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Indoor storage of honey bees (Apis mellifera L.) during winter months has been practiced for decades to protect colonies from the adverse effects of long, harsh winter months. Beekeepers have recently employed indoor storage to reduce labor, feeding costs, theft, and woodenware degradation. Despite the growing number of colonies stored indoors, national survey results still reveal high losses. Varroa mites (Varroa destructor Anderson and Trueman) are the most critical threat to colony winter survival and health of colonies because they contribute to the transmission of viruses and colony mortality. To investigate the effect of high CO2 on varroa mites during the indoor storage of honey bees, 8-frame single deep colonies were stored in two separate environmental chambers at 4°C each. One environmental chamber was set at 8.5% CO2 (high CO2), while the other was set at low CO2 (0.12%). Dead and falling mites were collected and counted from the bottom of individual colonies weekly during the experiment. There was a significant difference in mite mortality of colonies with high CO2 compared to colonies held at low CO2. These results indicated that high CO2 could increase mite mortality during the period of indoor storage, potentially improving honey bee health coming out of the winter months. Our research offers a critical addition to beekeepers’ tools for managing varroa mite populations.

Entomopathogenic fungi show great promise as pesticides in terms of their relatively high target specificity, low non-target toxicity, and low residual effects in agricultural fields and the environment. However, they also frequently have characteristics that limit their use, especially concerning tolerances to temperature, ultraviolet radiation, or other abiotic factors. The devastating ectoparasite of honey bees, Varroa destructor, is susceptible to entomopathogenic fungi, but the relatively warm temperatures inside honey bee hives have prevented these fungi from becoming effective control measures. Using a combination of traditional selection and directed evolution techniques developed for this system, new strains of Metarhizium brunneum were created that survived, germinated, and grew better at bee hive temperatures (35 °C). Field tests with full-sized honey bee colonies confirmed that the new strain JH1078 is more virulent against Varroa mites and controls the pest comparable to current treatments. These results indicate that entomopathogenic fungi are evolutionarily labile and capable of playing a larger role in modern pest management practices.

Global decline in insect pollinators, especially bees, have resulted in extensive research into understanding the various causative factors and formulating mitigative strategies. For commercial beekeepers in the United States, overwintering honey bee colony losses are significant, requiring tactics to overwinter bees in conditions designed to minimize such losses. This is especially important as overwintered honey bees are responsible for colony expansion each spring, and overwintered bees must survive in sufficient numbers to nurse the spring brood and forage until the new ‘replacement’ workers become fully functional. In this study, we examined the physiology of overwintered (diutinus) bees following various overwintering storage conditions. Important physiological markers, i.e., head proteins and abdominal lipid contents were higher in honey bees that overwintered in controlled indoor storage facilities, compared with bees held outdoors through the winter months. Our findings provide new insights into the physiology of honey bees overwintered in indoor and outdoor environments and have implications for improved beekeeping management.

Cryopreservation is an advanced method used to protect germplasm in liquid nitrogen. Honey bees are of special interest to protect because of their pollination activity and critical role in agriculture. There has been important progress in the cryopreservation of honey bee germplasm in recent years, leading to practical recovery of genetic material for breeding purposes following freezing. However, there remains room for improvement and the goal of the present study was to evaluate the effect of different “extenders” added post-thaw on the fertilization rate of cryopreserved honey bee semen. The purpose of adding extender post-thaw was to dilute the cryoprotectant to remove chemicals after centrifugation because of potential adverse effects. The control consisted of frozen-thawed semen without the addition of an extender; treatment groups included the addition of one of the following extenders: glucose solution, fresh ram semen plasma, fresh honey bee semen plasma, extender solution. All of the above treatments and frozen-thawed control were compared to fresh semen. For each group, 15 virgin queens were instrumentally inseminated with the semen-diluent solution and introduced into nucleus colonies to determine the brood patterns of the queens. Percentages of worker brood produced in the fresh semen, frozen-thawed semen control, glucose, fresh ram semen plasma, fresh honey bee semen plasma, and extender solution supplemented groups were 98.±1.1%, 47.0 ± 0.9%, 3.0 ± 0.8%, 0.3 ± 0.1%, 48.1 ± 4.1% and 40.3 ± 2.4%, respectively. Similiarly, spermatozoa numbers in the spermathecae of the same treatment groups were 3.6 × 10 6 , 1.6 × 10 6 , 7.3 × 10 5 , 4.7 × 10 5 , 8.1 × 10 5 , and 4.6 × 10 5 spermatozoa for the same treatment, respectively. The differences in both worker brood percentage and sperm count in the spermatheca were statistically significant (P

Patterns of genetic structure and diversity are largely mediated by a species’ ecological niche and sensitivity to climate variation. Some species with narrow ecological niches have been found to exhibit increased population differentiation, limited gene flow across populations, and reduced population genetic diversity. In this study, we examine patterns of population genetic structure and diversity of four bumble bee species that are broadly sympatric, but do not necessarily inhabit the same ecological niche in the Pacific Northwest of the United States. Testing for the effect of isolation by geographic distance (IBD) with linearized F st and D est found that Bombus sylvicola and B. mixtus exhibited significant IBD across populations. In contrast, both B. melanopygus and B. flavifrons, two species that are distributed across a broad elevation gradient, exhibited no IBD, a result further corroborated by Bayesian a priori population assignment tests. Furthermore, we discovered that B. sylvicola populations distributed on the Olympic Peninsula have significantly less average allelic diversity than populations distributed in the Cascade Mountains. Our results suggest that populations distributed in the Olympic Mountains represent a distinct genetic cluster relative to the Cascade Mountains, with B. sylvicola and B. mixtus likely experiencing the greatest degree of population genetic differentiation relative to B. flavifrons and B. melanopygus. While bumble bees are known to co-exist across a diversity of habitats, our results demonstrate that underlying population genetic structure and diversity may not necessarily be similar across species, and are largely governed by their respective niches.

Global climate change is the greatest environmental challenge of the modern era. The impacts of climate change are increasingly well understood, and have already begun to materialize across diverse ecosystems and organisms. Bumble bees (Bombus) are suspected to be highly sensitive to climate change as they are predominately adapted to temperate and alpine environments. In this study, we determine which bumble bee species are most vulnerable to climate change in the Pacific Northwest. The Pacific Northwest is a topographically complex landscape that is punctuated by two major mountain ranges and a labyrinth of offshore islands in the Salish Sea. Using standardized survey methods, our study documents the occurrence of 15 bumble bee species across 23 field sites in seven federal parks, historical sites, and monuments. Our results show that bumble bee community richness and diversity increases along an altitude gradient in these protected areas. Furthermore, NMDS analysis reveals that high altitude environments are composed of a unique group of bumble bee species relative to low altitude environments. Finally, based on an analysis of species distributions models that aggregate bioclimatic data from global circulation climate models with preserved specimen records, we discover that 80% of the bumble bee species detected in our survey are poised to undergo habitat suitability (HS) loss within the next 50 years. Species primarily found in high altitude environments namelyB. vandykei, B. sylvicola, andB. bifariusare projected to incur a mean HS loss of 63%, 59%, and 30% within the federally protected areas, respectively. While the implementation of climate change policies continue to be a significant challenge, the development of mitigation strategies to conserve the most vulnerable species may be a tractable option for land managers and stakeholders of protected areas. Our study meets this need by identifying which species and communities are most sensitive to climate change.

Bumble bees (Hymenoptera: Apidae, Bombus) are cold-adapted insects, primarily known for their importance in providing ecosystem services to wild and cultivated flowering plants. Recent expeditions into the wilderness regions of the Olympic Mountains of Olympic National Park, USA discovered undocumented populations of two bumble bee species: Bombus sylvicola and B. vandykei. Application of species distribution models with range-wide locality records identified the Olympic Mountains to have high habitat suitability for B. sylvicola and low habitat suitability for B. vandykei. Our results suggest that Olympic National Park is a habitat island for B. sylvicola, isolated from the relatively contiguous distribution of the species in the Cascade and Sierra Nevada mountain ranges. Bumble bees are sensitive to environmental change, thus our discoveries will likely stimulate conservation-oriented investigations on these charismatic pollinators on the Olympic Peninsula and throughout the Pacific Northwest.

A comparative assessment of apiaries in urban, rural, and agricultural areas was undertaken in 2013 and 2014 to examine potential honey bee colony exposure to neonicotinoid insecticides from pollen foraging. Apiaries ranged in size from one to hundreds of honey bee colonies, and included those operated by commercial, sideline (semicommercial), and hobbyist beekeepers. Residues in and on wax and beebread (stored pollen in the hive) were evaluated for the nitro-substituted neonicotinoid insecticides imidacloprid and its olefin metabolite and the active ingredients clothianidin, thiamethoxam, and dinotefuran. Beebread and comb wax collected from hives in agricultural landscapes were more likely to have detectable residues of thiamethoxam and clothianidin than that collected from hives in rural or urban areas (∼50% of samples vs.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

International audience; Honey bees (Apis mellifera L.) provide essential pollination services for agricultural production worldwide. Recent declines in honey bee populations have been attributed to a multitude of potential stressors, such as pest pressure, nutritional stress, and exposure to pesticides (vanEngelsdorp et al. 2009b). One possible explanation for bee decline is the accumulation of pesticide residues in honey bee brood comb, resulting in chronic exposure to a suite of agrochemicals which may detrimentally influence honey bee health. Wu et al. (2011) demonstrated that bees reared in brood comb containing high levels of pesticide residues exhibit increased brood mortality and shorter adult longevity.

SummaryWe analyzed mitochondrial DNA variation in honey bee colonies in Jordan using DraI restriction profiles of the COI-COII intergenic region. Seven different haplotypes were observed, three of which belonged to the C mitochondrial lineage. Samples displaying haplotypes of the C lineage were concentrated in the north of the country where frequent importations of European bees have occurred. We conclude that the C mitochondrial haplotypes found in the study reflect past or recent importations of non-native honey bees. No C haplotypes were detected in areas that have been identified as possible source populations for the conservation of original A. m. syriaca in the south of the sampling region, thus confirming the existence of comparatively pure genetic resources of this subspecies in Jordan.

Scolytus schevyrewi Semenov, the banded elm bark beetle, and S. multistriatus Marsham, the smaller European elm bark beetle, are morphologically similar. Reliance on adult external morphological characters for identification can be problematic because of wide within-species variability and the need for good-quality specimens. The inability to identify developmental stages can also hamper early-detection programs. Using two character identification systems, genitalic (aedeagus) morphology, and DNA markers (random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)) to distinguish S. schevyrewi from S. multistriatus, we examined specimens from geographically distinct populations of both species collected from infested host trees or semiochemical-baited funnel traps. We found that aedeagus morphology can be used to identify the two species. The use of two oligonucleotide primers in the RAPD-PCR analysis yielded distinct DNA banding patterns for the two species. Species identification using RAPD-PCR analysis was validated by a blind test and used to make species identifications of larval specimens. These tools improve the ability to differentiate between S. schevyrewi and S. multistriatus at immature and adult stages, and could be developed and used for other scolytines as well.

A population of honey bees (Apis mellifera mellifera L.) with an annual colony brood cycle adapted to a locally abundant floral source in the Landes region of Southwest France is the subject of genetic conservation efforts. This population is maintained by local beekeepers in an area that experiences both an annual seasonal influx of non-local colonies and the permanent culture of imported stock. However, some colonies native to the Landes do not have the adapted brood cycle and their status as ecotypic are in question. The present study used morphology, mitochondrial DNA and microsatellites to characterize the endemic population and suggests further genetic conservation strategies. These methods yielded different degrees of discrimination of native and imported colonies and provided a powerful suite of tools for local resource managers. Colonies from the Landes could be differentiated from non-local French A. m. mellifera populations using morphometric analysis, and from non-native and reference populations using mtDNA and microsatellites. Seven morphological characters were identified by discriminant analysis as informative for delineating the Landes ecotype from other A. m. mellifera populations. Mitochondrial haplotypes for the population were characterized and five microsatellite loci were found to be informative in characterizing the Landes population. Asymmetric gene flow detected with microsatellite alleles was observed to be 5.5–5.9% from imported to native stocks of honey bees while introgression of native microsatellite alleles into imported colonies was 21.6%.

SummaryIt has been proposed that the Iberian Peninsula served as an important refuge for honey bees during periods of European glaciation. The discovery of genetic markers originating from both African and European honey bee lineages in extant Iberian populations led to hypotheses that the region may be a zone of hybridization or reflect the action of human-assisted translocations. In the present study, we investigated allozyme, mitochondrial and morphological variation in honey bee populations sampled from southern France to northern Morocco. Morphological data supported the interpretation that the bees of Iberia, south and west of a line from Zaragoza to Barcelona, can be characterized as the subspecies Apis mellifera iberica (A. m. iberiensis). There appears to be a narrow demarcation between this subspecies and A. m. mellifera, rather than a gradual cline. The persistence of significant subpopulation differentiation indicates that migratory beekeeping has not strongly influenced the genetic characteri...

The biogeography and intraspecific variability of the eastern cavity-nesting honeybee, Apis cerana, are not very well known. We studied the variability of this species in China using morphometrical methods together with restriction and sequence analysis of two different regions of mitochondrial DNA. Samples of A. cerana were collected from feral or traditionally managed colonies in 19 locations of the Chinese mainland, covering the main ecological regions. Worker bees from each sample were dissected and morphometric characters were measured. The data were analysed with multivariate statistical procedures. Data were supplemented by previously published Chinese samples from the Oberursel data bank and reference samples of A. cerana from adjacent countries. A mitochondrial DNA fragment containing a non-coding region was amplified and analysed with the restriction enzyme DraI. This fragment was sequenced for two samples. For a subset of samples, the subunit 2 of the mitochondrial NADH gene was amplified and sequenced. Morphometric analysis revealed a high degree of variation, strongly associated with ecological zones and correlated with geographical and climatic parameters. Two main clusters were apparent, one comprised the bees from the southern tropical seasonal rain forest region, showing strong associations to the bees of Vietnam, Thailand and Myanmar. The second main cluster included the bees from the temperate deciduous broad-leaved forest region, the subtropical evergreen broad-leaved forest zone, the high, cold meadow and steppe region and the North, and showed increasing similarity to the bees of Korea and Japan. In particular, the bees from Qingzang plateau, on the fringe of the Ganshu province, were set apart by their exceptional body size, darkness and pilosity. There was no variation in DraI restriction patterns within China. Sequence variation of the mitochondrial ND2 region was consistent with geographic patterns of morphological variation. Bees of the South Yunnan region were set apart by characteristically broad abdominal sterna and wax mirrors, with this locally restricted trait transcending the main transition line at the northern limit of the tropical seasonal rain forest region. This northern limit appears to correspond to the separation line between A. cerana indica and A. cerana cerana. © The Natural History Museum.

We characterized Apis mellifera in both native and introduced ranges using 1136 single-nucleotide polymorphisms genotyped in 341 individuals. Our results indicate that A. mellifera originated in Africa and expanded into Eurasia at least twice, resulting in populations in eastern and western Europe that are geographically close but genetically distant. A third expansion in the New World has involved the near-replacement of previously introduced “European” honey bees by descendants of more recently introduced A. m. scutellata (“African” or “killer” bees). Our analyses of spatial transects and temporal series in the New World revealed differential replacement of alleles derived from eastern versus western Europe, with admixture evident in all individuals.

Two different genomic regions (ND2 mitochondrial gene and EF1-α intron) were PCR amplified, cloned and sequenced for the ten known honey bee species collected within their natural range distribution. DNA sequences were analyzed using parsimony, distance and maximum likelihood methods to investigate phylogenetic relationships within Apis . The phylogenetic analyses strongly supported the basic topology recoverable from morphometric analysis, grouping the honey bees into three major clusters: giant bees ( A. dorsata , A. binghami , and A. laboriosa ), dwarf bees ( A. andreniformis and A. florea ), and cavity-nesting bees ( A. mellifera , A. cerana , A. koschevnikovi , A. nuluensis , and A. nigrocincta ). However, the clade of Asian cavity-nesting bees included paraphyletic taxa. Exemplars of Apis cerana collected from divergent portions of its range were less related to each other than were sympatric A. cerana , A. nuluensis , and A. nigrocincta taxa. Nucleotide sequence divergence between allopatrically distributed western ( A. mellifera ) and eastern ( A. cerana , A. koschevnikovi , A. nigrocincta , and A. nuluensis ) cavity-nesting species, around 18% for the mitochondrial gene and 10–15% for the nuclear intron, suggested an earlier divergence for these groups than previously estimated from morphometric and behavioral studies. This latter finding neccessitates reevaluation of the hypothesized origin of extant European, African, and west Asian Apis mellifera . Sequence divergence between A. laboriosa and A. dorsata was consistent with behavioral data and supports the species status of A. laboriosa .

The Nearctic leafhopper genus Errhomus Oman (Hemiptera: Cicadellidae) exhibits high host-plant selectivity and female brachyptery, which may limit gene flow. Two subgenera, Errhomus (Errhomus) and Errhomus (Erronus), are recognized based on morphological and behavioral differences. We used mitochondrial DNA sequencing to elucidate the phylogenetic relationships among members of the subgenus Errhomus (Erronus). Portions of the NADH dehydrogenase subunit 1 (ND1) and the leucine (CUN) transfer RNA (tRNA-Leu) genes were sequenced and were analyzed with parsimony and maximum likelihood. Our results generally verified the findings of Hamilton and Zack (1999); however, both optimality criteria revealed a paraphyletic Errhomus (Erronus). Errhomus (Erronus) calvus does not seem to be a member of the subgenus Errhomus (Erronus) but seems to be more closely aligned with the subgenus Errhomus (Errhomus). We tested two a priori null hypotheses: 1) that the phylogeny estimated from the molecular data matched a previous phylogeny based on morphology and biogeography from Hamilton and Zack (1999) and 2) that diagnostic characters of the male genitalia (serrated aedeagal processes and triangular aedeagal shaft tip) were synapomorphies that defined major clades. We rejected these hypotheses with both Bayesian tests and parametric bootstraps.

Polymerase chain reaction (PCR)-amplified mitochondrial DNA (mtDNA) assays have been used in studies of the Africanization process in neotropical feral and managed honey bee populations. The approach has been adopted, in conjunction with morphometric analysis, to identify Africanized bees for regulatory purposes in the United States such as in California. In this study, 211 Old World colonies, representing all known introduced subspecies in the United States, and 451 colonies from non-Africanized areas of the southern United States were screened to validate a rapid PCR-based assay for identification of Africanized honey bee mtDNA. This PCR-based assay requires a single enzyme digestion (BglII) of a single PCR-amplified segment of the cytochrome b gene. The BglII polymorphism discriminates the mitochondrial haplotype (mitotype) of Apis mellifera scutellata L. (ancestor of Africanized bees) from that of A. m. mellifera, A. m. caucasia, A. m. ligustica, A. m. carnica, A. m. lamarcki, A. m. cypria, A. m. syriaca, and some A. m. iberiensis, but not from that of A. m. intermissa and some A. m. iberiensis. Nonetheless, given the very low frequency (

International audience; Endemic honey bees of the Tien Shan Mountains in Central Asia are described as a new subspecies, Apis mellifera pomonella, on the basis of morphometric analyses. Principal component and discriminant analysis of the morphological characters measured clearly place these bees into the oriental evolutionary branch of honey bees, but also show that they are distinct from the other subspecies in this lineage. The existence of this newly described honey bee subspecies extends the range of endemic A. mellifera more than 2000 km eastward than previously estimated. Sequence analysis of mitochondrial DNA places A. m. pomonella within the C mitochondrial lineage (a group that is inclusive of both C and O morphological lineages). These findings support the conclusion that A. m. pomonella has a phylogeographic history shared with subspecies from the eastern limit of the previously known range.

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study outbreaks of Mediterranean fruit fly in California in the years 1992-94 and 1997-99. A total of 359 flies caught in monitoring traps during these years were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. Composite genotypes obtained through analysis of these markers indicate at least five independent introductions of medflies into California between 1992 and 1998. Whereas the majority of specimens displayed a single mtDNA haplotype (AAA), variation of microsatellite alleles among these flies suggests at least one additional introduction in 1993 into southern California. Flies displaying the AAB haplotype sampled in 1992 both in northern and southern California shared microsatellite alleles absent in AAA flies although lacking others commonly found in AAA specimens, thus supporting the hypothesis of an independent introduction of these flies from a different source. In contrast to earlier infestations, a few specimens caught in southern California in 1993 and again in 1998 showed both mtDNA and microsatellite patterns consistent with a Hawaiian origin. Single flies collected in Santa Clara County in 1997 and in El Monte, Los Angeles County & in 1999 most likely represent a sixth and seventh distinct introduction, respectively.

Seven treatments for the control of Varroa destructor (Anderson & Trueman) were tested to determine the optimum timing of miticide application. Threshold mite levels indicating miticide application were determined for three possible treatment dates: April, August, and October. The treatments were as follows: (1) fluvalinate in April, (2) fluvalinate in August, (3) fluvalinate in October, (4) fluvalinate in April and October, (5) fluvalinate applied continuously (except during honey flow) with replacement every 42 d, (6) control (no treatment), and (7) coumaphos in April. The number of miticide applications in a season had no effect on brood area or colony bee population a year after initiating the experiment. However, the absence of any treatment significantly reduced brood area and colony bee population and significantly increased colony mite population. Date of treatment had significant effects on colony mortality rates, mite levels, and brood area the following spring. When coupled with sampling and threshold recommendations, a single, late-season application of fluvalinate is as effective for the control of V. destructor as semiannual or continuous miticide applications. Treatment thresholds were recommended for ether roll and 48-h sticky board sampling methods in April (three and 24 mites, respectively) and August (14 and 46 mites, respectively) and for ether rolls in October (three mites) in cold climates.

In 1956, honey bees of the African subspecies A. m. scutellata were imported to Brazil, and their descendents subsequently spread to South, Central, and North America. This invasion sparked significant academic controversy, particularly concerning the genetic composition of the expanding population. We review the biogeography and intraspecific phylogeny of Apis mellifera in the Old World as it pertains to African-derived bees in the Americas, the methods used to study gene flow between European-derived and African-derived populations in the New World, and the techniques used in identification of African-derived bees.

International audience; Thirty-nine samples of Apis mellifera monticola and A. m. scutellata from three different regions of Kenya were analyzed for mitochondrial DNA (mtDNA) variation using 6-base and 4-base restriction enzymes. Restriction with HpaII and AluI resulted in distinct patterns that together produced three different haplotypes. While haplotypes 2 and 3 were restricted to samples from the mountain forest, haplotype 1 was found in A. m. scutellata and in all samples from the Ngong Hills. No introgression of A. m. scutellata mtDNA was detected in bees collected in mountain environments, but a few samples from the savanna had A. m. monticola morphology, or mtDNA, or both. These results support the hypothesis that A. m. monticola is a distinct subspecies and not an ecotype of A. m. scutellata. The polymorphic restriction sites were mapped. Ten samples of A. m. litorea from the coast of Kenya were analyzed using polymerase chain reaction (PCR) amplification and subsequent restriction analysis. All samples of A. m. litorea shared the A. m. scutellata haplotype.; Polymorphisme de l'ADNmt chez les sous-espèces d'abeilles domestiques du Kenya. La variabilité de l'ADNmt a été étudiée sur 39 échantillons d'Apis mellifera monticola et d'A. m. scutellata identifiés morphologiquement et provenant de trois régions montagneuses du Kenya et des savanes adjacentes. L'ADN total a été digéré par les enzymes AccI, AluI, BclI, BglII, EcoRI, HpaII, MboI, RsaI et XbaI, reconnaissant des sites de restriction à quatre ou six bases. Les fragments ont été séparés par électrophorèse sur gel d'agarose à 1,2 % , colorés au bromure d'éthidium, photographiés et transférés sur des membranes de nitrocellulose. Ensuite, les membranes ont été préhybridées et hybridées à 50 $^{\circ}$C avec une sonde d'ADN radiomarquée produite à partir d'ADNmt purifié d'abeilles. Les sites de restrictions polymorphes ont été cartographiés en effectuant des digestions doubles avec EcoRV, XhoI et AccI. D'autre part, le polymorphisme de restriction de dix échantillons d'A. m. litorea récoltés sur la côte du Kenya a été analysé à l'aide d'amplification par PCR suivie de digestions de restriction. Tous les échantillons de l'étude avaient en commun les profils de restriction produits par les enzymes AccI, BclI, BglII, EcoRI, RsaI et XbaI. La restriction avec les enzymes à quatre bases, AluI, HpaII, a permis de mettre en évidence au sein des deux races monticola et scutellata (Fig. 1) trois haplotypes différents (Tab. I), qui présentent une répartition géographique caractéristique (Fig. 2). La figure 3 montre les positions des sites de restriction polymorphes cartographiés pour AluI et HpaII. Tandis que les haplotypes 2 et 3 sont limités aux échantillons de A. m. monticola du Mont Elgon et du Mont Kenya, l'haplotype 1 se trouve chez A. m. scutellata et dans tous les échantillons des Ngong Hills, région sujette à une urbanisation croissante. C'est la première fois que l'on met en évidence des différences diagnostiques dans la variation des profils d'ADNmt parmi les races d'abeilles mellifères d'Afrique orientale. Nos résultats prouvent clairement la validité de l'hypothèse selon laquelle A. m. monticola ne peut être considérée comme l'écotype d'A. m. scutellata, mais est le résultat de sa propre histoire évolutive. à la lumière des changements climatiques survenus pendant et après le Pléistocène, les populations actuellement disjointes d'A. m. monticola représentent les reliques d'une plus grande population d'abeilles du Pléistocène actuellement limitées à des zones refuges dans les montagnes. Nous n'avons pas détecté d'introgression chez A. m. scutellata parmi les échantillons récoltés dans les régions montagneuses au travers des études de la morphologie et de la variation de l'ADNmt. Ceci est en contradiction avec l'hypothèse des migrations verticales à grande échelle d'A. m. scutellata depuis les savanes vers les montagnes. Au contraire, quelques colonies prélevées dans la savane soit avaient la morphologie et l'ADNmt d'A. m. monticola, soit combinaient la morphologie d'une sous-espèce avec l'ADNmt de l'autre (Fig. 2). Alors que les populations d'abeilles voisines du Mont Elgon et celles voisines du Mont Kenya présentaient des différences prononcées, les abeilles des Ngong Hills n'avaient qu'un seul haplotype typique d' A.m. scutellata. Nous émettons l'hypothèse que ceci est le résultat d'une hybridation entre les abeilles de montagne et celles de savane, conséquence d'une déforestation et d'une urbanisation grandissantes de cette région. Tous les échantillons d'A. m. litorea de cette étude partageaient l'haplotype d'ADNmt typique d' A. m. scutellata. Ce résultat ne fournit pas de nouvel élément de réponse à la question : A. m. litorea doit elle être considérée comme un écotype d' A. m. scutellata ou comme une sous-espèce distincte, comme le suggère l'analyse morphométrique ?

The development times of daughter queens from African and European matrilines mated to both African and European drones were recorded. Regardless of the matriline, African patriline queens completed their development and emerged 8–12 h before those with European paternity. A probability distribution function derived from the emergence time data indicated that because of differences in development times between patrilines, the probability that an African patriline queen will emerge 1st can be 2–3 times greater than the proportion of the African patrilines in the colony population. Because the 1st queen to emerge has the best chance of becoming the colony's new queen, differences in queen development times between Africanand European patrilines might be a factor contributing to the asymmetrical gene flow between African and European honey bee, Apis mellifera L., populations, and the eventual loss of European nuclear markers and behavioral attributes in European honey bee populations where African bees have migrated.

Variation in mitochondrial DNA (mtDNA) was assessed for >100 populations of Mediterranean fruit flies Ceratitis capitata (Weidemann), collected from the New and Old World using 3 restriction enzymes, Eco RV, Mnl I, and Xba I. Previously reported variation for some populations was based on Southern blot analysis of nucleic acid extractions digested with Eco RV and Xba I. Mnl I variation for these populations and complete restriction enzyme profiles for many new populations were obtained with a polymerase chain reaction amplification-based method that revealed polymorphism in portions of the mitochondrial genes for NADH dehydrogenase subunits 4 and 5. Highest levels of mtDNA diversity were found in samples from sub-Saharan Africa. Lower levels of mtDNA polymorphism were found in the countries of the Mediterranean region, where the Mediterranean fruit fly has been established for >150 yr. Examination of South and Central American populations revealed a disjunct pattern of mtDNA haplotypes with relatively low intrapopulational variation, reflecting multiple introductions into the New World. Our approach provides improved resolution of possible source countries for historical infestations in the United States.

Les abeilles endemiques de l'ile de Malte sont decrites comme une race geographique distincte, A m ruttneri, sur la base d'une analyse morphologique discriminante. Les abeilles de Malte sont semblables en taille a A m sicula et a A m intermissa, mais possedent des pattes et des ailes plus courtes ; les ailes sont egalement beaucoup plus etroites. L'abdomen des abeilles trouvees a Malte est beaucoup plus large (index du sternum (SI) = 77,74) que celui de la race apparentee A m intermissa (SI = 81,52). Ainsi A m ruttneri est la sous-espece d'A mellifera la plus large ; elle est meme plus large que A m mellifera (SI = 78,61) (Ruttner, 1992). Le tableau I donne les moyennes et les deviations standard de certains caracteres pour A m ligustica, A m sicula, A m intermissa et pour les echantillons maltais. L'haplotype mitochondrial trouve chez l'abeille maltaise est typique des sous-especes africaine et sicilienne. Les caracteristiques de l'ADN et celles du comportement plaident en faveur d'une relation plus etroite d'A m ruttneri avec A m intermissa d'Afrique du Nord qu'avec les sous-especes europeennes, situation semblable a celle des abeilles endemiques de Sicile (A m sicula). Ces resultats suggerent que les abeilles qui ont peuple les iles de la Mediterranee centrale ont connu un developpement commun au cours de l'evolution. La sous-espece est nommee ruttneri en hommage au professeur F Ruttner qui a tant contribue a la connaissance de la taxinomie infraspecifique de l'abeille mellifere. Malheureusement, en raison de l'etendue restreinte de la race, de la perte causee aux populations locales par l'acarien Varroa jacobsoni et de l'importation de souches de races etrangeres, la situation de cette race est en grand danger. La probabilite de la voir disparaitre est tres elevee.

A mitochondrial DNA region encompassing part of the NADH dehydrogenase subunit 2 and isoleucine transfer RNA genes was PCR amplified, cloned, and sequenced for 14 morphometrically identified Apis mellifera subspecies and the New World "Africanized" honeybee. Twenty different haplotypes were detected and phylogenetic analyses supported the existence of 3 or 4 major subspecies groups similar to those based on morphometric measurements. However, some discrepancies are reported concerning the subspecies composition of each group. Based on the sequence divergence of Drosophila (2% per Myr) we found that the four lineages may have diverged around 0.67 Myr. The variability found in this region enables us to infer phylogenetic relationships and test hypotheses concerning subspecies origin, dispersion, and biogeography.

Approximately 3.2 million honey bee colonies are maintained by beekeepers in the United States and many are headed by commercially bred queens. We used mitochondrial DNA (mtDNA) and allozyme variation to characterize 142 breeder queen colonies from 22 apiaries in the southeastern United States that produced ≈483,900 commercial honey bee queens in 1993. Analysis of mtDNA haplotypes showed that 4% of the 142 commercial breeder queen colonies were maternal descendants of Apis mellifera mellifera , a subspecies that was imported into the United States by the 17th century but is no longer used commercially. The other 96% were probably descendants of A. m. ligustica . or A. m. carnica , subspecies imported in the 19th century which are still sold as commercial strains. Malate dehydrogenase allele frequencies for the 142 breeder queen colonies were determined to be Mdh 65 = 0.50, Mdh 80 = 0.23, and Mdh 100 = 0.27. Five other enzymes known to be polymorphic in adult honey bees were invariant. Significant genetic differences between commercial and feral populations suggest that the feral population may represent a novel source of genetic variation for breeding programs.

Africanized honeybees are presently expanding their geographic range within the USA and are considered undesirable due to their substantial defensive behaviour. Africanized honeybees can be differentiated from honeybees of European ancestry using discriminant analysis of morphological characters, based on a minimum of ten intact specimens per colony. In this paper we report a PCR-based method suitable for the identification of African or European mtDNA from sting remnants, such as typically remain on victims following a stinging incident. We experimentally simulated collection and shipment conditions with dried and alcohol preservation of stings, and also report that the method was suitable for sting remnants stored with a victim sample for over one year in alcohol. The determination of mtDNA haplotypes from stinger remnants must be tempered by the constraints inherent in interpretation of mtDNA haplotype data. Such data do not provide any information regarding the genetic contribution of the paternal lineage. However, in geographic areas where baseline information regarding European haplotype frequencies is known, the detection of a different mtDNA haplotype in stingers from a victim, especially when associated with observations of extreme defensive behaviour, would certainly be suggestive of Africanization.

Mitochondrial DNA (mtDNA) and allozyme analyses of 692 feral honey bee, Apis mellifera L., colonies revealed significant genetic population structure (heterogeneity) in the southern tier of the United States. Analysis of mtDNA haplotypes showed that 61% of the colonies were maternal descendants of the European races most commonly used for commercial beekeeping and 37% were descendants of a race ( A. m. mellifera L.) that was imported between 1620 and the 1860s, but is no longer used commercially. Twelve colonies from five states exhibited an African haplotype associated with the race A. m. lamarckii Cockerell introduced from Egypt >100 yr ago. Several electrophoretic alleles not previously reported from U.S. populations were detected, and malate dehydrogenase allele frequencies for the 692 colonies were determined to be Mdh65 = 0.47, Mdh80 = 0.33, and Mdh100 = 0.20. Despite the significant heterogeneity of allele frequencies, no significant deviations from Hardy-Weinberg expectations were detected when all bees sampled were artificially pooled into one population or by individual states. The Mdh80 frequency was higher than that found in previous studies of commercial populations, suggesting a disproportionately higher genetic contribution of A. m. mellifera to the feral population. The discovery of significant heterogeneity among U.S. feral honey bees implies that this population may represent a source of genetic variation for breeding programs.

Nosema ceranae and pesticide exposure can contribute to honey bee health decline. Bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with N. ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores+syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection.

BACKGROUND: Numerous surveys reveal high levels of pesticide residue contamination in honey bee comb. We conducted studies to examine possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan. METHODOLOGY/PRINCIPAL FINDINGS: Worker bees were reared in brood comb containing high levels of known pesticide residues (treatment) or in relatively uncontaminated brood comb (control). Delayed development was observed in bees reared in treatment combs containing high levels of pesticides particularly in the early stages (day 4 and 8) of worker bee development. Adult longevity was reduced by 4 days in bees exposed to pesticide residues in contaminated brood comb during development. Pesticide residue migration from comb containing high pesticide residues caused contamination of control comb after multiple brood cycles and provided insight on how quickly residues move through wax. Higher brood mortality and delayed adult emergence occurred after multiple brood cycles in contaminated control combs. In contrast, survivability increased in bees reared in treatment comb after multiple brood cycles when pesticide residues had been reduced in treatment combs due to residue migration into uncontaminated control combs, supporting comb replacement efforts. Chemical analysis after the experiment confirmed the migration of pesticide residues from treatment combs into previously uncontaminated control comb. CONCLUSIONS/SIGNIFICANCE: This study is the first to demonstrate sub-lethal effects on worker honey bees from pesticide residue exposure from contaminated brood comb. Sub-lethal effects, including delayed larval development and adult emergence or shortened adult longevity, can have indirect effects on the colony such as premature shifts in hive roles and foraging activity. In addition, longer development time for bees may provide a reproductive advantage for parasitic Varroa destructor mites. The impact of delayed development in bees on Varroa mite fecundity should be examined further.

SUMMARYAt Chanthaburi, Thailand, four species of Apis, A. andreniformis, A. florea, A. cerana and A. dorsata, are sympatric. Observations were carried out on three wild colonies of each species on various days in February 1992. The daily drone flight periods were only partially specific: A. andreniformis from 12.15 h to 13.45 h; A. florea from 14.00 h to 16.45 h; A. cerana from 15.15 h to 17.30 h; and A dorsata from 18.15 h to 18.45 h. The significance of these partially separate drone flight periods is discussed in terms of both reproductive isolation and evolution.

A genetic stock certification assay was developed to distinguish Russian honey bees from other European (Apis mellifera L.) stocks that are commercially produced in the United States. In total, 11 microsatellite and five single-nucleotide polymorphism loci were used. Loci were selected for relatively high levels of homogeneity within each group and for differences in allele frequencies between groups. A baseline sample consisted of the 18 lines of Russian honey bees released to the Russian Bee Breeders Association and bees from 34 queen breeders representing commercially produced European honey bee stocks. Suitability tests of the baseline sample pool showed high levels of accuracy. The probability of correct assignment was 94.2% for non-Russian bees and 93.3% for Russian bees. A neighbor-joining phenogram representing genetic distance data showed clear distinction of Russian and non-Russian honey bee stocks. Furthermore, a test of appropriate sample size showed a sample of eight bees per colony maximizes accuracy and consistency of the results. An additional 34 samples were tested as blind samples (origin unknown to those collecting data) to determine accuracy of individual assignment tests. Only one of these samples was incorrectly assigned. The 18 current breeding lines were represented among the 2009 blind sampling, demonstrating temporal stability of the genetic stock identification assay. The certification assay will be used through services provided by a service laboratory, by the Russian Bee Breeders Association to genetically certify their stock. The genetic certification will be used in conjunction with continued selection for favorable traits, such as honey production and varroa and tracheal mite resistance.

Restriction enzyme cleavage sites of mitochondrial DNA (mtDNA) from the Mediterranean fruit fly were found to vary among introduced populations in the Neotropics. The survey included samples from 15 established natural populations and 5 laboratory cultures from Hawaii, Central America, South America and West Africa and samples from recent California infestations (1989, 1991). Based on restriction fragment length polymorphisms from 2 enzymes, Hawaii is an unlikely source for the 1989 and 1991 California infestations. Interpopulational variation in mtDNA demonstrates the potential for the technique to trace the process of colonization (geographic spread) by this insect.

On Kangaroo Island, South Australia, an introduced population of Apis mellifera ligustica has reputedly not hybridized with other A. mellifera subspecies due to isolation. Bees were collected from ...

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.