae: Bombini)

Bumblebees are widely distributed across the world and have great economic and ecological importance as pollinators in the forest as well as in agriculture. The insect midgut consists of three cell types, which play various important roles in digestion, absorption, and hormone production. The present study characterized the anterior and posterior midgut regions of the bumblebee, Bombus morio. The digestive, regenerative and endocrine cells in the midgut showed regional differences in their number, nuclear size, as well as the size of the striated border. Ultrastructurally, the digestive cells contained many mitochondria and long microvilli; however, in the anterior midgut region, these cells showed dilated basal labyrinths with a few openings for the hemocoel, whereas the labyrinths of the basal posterior region remained inverse characteristics. Thus, the characterization of the midgut of B. morio supported an ecto-endoperitrophic circulation, contributing to a better understanding of the digestive process in this bee.     

Detection of aerosolized bacterial spores (Bacillus atrophaeus) using free-flying honey bees (Hymenoptera: Apidae) as collectors

An aerosol cloud of Bacillus atrophaeus (previously B. subtilis variety niger) spores, an anthrax surrogate, was created in a large 0.4 ha (1 ac), bee-containing, open-mesh tent. Bees from a B. atrophaeus uncontaminated hive flying through the cloud adsorbed the spores in statistically significant quantities. After removal of the B. atrophaeus contaminated hive and introduction of another B. atrophaeus uncontaminated hive, the bees again were monitored for the next few days for B. atrophaeus spores. B. atrophaeus spores accumulated on the bees bodies following their exposure to the residual B. atrophaeus contamination in the tent. The spore loads on the bees quickly returned to background levels after the hives were removed from the contaminated tent area. It may therefore be practical to use honey bee colonies to monitor foraging areas for disease-causing spores.     

Cophylogeny of Nosema (Microsporidia: Nosematidae) and bees (Hymenoptera: Apidae) suggests both cospeciation and a host-switch

Some microsporidian parasites belonging to the genus Nosema infect bees. Previous phylogenies of these parasites have produced alternative, conflicting relationships. We analyzed separately, and in combination, large and small subunit ribosomal DNA sequences of Nosema species infecting bees under neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian frameworks. We observed a sister relationship between Nosema ceranae and Nosema bombi, with Nosema apis as a basal member to this group. When compared to their respective hosts (Apis cerana, Bombus spp., and A. mellifera), 2 plausible evolutionary scenarios emerged. The first hypothesis involves a common ancestor of N. bombi host-switching from a historical Bombus lineage to A. cerana. The second suggests an ancestral N. ceranae host-switching to a species of Bombus. The reported events offer insight into the evolutionary history of these organisms and may explain host specificity and virulence of Nosema in these economically important insects.     

Microsatellite loci for Euglossa annectans (Hymenoptera: Apidae) and their variability in other orchid bees

Orchid or euglossine bees are conspicuous Hymenoptera of the Neotropics, where they pollinate numerous plants, including orchids. Allozyme-based analyses have suggested that their populations suffer from inbreeding, as evidenced by so-called diploid male production. We have developed nine polymorphic microsatellite loci for the widespread Euglossa annectans, with observed heterozygosities ranging from 0.143 to 0.952 and between 2 and 9 alleles per species. These loci will be useful for analysis of relatedness, population genetic structure and diploid male production in this and related species.     

The behavioural and structural aspects of grooming and related activities in euglossine bees (Hymenoptera: Apidae)

The movements and structures involved in grooming, resin and pollen manipulation, and collection of aromatic material are described for members of the tribe Euglossini. These bees typically groom with each leg while hanging by their mandibles, working from the head backwards. Collection and transfer of aromatic material is described after re-evaluation using fluorescent powder. Resin collection involves asynchronous ipsilateral transfer of the resin to the hindtibia. Pollen collection, in contrast, involves synchronous ipsilateral transfer. Areas of the body that are not groomed are illustrated, and the various plants and animals that exploit these regions discussed. The similarities between the behaviours of euglossines and bumblebees supports the idea that they are closely related.     

Historical biogeography, divergence times, and diversification patterns of bumble bees (Hymenoptera: Apidae: Bombus)

Bumble bees (Bombus) are a cold-adapted, largely alpine group that can elucidate patterns of Holarctic historical biogeography, particularly in comparison to the alpine plants with which they likely coevolved. A recently published molecular phylogeny of bumble bees provides uniquely comprehensive species sampling for exploring historical patterns of distribution and diversification. Using this phylogeny and detailed data on extant distributions, I reconstruct the historical distribution of bumble bees in a temporal framework, estimating divergence times using fossil data and molecular rates derived from the literature. The nearly comprehensive phylogeny allows assessment of the tempo of diversification within the bumble bees using lineage-through-time plots and diversification statistics, which have been performed with special consideration to confidence intervals. These analyses reveal movements of Bombus concordant with geographic and climatic events of the late Cenozoic. The initial diversification of extant bumble bee lineages was estimated at around 25 to 40 Ma, near the Eocene-Oligocene boundary 34 Ma, a period of dramatic global cooling. Dispersal-vicariance analysis (DIVA) predicted an Old World Bombus ancestor, with early diversification events largely restricted to the eastern Old World. The numerous intercontinental dispersal events occurred mostly in the direction of Old World to New World and North America to South America. Early movements from the Palearctic into the Nearctic most likely took place after 20 Ma and may have coincided with a period of Miocene cooling that gave rise to taiga habitat across Beringia. Subsequent dispersal between these regions is estimated to have occurred among boreal and tundra-adapted species mostly in the last 5 million years. Radiations are estimated in both Nearctic and Neotropical regions at approximately 6 to 8 Ma and after 3.5 Ma, concordant with the opening of land corridors between the continents.