Heavy Metal (Hg,Cr, Cd,and Pb) Contamination in Urban Areas and Wildlife Reserves: Honeybees as Bioindicators

The degree of heavy metal (Hg, Cr, Cd, and Pb) pollution in honeybees (Apis mellifera) was investigated in several sampling sites around central Italy including both polluted and wildlife areas. The honeybee readily inhabits all environmental compartments, such as soil, vegetation, air, and water, and actively forages the area around the hive. Therefore, if it functions in a polluted environment, plant products used by bees may also be contaminated, and as a result, also a part of these pollutants will accumulate in the organism. The bees, foragers in particular, are good biological indicators that quickly detect the chemical impairment of the environment by the high mortality and the presence of pollutants in their body or in beehive products. The experiment was carried out using 24 colonies of honeybees bred in hives dislocated whether within urban areas or in wide countryside areas. Metals were analyzed on the foragers during all spring and summer seasons, when the bees were active. Results showed no presence of mercury in all samples analyzed, but honeybees accumulated several amounts of lead, chromium, and cadmium. Pb reported a statistically significant difference among the stations located in urban areas and those in the natural reserves, showing the highest values in honeybees collected from hives located in Ciampino area (Rome), next to the airport. The mean value for this sampling station was 0.52 mg kg⁻¹, and July and September were characterized by the highest concentrations of Pb. Cd also showed statistically significant differences among areas, while for Cr no statistically significant differences were found.     

Farming with native bees (<Emphasis Type="Italic">Apis mellifera</Emphasis> subsp. <Emphasis Type="Italic">capensis</Emphasis> Esch.) has varied effects on nectar-feeding bird communities in South African fynbos vegetation

Outside their natural range, honeybees (Apis mellifera) are known to have detrimental effects on indigenous pollinators through exploitative or interference competition, but little is known about the effect of honeybee farming in areas where honeybees occur naturally. In the Cape Floristic Region of South Africa, where honeybees are indigenous, managed hives potentially elevate the abundance of honeybees far above natural levels, but impacts on other floral resource-dependent species have not been studied. Here we use experimental manipulation of honeybee density to test whether honeybee farming affects nectar-feeding birds. We selected the common sugarbush (Protea repens), utilized by both birds and bees, and analysed the time (before/after) by treatment (control/experiment) interaction to explore changes in bee abundance, nectar availability and bird abundance at three sites. Hive addition increased honeybee abundance in inflorescences of P. repens above expected levels. Despite experimental increase in honeybee numbers, there is no reduction in nectar sugar availability relative to the control areas. Where honeybee density was highest, sugarbird (Promerops cafer) numbers declined relative to expected, but sunbirds (Nectarinidae) were not affected at any of the sites. We conclude that stocking rates of more than one honey bee per P. repens inflorescence have detrimental effects on bird abundance due to interference, rather than resource competition.     

Gut microbiome helps honeybee (Apis mellifera) resist the stress of toxic nectar plant (Bidens pilosa) exposure: Evidence for survival and immunity

Honeybee (Apis mellifera) ingestion of toxic nectar plants can threaten their health and survival. However, little is known about how to help honeybees mitigate the effects of toxic nectar plant poisoning. We exposed honeybees to different concentrations of Bidens pilosa flower extracts and found that B. pilosa exposure significantly reduced honeybee survival in a dose-dependent manner. By measuring changes in detoxification and antioxidant enzymes and the gut microbiome, we found that superoxide dismutase, glutathione-S-transferase and carboxylesterase activities were significantly activated with increasing concentrations of B. pilosa and that different concentrations of B. pilosa exposure changed the structure of the honeybee gut microbiome, causing a significant reduction in the abundance of Bartonella (p < 0.001) and an increase in Lactobacillus. Importantly, by using Germ-Free bees, we found that colonization by the gut microbes Bartonella apis and Apilactobacillus kunkeei (original classification as Lactobacillus kunkeei) significantly increased the resistance of honeybees to B. pilosa and significantly upregulated bee-associated immune genes. These results suggest that honeybee detoxification systems possess a level of resistance to the toxic nectar plant B. pilosa and that the gut microbes B. apis and A. kunkeei may augment resistance to B. pilosa stress by improving host immunity.     

The impact of apiculture on the genetic structure of wild honeybee populations (<Emphasis Type="Italic">Apis mellifera</Emphasis>) in Sudan

Apiculture often relies on the importation of non-native honeybees (Apis mellifera) and large distance migratory beekeeping. These activities can cause biodiversity conflicts with the conservation of wild endemic honeybee subspecies. We studied the impact of large scale honeybee imports on managed and wild honeybee populations in Sudan, a centre of biodiversity of A. mellifera, using as set of linked microsatellite DNA loci and mitochondrial DNA markers. The mitochondrial DNA analyses showed that all wild honey bees exclusively belonged to African haplotypes. However, we revealed non-native haplotypes in managed colonies on apiaries reflecting unambiguous evidence of imports from European stock. Moreover, we found significantly higher linkage disequilibria for microsatellite markers in wild populations in regions with apiculture compared to wild populations which had no contact to beekeeping. Introgression of imported honeybees was only measurable at the population level in close vicinity to apicultural activities but not in wild populations which represent the vast majority of honeybees in Sudan.     

A SNP test to identify Africanized honeybees via proportion of ‘African’ ancestry

The honeybee, Apis mellifera, is the world's most important pollinator and is ubiquitous in most agricultural ecosystems. Four major evolutionary lineages and at least 24 subspecies are recognized. Commercial populations are mainly derived from subspecies originating in Europe (75–95%). The Africanized honeybee is a New World hybrid of A. m. scutellata from Africa and European subspecies, with the African component making up 50–90% of the genome. Africanized honeybees are considered undesirable for bee‐keeping in most countries, due to their extreme defensiveness and poor honey production. The international trade in honeybees is restricted, due in part to bans on the importation of queens (and semen) from countries where Africanized honeybees are extant. Some desirable strains from the United States of America that have been bred for traits such as resistance to the mite Varroa destructor are unfortunately excluded from export to countries such as Australia due to the presence of Africanized honeybees in the USA. This study shows that a panel of 95 single nucleotide polymorphisms, chosen to differentiate between the African, Eastern European and Western European lineages, can detect Africanized honeybees with a high degree of confidence via ancestry assignment. Our panel therefore offers a valuable tool to mitigate the risks of spreading Africanized honeybees across the globe and may enable the resumption of queen and bee semen imports from the Americas.     

Hymenopteran specificity of Bacillus thuringiensis strain PS86Q3

The biological activity of Bacillus thuringiensis (Bt) strain PS86Q3 against five Hymenopteran species was determined by means of bioassays adapted to each species. Four species of sawfly that are important pests of conifers (Diprion pini, Gilpinia hercyniae and Pristiphora abietina) or ornamental plants (Arge rosae), as well as the non-target honeybee, Apis mellifera, were studied. Two out of the four sawfly species tested were found to be sensitive to PS86Q3 crystals or spore/crystal suspensions. A sporulated culture of this strain was moderately active on D. pini, and a complete bioassay with solubilized crystals was performed to estimate the LC₅₀ of 4.9 mg/ml. Pristiphora abietina was also found to be sensitive to PS86Q3, with an LC₅₀ of 1.6 mg/ml. By contrast, at the concentrations tested, PS86Q3 did not prove active on the remaining sawflies, G. hercyniae and A. rosae. The strain was administered orally to check its effects on honeybees which were fed sucrose solutions supplemented with a PS86Q3 sporulated suspension, in a field assay using commercial beehives. No significant differences in larval mortality (as deduced by comparing the number of larvae, pupae and empty cells) were found between the Bt and control treatments. On the basis of the results presented here, the suitability of PS86Q3 for the control of Hymenopteran pests, particularly sawflies, in terms of both potency and environmental safety, is discussed.     

Evidence for competition between honeybees and bumblebees; effects on bumblebee worker size

Numerous studies suggest that honeybees may compete with native pollinators where introduced as non-native insects. Here we examine evidence for competition between honeybees and four bumblebee species in Scotland, a region that may be within the natural range of honeybees, but where domestication greatly increases the honeybee population. We examined mean thorax widths (a reliable measure of body size) of workers of Bombus pascuorum, B. lucorum, B. lapidarius and B. terrestris at sites with and without honeybees. Workers of all four species were significantly smaller in areas with honeybees. We suggest that reduced worker size is likely to have implications for bumblebee colony success. These results imply that, for conservation purposes, some restrictions should be considered with regard to placing honeybee hives in or near areas where populations of rare bumblebee species persist.     

Synthetic Nasonov gland pheromone enhances abundance and visitation of honeybee,Apis mellifera,in Korla fragrant pear,Pyrus sinkiangensis

1. Korla fragrant pear (Pyrus sinkiangensis Yü) depends on cross-pollination by honeybees (Apis mellifera) but may suffer from low honeybee visitation.
2. We assessed whether honeybee abundance and visitation frequency are enhanced by using synthetic Nasonov gland pheromone (NGP), which is naturally produced by worker bees to stimulate the aggregation of bees to food resources or nesting sites.
3. The response of honeybees to synthetic NGP was firstly assessed using Y-tube olfactometer tests in the laboratory, and subsequently in the field, by placing NGP lures on Korla fragrant pear trees in orchards with and without beehives. Honeybee abundance was assessed using coloured pan traps while honeybee visits were assessed by visual observations on pear flowers.
4. Y-tube olfactometer tests showed a significant preference of honeybees for NGP. In pear orchards with beehives, honeybee abundance was 2.5-fold higher on trees with NGP lures than on trees without NGP, and 2.2-fold higher in orchards in which all trees contained NGP lures than in orchards without NGP lures. Such positive effects were not observed in orchards without beehives.
5. Flower visitation by honeybees was significantly higher in trees with NGP lures than without NGP lures, irrespective of the presence (5.7-fold higher) or absence of beehives (27.6-fold higher).
6. In mixed pear-apricot orchards, honeybee abundance was higher in pear trees with NGP lures than without lures.
7. Our results show that NGP lures attract honeybees to flowering pear trees in monoculture pear and mixed pear-apricot orchards, and that this effect is greatest in orchards with beehives.

     

Environmental factors have a strong impact on the composition and diversity of the gut bacterial community of Chinese black honeybees

The Chinese black honeybees is an ecotype of the European honeybee that is formed by the natural hybridization of Apis mellifera mellifera and A. m. carnica. It is distributed in nature reserves in North China and has been an important breeding resource for disease resistance and other desirable traits. Compared to the areas outside of reserves, the nature reserves offer significant biodiversity benefits not only to the Chinese black honey bees but also to the other valuable plants and animals. In recent years there has been growing evidence that environmental factors including food choices play an important role in shaping the composition and activity of gut microbiota, which in turn can impact host health. In the previous studies on Chinse black honeybees, little attention has been paid to the diverse population of microbes in the gut that play a vital role in host health. In order to achieve a better understanding on the role of environmental factors in diversity and composition of gut microbiota of honey bees, in the present study, we analyzed the gut bacterial communities of Chinese black honeybees using terminal restriction fragment length polymorphism (T-RFLP). The results showed that the samples from the national nature reserves that are protected and managed so as to preserve and enrich their natural condition and resources for Chinese black honeybees had higher variety and richness of gut bacteria than that collected from unreserved regions that also harbor populations of Chinese black honeybees. The four terminal restriction fragments (T-RFs), 201, 223, 247 and 320 bp, were identified to be the dominant bacteria of Chinese black honeybees. Of which 247 and 320 bp had greater differences between bee groups sampled in different regions and therefore could be used as genetic markers to separate samples collected from the national nature reserves to samples collected from unreserved regions. The results clearly indicate that national nature reserve protects biological diversity and ecological and evolutionary processes which have had a significant influence on the diversity of gut bacteria of Chinese black honeybees. The ubiquity of gut symbiotic bacteria identified in Chinese black honeybee suggests that environmental factors could play an important role in diversity and composition of gut bacteria and warrant further investigation into the functional significance of these gut bacteria for the honeybee health.     

Gut microbiome dysbiosis and honeybee health

Since a few decades, apiculture is facing important economic losses worldwide with general major consequences in many areas of agriculture. A strong attention has been paid towards the phenomenon named Colony Collapse Disorder in which colonies suddenly disappear with no clear explanations. Honeybee colonies can be affected by abiotic factors, such as environmental pollution or insecticide applications for agricultural purposes. Also biotic stresses cause colony losses, including bacterial (e.g. Paenibacillus larvae) and fungal (e.g. Ascosphaera apis) pathogens, microsporidia (e.g. Nosema apis), parasites (i.e. Varroa destructor) and several viruses. In the light of recent research, intestinal dysbiosis, considered as the relative disproportion of the species within the native microbiota, has shown to affect human and animal health. In arthropods, alteration of the gut microbial climax community has been shown to be linked to health and fitness disequilibrium, like in the medfly Ceratitis capitata for which low mate competitiveness is determined by a gut microbial community imbalance. According to these observations, it is possible to hypothesize that dysbiosis may have a role in disease occurrence also in honeybees. Here we aim to discuss the current knowledge on dysbiosis in the honeybee and its relation with honeybee health by reviewing the investigations of the microbial diversity associated to honeybees and the recent experiments performed to control bee diseases by microbial symbionts. We conclude that, despite the importance of a good functionality of the associated microbiota in preserving insect health has been proved, the mechanisms involved in honeybee gut dysbiosis are still unknown. Accurate in vitro, in vivo and in field investigations are required under healthy, diseased and stressed conditions for the host.     

Effect of Some Honeybee Diseases on Seasonal Mortality of <Emphasis Type="Italic">Apis mellifera intermissa</Emphasis> in Algeria Apiaries

With a view to identify the pathogens and to establish the role of these pathogens in regulation of the density of honey bee population occurring in the apiaries of the area concerned samples of honeybee were collected from the beekeepers in some parts of central Algeria It is revealed that Nosema sp., Varroa destrutor, Peanibacillus larvae are associated with the disease manifestation in honey bees. The presence of Nosema sp., Varroa destrutor, Peanibacillus larvae was analyzed using standard OIE methods. Spores of Paenibacillus larvae were detected in 56.6 % in winter 52.32 % in spring. 29.33 % in autumn and 11.25 % in summer. Nosema infestation was recorded in 47.91 % bee individuals during spring. Varroa infestation rate was maximum 12.57 % in summer and lowest 3.44 % in spring. Analysis of data indicates that Boumerdes and Tipaza, diseases induced mortality exceeds 10 % in honeybee. There exists a significant correlation between Nosema disease and mortalities in honeybees. Seasons play significant role, irrespective of pathogens, in disease manifestation.     

Preparation for disturbance-induced absconding of Cape honeybee colonies (Apis mellifera capensis Esch.)

African honeybees, Apis mellifera, are characterised by frequent disturbance-induced absconding. However, the effectiveness in preparation before such disturbance-induced absconding has not been rigorously quantified yet. We investigated the effectiveness of preparation for disturbance-induced absconding by evaluating colony phenotypes prior to and after absconding in ten colonies of the Cape honeybee, A. m. capensis. Seven non-absconding colonies at the same apiary were used as controls. While seven absconded colonies left neither stores nor brood behind, three colonies abandoned only a small area of honey, pollen, open or capped brood. At the end of the observations, the control colonies still had pollen and honey stores and brood. The mean reduction rate between a major disturbance and the absconding event was 0.052 ± 0.018 cm² stores and open brood per worker per day. Our results demonstrate that disturbance-induced absconding can also occur with preparation, if the disturbance is not highly destructive and enough time for preparation is available. We conclude that Cape honeybee colonies can show a considerable high effectiveness in their preparation before disturbance-induced absconding, which limits the loss of colony resources. In light of the general high mobility of African honeybee colonies such an efficient behaviour is probably adaptive. Received 22 December 2004; revised 3 June 2005; accepted 13 June 2005.     

Infections of Nosema ceranae in four different honeybee species

The microsporidium Nosema ceranae is detected in honeybees in Thailand for the first time. This endoparasite has recently been reported to infect most Apis mellifera honeybee colonies in Europe, the US, and parts of Asia, and is suspected to have displaced the endemic endoparasite species, Nosema apis, from the western A. mellifera. We collected and identified species of microsporidia from the European honeybee (A. mellifera), the cavity nesting Asian honeybee (Apis cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) from colonies in Northern Thailand. We used multiplex PCR technique with two pairs of primers to differentiate N. ceranae from N. apis. From 80 A. mellifera samples, 62 (77.5%) were positively identified for the presence of the N. ceranae. Amongst 46 feral colonies of Asian honeybees (A. cerana, A. florea and A. dorsata) examined for Nosema infections, only N. ceranae could be detected. No N. apis was found in our samples. N. ceranae is found to be the only microsporidium infesting honeybees in Thailand. Moreover, we found the frequencies of N. ceranae infection in native bees to be less than that of A. mellifera.     

Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L

We previously reported preferential expression of genes for ecdysteroid signaling in the mushroom bodies of honeybee workers, suggesting a role of ecdysteroid signaling in regulating honeybee behaviors. The organs that produce ecdysteroids in worker honeybees, however, remain unknown. We show here that the expression of neverland and Non-molting glossy/shroud, which are involved in early steps of ecdysteroid synthesis, was enhanced in the ovary, while the expression of CYP306A1 and CYP302A1, which are involved in later steps of ecdysone synthesis, was enhanced in the brain, and the expression of CYP314A1, which is involved in converting ecdysone into active 20-hydroxyecdysone (20E), was enhanced in the brain, fat body, and ovary. In in vitro organ culture, a significant amount of ecdysteroids was detected in the culture medium of the brain, fat body, and hypopharyngeal glands. The ecdysteroids detected in the culture medium of the fat body were identified as ecdysone and 20E. These findings suggest that, in worker honeybees, cholesterol is converted into intermediate ecdysteroids in the ovary, whereas ecdysone is synthesized and secreted mainly by the brain and converted into 20E in the brain and fat body.     

澜沧江流域北部中华蜜蜂有毒蜂蜜孢粉学和营养生态位分析

为分析澜沧江流域北部人食用蜂蜜中毒的原因,于2013年6—9月份,对该区域蜜蜂和蜜源植物的分布情况进行了调查,观察蜜蜂采集有毒蜜源植物的行为,并进一步调查了蜜蜂巢内蜂蜜、蜂花粉的储存情况,采集了中华蜜蜂蜂蜜样品,进行蜂蜜孢粉学与营养生态位分析。该区域有大量采用传统方式进行人工饲养的中华蜜蜂(Apis cerana cerana),及少量野生中华蜜蜂、黑色小蜜蜂(Apis audreniformis)、黑色大蜜蜂(Apis laboriosa smith)群体分布;人工饲养的中华蜜蜂蜂巢内部结构与野生中华蜜蜂蜂巢相似,为自然蜂巢,内有充足的蜜粉储存,部分蜂群蜂巢内虫害严重。该区域内主要蜜源植物为荞麦(Fagopyrum esculentum Moench),其他零星辅助蜜源较多,部分地点南烛(Vaccinium bracteatum Thunb)、昆明山海棠(Tripterygium hypoglaucum(Levl.)Hutch)连片集中分布。对中华蜜蜂蜂蜜进行孢粉学和营养生态位分析,结果表明:中华蜜蜂蜂蜜标本中含有有毒蜜源植物南烛、昆明山海棠花粉,部分样品中南烛、昆明山海棠的花粉含所占比例较高;中华蜜蜂的营养生态位宽度为0.22,比其他地区中华蜜蜂生态位指数小,推测澜沧江水电枢纽的修建等人为原因已对蜜蜂种类、蜜源植物的物种组成、群落结构造成了较大影响。     

What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.     

Seasonal variation in the activity of selected antioxidant enzymes and malondialdehyde level in worker honey bees

The recent decline in managed honey bee populations, Apis mellifera L. (Hymenoptera: Apidae), has caused scientific, ecological, and economic concern. Research into the formation of reactive oxygen species (ROS), antioxidative defense mechanisms, and oxidative stress can contribute to our understanding of bee survival and conservation of this species. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S‐transferase (GST) enzymes together with levels of malondialdehyde (MDA) were measured in summer and winter honey bees sampled from three colonies. One colony was stationary (C1), entering the winter period having accumulated Robinia pseudoacacia L. (Fabaceae) honey, and two were migratory (C2 and C3), entering the winter period with mainly Tilia (Malvaceae) and Brassica (Brassicaceae) honey, respectively. Compared to summer workers, winter worker bees had decreased SOD and GST activity, and MDA level, whereas CAT activity increased in all three colonies. We also demonstrated that seasonality is the main factor responsible for changes in antioxidant enzymes and MDA levels in worker honey bees. Overall, our results indicate a difference between summer and winter worker bees, pointing at a reduced level of antioxidant enzyme defenses during overwintering which may be due to a decrease in production of ROS. The decreased levels of MDA measured in winter honey bees confirm this. As ROS are actively used by insects as a defense mechanism to fight pathogens, we suggest that reduced production of ROS contributes to higher susceptibility of winter honey bees to infections and reduced overwinter survival.