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.     

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.     

Honeybee glands as possible infection reservoirs of Nosema ceranae and Nosema apis in naturally infected forager bees

Aims: To determine whether Nosema ceranae and Nosema apis are present in different gland tissues of honeybee, Apis mellifera L. and to monitor spore presence and quantity in these glands in naturally infected hives from July 2009 to July 2010 in Quebec, Canada. Methods and Results: Nosema spp. were quantified using duplex quantitative real‐time PCR in the thoracic salivary, hypopharyngeal, mandibular glands, and venom sac and glands of A. mellifera over a period of 8 months. Both Nosema species were present in all the glands as single or mixed species; however, N. apis was not present as single‐species detections in the salivary glands (see Table 2). Nosema ceranae was more prevalent throughout the 8 months. Significant correlative relationships were established for N. ceranae and N. apis levels in the honeybee glands and those found within the intestines of forager honeybees. Overall, the seasonality of N. ceranae and N. apis in the different glands tightly followed the seasonal patterns in the honeybee guts. Conclusions: Nosema ceranae and N. apis are not tissue specific, and honeybee glands have potential to become a useful indicator of the extent of disease in the colony and may represent a potential infection reservoir. Significance and Impact of the Study: First report of spore load quantification of Nosema spp. in different honeybee glands.     

Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera)

Global pollinators, like honeybees, are declining in abundance and diversity, which can adversely affect natural ecosystems and agriculture. Therefore, we tested the current hypotheses describing honeybee losses as a multifactorial syndrome, by investigating integrative effects of an infectious organism and an insecticide on honeybee health. We demonstrated that the interaction between the microsporidia Nosema and a neonicotinoid (imidacloprid) significantly weakened honeybees. In the short term, the combination of both agents caused the highest individual mortality rates and energetic stress. By quantifying the strength of immunity at both the individual and social levels, we showed that neither the haemocyte number nor the phenoloxidase activity of individuals was affected by the different treatments. However, the activity of glucose oxidase, enabling bees to sterilize colony and brood food, was significantly decreased only by the combination of both factors compared with control, Nosema or imidacloprid groups, suggesting a synergistic interaction and in the long term a higher susceptibility of the colony to pathogens. This provides the first evidences that interaction between an infectious organism and a chemical can also threaten pollinators, interactions that are widely used to eliminate insect pests in integrative pest management.     

Genetic dissection of honeybee (Apis mellifera L.) foraging behavior

We demonstrate the effects of a new quantitative trait locus (QTL), designated pln3, that was mapped in a backcross population derived from strains of bees selected for the amount of pollen they store in combs. We independently confirmed pln3 by demonstrating its effects on individual foraging behavior, as we did previously for QTLs pln1 and pln2 (Hunt et al. 1995). QTL pln2 is very robust in its effects on foraging behavior. In this study, pln2 was again shown to affect individual foraging behavior of workers derived from a hybrid backcross of the selected strains. In addition, pln2 was shown to affect the amount of pollen stored in combs of colonies derived from a wide cross of European and Africanized honeybees. This is noteworthy because it demonstrates that we can map QTLs for behavior in interstrain crosses derived from selective breeding and study their effects in unselected, natural populations. The results we present also demonstrate the repeatability of finding QTLs with measurable effects, even after outcrossing selected strains, suggesting that there is a relatively small subset of QTLs with major effects segregating in the population from which we selected our founding breeding populations. The different QTLs, pln1, pln2, and pln3, appear to have different effects, revealing the complex genetic architecture of honeybee foraging behavior.     

Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc × Chinese Erhualian intercross resource population

The number of vertebrae is associated with body size and meat production in pigs. To identify quantitative trait loci (QTL) for the number of vertebrae, phenotypic values were measured in 1029 individuals from a White Duroc × Chinese Erhualian intercross F₂ population. A whole genome scan was performed with 194 microsatellite markers in the F₂ population. Four genome‐wide significant QTL and eight chromosome‐wide significant QTL for the number of vertebrae were identified on pig chromosomes (SSC) 1, 2, 6, 7, 10 and 12. The most significant QTL was detected on SSC7 with a confidence interval of 1 cM, explaining 42.32% of the phenotypic variance in the thoracic vertebral number. The significant QTL on SSC1, 2 and 7 confirmed previous reports. A panel of 276 animals representing seven Western and Chinese breeds was genotyped with 34 microsatellite markers in the SSC7 QTL region. No obvious selective sweep effect was observed in the tested breeds, indicating that intensive selection for enlarged body size in Western commercial breeds did not wipe out the genetic variability in the QTL region. The Q alleles for increased vertebral number originated from both Chinese Erhualian and White Duroc founder animals. A haplotype block of approximately 900 kb was found to be shared by all Q‐bearing chromosomes of F₁ sires except for one distinct Q chromosome. The critical region harbours the newly reported VRTN gene associated with vertebral number. Further investigations are required to confirm whether VRTN or two other positional candidate genes, PROX2 and FOS, cause the QTL effect.     

A quantitative trait locus for cold tolerance at the booting stage on rice chromosome 8

A quantitative trait locus (QTL) for cold tolerance at the booting stage of a cold-tolerant rice breeding line, Hokkai-PL9, was analyzed. A total of 487 simple sequence repeat (SSR) markers distributed throughout the genome were used to survey for polymorphism between Hokkai-PL9 and a cold-sensitive breeding line, Hokkai287, and 54 markers were polymorphic. Single marker analysis revealed that markers on chromosome 8 are associated with cold tolerance. By interval mapping using an F₂ population between Hokkai-PL9 and Hokkai287, a QTL for cold tolerance was detected on the short arm of chromosome 8. The QTL explains 26.6% of the phenotypic variance, and its additive effect is 11.4%. Substitution mapping suggested that the QTL is located in a 193-kb interval between SSR markers RM5647 and PLA61. We tentatively designated the QTL as qCTB8 (quantitative trait locus for cold tolerance at the booting stage on chromosome 8).     

Social parasitism by Cape honeybee workers in colonies of their own subspecies (Apis mellifera capensis Esch.)

Social parasitism is widespread in the eusocial insects. Although social parasites often show a reduced worker caste, unmated workers can also parasitize colonies. Cape honeybee workers, Apis mellifera capensis, can establish themselves as social parasites in host colonies of other honeybee subspecies. However, it is unknown whether social parasitism by laying workers also occurs among Cape honeybee colonies. In order to address this question we genotyped worker offspring of six queenless A. m. capensis colonies and determined the maternity of the reproducing workers. We found that three non-nestmate workers dominated reproduction in a host colony and produced 62.5% of the progeny. Our results show that social parasitism by laying workers is a naturally occurring part of the biology of Cape honeybees. However, such social parasitism is not frequently found (6.41% of the total worker offspring) probably due to co-evolutionary processes among A. m. capensis resulting in an equilibrium between selection for reproductive dominance in workers, colony maintenance and queen adaptation. Received 28 July 2005; revised 19 September and 11 November 2005; accepted 16 November 2005.     

Using the SSU,ITS, and Ribosomal DNA Operon Arrangement to Characterize Two Microsporidia Infecting Bruce Spanworm,Operophtera bruceata (Lepidoptera: Geometridae)

Research pertaining to the two closely‐related microsporidian genera Nosema and Vairimorpha is hindered by inconsistencies in species differentiation within and between the two clades. One proposal to better delimit these genera is to restructure the Nosema around a “True Nosema” clade, consisting of species that share a characteristic reversed ribosomal DNA operon arrangement and small subunit (SSU) ribosomal DNA sequences similar to that of the Nosema type species, N. bombycis. Using this framework, we assess two distinct microsporidia recovered from the forest insect Bruce spanworm (Operophtera bruceata) by sequencing their SSU and internal transcribed spacer regions. Phylogenetic analyses place one of our isolates within the proposed True Nosema clade close to N. furnacalis and place the other in the broader Nosema/Vairimorpha clade close to N. thomsoni. We found that 25% of Bruce spanworm cadavers collected over the four‐year study period were infected with microsporidia, but no infections were detected in cadavers of the Bruce spanworm's invasive congener, the winter moth (O. brumata), collected over the same period. We comment on these findings as they relate to the population dynamics of the Bruce spanworm‐winter moth system in this region, and more broadly, on the value of ribosomal DNA operon arrangement in Nosema systematics.     

Geographically widespread honeybee‐gut symbiont subgroups show locally distinct antibiotic‐resistant patterns

How long‐term antibiotic treatment affects host bacterial associations is still largely unknown. The honeybee‐gut microbiota has a simple composition, so we used this gut community to investigate how long‐term antibiotic treatment affects host‐associated microbiota. We investigated the phylogenetic relatedness, genomic content (GC percentage, genome size, number of genes and CRISPR) and antibiotic‐resistant genes (ARG) for strains from two abundant members of the honeybee core gut microbiota (Gilliamella apicola and Snodgrassella alvi). Domesticated honeybees are subjected to geographically different management policies, so we used two research apiaries, representing different antibiotic treatment regimens in their apiculture: low antibiotic usage (Norway) and high antibiotic usage (Arizona, USA). We applied whole‐genome shotgun sequencing on 48 G. apicola and 22 S. alvi. We identified three predominating subgroups of G. apicola in honeybees from both Norway and Arizona. For G. apicola, genetic content substantially varied between subgroups and distance similarity calculations showed similarity discrepancy between subgroups. Functional differences between subgroups, such as pectin‐degrading enzymes (G. apicola), were also identified. In addition, we identified horizontal gene transfer (HGT) of transposon (Tn10)‐associated tetracycline resistance (Tet B) across the G. apicola subgroups in the Arizonan honeybees, using interspace polymorphisms in the Tet B determinant. Our results support that honeybee‐gut symbiont subgroups can resist long‐term antibiotic treatment and maintain functionality through acquisition of geographically distinct antibiotic‐resistant genes by HGT.     

Parasites and Pathogens of the Honeybee (Apis mellifera) and Their Influence on Inter-Colonial Transmission

Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony (“drifting”). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and Nosema spp., and Varroa mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or Nosema spp. were associated with the frequency of drifting. Only colony infestation with Varroa was associated with significantly enhanced drifting. More specifically, colonies with high Varroa infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since Varroa-infested colonies show an enhanced attraction of drifting workers, and not only those infected with Varroa and its associated pathogens, infestation by Varroa may also facilitate the uptake of other pests and parasites.     

Saturation and comparative mapping of a major Fusarium head blight resistance QTL in tetraploid wheat

Fusarium head blight (FHB) is a devastating disease of cultivated wheat worldwide. Partial resistance to FHB has been identified in common wheat (Triticum aestivum L.). However, sources of effective FHB resistance have not been found in durum wheat (T. turgidum L. var. durum). A major FHB resistance quantitative trait loci (QTL), Qfhs.ndsu-3AS, was identified on chromosome 3A of T. dicoccoides, a wild relative of durum wheat. Here, we saturated the genomic region containing the QTL using EST-derived target region amplified polymorphism (TRAP), sequence tagged site (STS), and simple sequence repeat (SSR) markers. A total of 45 new molecular marker loci were detected on chromosome 3A and the resulting linkage map consisted of 55 markers spanning a genetic distance of 277.2 cM. Qfhs.ndsu-3AS was positioned within a chromosomal interval of 11.5 cM and is flanked by the TRAP marker loci, Xfcp401 and Xfcp397.2. The average map distance between the marker loci within this QTL region was reduced from 4.9 cM in the previous study to 3.5 cM in the present study. Comparative mapping indicated that Qfhs.ndsu-3AS is not homoeologous to Qfhs.ndsu-3BS, a major FHB QTL derived from the common wheat cultivar Sumai 3. These results facilitate our efforts toward map-based cloning of Qfhs.ndsu-3AS and utilization of this QTL in durum wheat breeding via marker-assisted selection.     

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.     

Knock‐on community impacts of a novel vector: spillover of emerging DWV‐B from Varroa‐infested honeybees to wild bumblebees

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock‐on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re‐emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV‐B overtaking DWV‐A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock‐on effects.     

Varroa treatment with bromfenvinphos markedly suppresses honeybee biochemical defence levels

We examined the influence of bromfenvinphos, a commonly used acaricide, on activities of many metabolic enzymes affecting the biochemical defences/physiology of the western honeybee, Apis mellifera L. (Hymenoptera: Apidae), as well as on some metabolic compound concentrations, percentage of global DNA methylation, and Nosema spp. infection levels. Bromfenvinphos‐treated workers had decreased haemolymph volumes and higher protein concentrations on their cuticle but lower protein concentrations in the haemolymph. They had higher global DNA methylation levels independent of the age‐related variants. Bromfenvinphos decreased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), acidic, neutral, and alkaline protease inhibitors and enzymatic physiological markers (AST, ALT, ALP), and concentrations of urea, uric acid, creatinine, cholesterol, glucose, Mg²⁺, and Ca²⁺ in worker haemolymph, depending on the age of the bees. Protease activities were higher only in the haemolymph of young bromfenvinphos‐treated bees in comparison with untreated bees. This compound decreased the activities of alkaline proteases and neutral protease inhibitors on the cuticle. Unexpectedly, in the treated bees, the activities of acidic and neutral proteases, and acidic and alkaline protease inhibitors, were higher in the young bees and lower in the older workers in comparison to the untreated group. The bromfenvinphos‐treated workers were more heavily infested with Nosema spp. Thus, bromfenvinphos not only supressed many levels of biochemical defences, and therefore stress‐resistance‐related biochemical pathways but also visibly increased the Nosema spp. infection levels.     

Biodiversity of Apis mellifera populations from Tenerife (Canary Islands) and hybridisation with East European races

The biodiversity of honeybee (Apis mellifera) populations from Tenerife (Canary Islands, Spain) has been assessed by restriction analysis of a mitochondrial non-coding intergenic region. Seventy-nine colonies were analysed from thirteen apiaries in six populations that have been kept from recent queen introduction. The length and restriction pattern of the PCR amplified products of the intergenic region identified four mitochondrial haplotypes. One of these haplotypes shows the same restriction pattern and composition of the intergenic region carried by honeybees belonging to the African lineage. Two haplotypes are characterised by a particular intergenic region found with high frequency in the Canarian populations. The haplotype representative of the East European honeybee lineage shows a frequency of 35%, thus indicating introduction of queen honeybees. The finding of this haplotype in Canarian honeybees suggests that hybridisation between the endemic Apis mellifera populations and imported bees is occurring in Tenerife.     

The Impact of Recent Queenloss and Colony Pheno-type on the Removal of Small Hive Beetle (Aethina tumida Murray) Eggs and Larvae by African Honeybee Colonies (Apis mellifera capensis Esch.)

The removal of small hive beetle [=SHB] eggs and larvae was studied in queenright and recently queenless Cape honeybee, Apis mellifera capensis, colonies over a range of phenotypes. The overall removal efficiency was not influenced by phenotypes or queenstate, because all introduced eggs and larvae were removed within 24 hours. Queenless colonies removed them merely slower than queenright ones. The latter ones rejected up to 300 larvae within one hour. However, colonies undergoing preparation for absconding did not completely remove SHB offspring, suggesting that removal efficiency was reduced. Since even small and recently queenless colonies effectively removed immature SHB, and no differences in the overall efficiency was found compared to A. m. scutellata we conclude that this defense behavior is well developed in African honeybees.     

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.     

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.     

Identification of X-linked quantitative trait loci affecting cold tolerance in Drosophila melanogaster and fine mapping by selective sweep analysis

Drosophila melanogaster is a cosmopolitan species that colonizes a great variety of environments. One trait that shows abundant evidence for naturally segregating genetic variance in different populations of D. melanogaster is cold tolerance. Previous work has found quantitative trait loci (QTL) exclusively on the second and the third chromosomes. To gain insight into the genetic architecture of cold tolerance on the X chromosome and to compare the results with our analyses of selective sweeps, a mapping population was derived from a cross between substitution lines that solely differed in the origin of their X chromosome: one originates from a European inbred line and the other one from an African inbred line. We found a total of six QTL for cold tolerance factors on the X chromosome of D. melanogaster. Although the composite interval mapping revealed slightly different QTL profiles between sexes, a coherent model suggests that most QTL overlapped between sexes, and each explained around 5–14% of the genetic variance (which may be slightly overestimated). The allelic effects were largely additive, but we also detected two significant interactions. Taken together, this provides evidence for multiple QTL that are spread along the entire X chromosome and whose effects range from low to intermediate. One detected transgressive QTL influences cold tolerance in different ways for the two sexes. While females benefit from the European allele increasing their cold tolerance, males tend to do better with the African allele. Finally, using selective sweep mapping, the candidate gene CG16700 for cold tolerance colocalizing with a QTL was identified.