Further Studies of the Cap Pushing Response in Honey Bees (Apis mellifera)

The Cap Pushing Response (CPR) is a free-flying technique used to study learning and memory in honey bees (Apis mellifera). The series of experiments outlined in this paper aimed to test whether honey bees exhibit the cognitive concept of “expectancy” utilizing the CPR in a weight differentiation paradigm. Five previous experiments in our laboratory have explored whether the concept of expectancy can account for honey bee performance and have all failed to support the cognitive interpretation. The first experiment examined if bees could differentiate between the two caps in the amount of force they used to push the cap and the distance the cap was pushed when the caps were presented one at a time. The second experiment explored cap weight preference by presenting bees with a choice between the two caps. The third and fourth experiments tested the bee’s ability to expect reward or punishment based on cap weight. Results revealed that bees were found to have a strong preference for the light cap and therefore were not able to expect reward or punishment based on cap weight. These experiments contribute to the debate on whether bees have “cognitive” representations and continue to support the behaviorist interpretation.

The Formamidine Amitraz as a Hyperglycemic α-Agonist in Worker Honeybees(Apis mellifera mellifera L.)in Vivo

Intra-abdominal injection of amitraz(0.25 nmol per honeybee,i.e.,approx 2.3 nmol/g)to emerging worker bees,in vivo,led to a significant hypertrehalosemia(300-400%)followed by a hyperglucosemia(≈600%).Maxima were reached at 0.5 and 2h,respectively.A strong negative correlation between glucosemia and trehalosemia appeared after injection of pure phentolamine (1 nmol per bee),suggesting stimulation of trehalase activities.Simultaneous administration of the α-blocker at≥0.25 nmol per individual suppressed the hyperglycemic response of amitraz. The formamidine pesticide thus likely acts on the honeybee α-aminergic system.1989 Academic Press,Inc.

Cloning and Sequence Analysis of Carbonic Anhydrase-related Protein 10-like in Apis mellifera

[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like （ CARP X） from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through RT-PCR, and then analyzed with bioinformatic method. [Result] The full-length cDNA sequence of CARPX was 972 bp long and encoded 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoeleetric point was 7.458. The CARP X from A. mellifera shared close relationship with proteins from Apisflorae, Bombtas impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may include two subfamilies. [ Conclusion] The results pro- vide basis for studying CARPs family.

Evaluation of Morphometric Characters of Honeybee (Apis mellifera L.) Populations in the Lake Chad Basin in Central Africa

Samples of workers of honeybee were collected from 42 colonies in 13 localities in the Lake Chad Basin (parts of Cameroon, Chad, and Nigeria) and analysed using classical morphometry. Measurements of 35 morphological characters of body size, colour and pilosity were taken from 10 workers per colony and the data subjected to one-way analysis of variance (ANOVA), principal components analysis (PCA), hierarchical cluster analysis (HCA), stepwise discriminant analysis (DA) and Pearson’s product-moment correlation analysis. A one-way ANOVA revealed that means of 21 of the morphometric characters differed significantly (p 0.05). The bees formed one cluster in a PCA. However, scatter plots of altitude against principal component 1 of PCA (loaded with characters of body size) revealed an increase of size of the bees along the gradient of the Lake Chad Basin. The coefficient of determination (R2) indicated that 88% and 77% of the variation in size might be explained by altitude in the southeastern and southwestern parts of the basin, respectively. Additionally, there was a very highly significant strong positive relationship between principal component 1 and altitude (r(30) = 0.618, p < 0.0005). Similarly, HCA and DA classified the colonies into three morphoclusters whose distribution closely followed the altitude of the area.

Effect of Apis mellifera on community composition of local pollinator bees and their pollination network in Qinling Mountains and surrounding areas

The Qinling Mountains, known for their rich vegetation and diverse pollinating insects, have seen a significant decline in bee species richness and abundance over recent decades, largely due to the introduction and spread of Apis mellifera. This decline has caused cascading effects on the region's community structure and ecosystem stability. To improve the protection of native bees in the natural and agricultural landscape of the Qinling Mountains and its surrounding areas, we investigated 33 sampling sites within three habitats: forest, forest-agriculture ecotones, and farmland. Using a generalized linear mixing model, t-test, and other data analysis methods, we explored the impact of Apis mellifera on local pollinator bee richness, abundance, and the pollination network in different habitats in these regional areas. The results show that(1)Apis mellifera significantly negatively affects the abundance and richness of wild pollinator bees,while Apis cerana abundance is also affected by beekeeping conditions.(2)There are significant negative effects of Apis mellifera on the community structure of pollinator bees in the Qinling Mountains and its surrounding areas: the Shannon-Wiener diversity index, Pielou evenness index, and Margalef richness index of bee communities at sites with Apis mellifera influence were significantly lower than those at sites without Apis mellifera influence.(3)The underlying driver of this effect is the monopolization of flowering resources by Apis mellifera. This species tends to visit flowering plants with large nectar sources, which constitute a significant portion of the local plant community. By maintaining a dominant role in the bee-plant pollination network, Apis mellifera competitively displaces native pollinator bees, reducing their access to floral resources. This ultimately leads to a reduction in local bee-plant interactions, decreasing the complexity and stability of the pollination network. These findings highlight the need for targeted conservation efforts to protect native pollinator species and maintain the ecological balance in the Qinling Mountains.

Daily number of bee louse （Braula coeca） in honey bee （Apis mellifera carnica and A. m. syriaca） colonies maintained under semi-arid conditions

Experimental work was conducted at two apiaries located in Irbid district and in Shuna North, Jordan, during the years 2004-2006. The aims of these investigations were to estimate the seasonal changes in the infestation rates of the bee louse （Braula sp.） and to develop an easy and rapid method of estimating the infestation rate on workers with bee Braula. Two major honey bee subspecies are reared in Jordan; Apis mellifera carnica and Apis mellifera syriaca were used in this study. The results showed that the infestation rate began to increase rapidly in May, reaching the season＇s maximum rate of 16.2%, 15.8% and 17.4% forA. m. carnica and 22.6%, 23.9% and 22.9% forA. m. syriaca in December of 2004, 2005 and 2006, respectively. The maximum adult numbers of bees were found in April and June, whereas the minimum for the year was in January in both honey bee subspecies colonies during the study period. The actual population of the bee louse could be estimated by counting the daily dropped lice and multiplying by a factor of 158. This factor is valid for the experimental colonies of both subspecies kept for 3 years under semi-arid Mediterranean conditions.

The integrative analysis of microRNA and mRNA expression in Apis mellifera following maze-based visual pattern learning

The honeybee （Apis mellifera） is a social insect with strong sensory capacity and diverse behavioral repertoire and is recognized as a good model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the changes in microRNA （miRNA） and messenger RNA （mRNA） following maze-based visual learning using next-generation small RNA sequencing and Solexa/lllumina Digital Gene Expression tag profiling （DGE）. For small RNA sequencing, we obtained 13 367 770 and 13 132 655 clean tags from the maze and control groups, respectively. A total of 40 differentially expressed known miRNAs were detected between these two samples, and all of them were up-regulated in the maze group compared to the control group. For DGE, 5 681 320 and 5 939 855 clean tags were detected from the maze and control groups, respectively. There were a total of 388 differentially expressed genes between these two samples, with 45 genes up-regulated and 343 genes down-regulated in the maze group, compared to the control group. Additionally, the expression levels of 10 differentially expressed genes were confirmed by quantitative reverse transcription polymerase chain reaction （qRT-PCR） and the expression trends of eight of them were consistent with the DGE result, although the degree of change was lower in amplitude. The integrative analysis of miRNA and mRNA expression showed that, among the 40 differentially expressed known miRNAs and 388 differentially expressed genes, 60 pairs of miRNA/mRNA were identified as co-expressed in our present study. These results suggest that both miRNA and mRNA may play a pivotal role in the process of learning and memory in honeybees. Our sequencing data provide comprehensive miRNA and gene expression information for maze-based visual learning, which will facilitate understanding of the molecular mechanisms of honeybee learning and memory.

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing （NGS） technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses char- acteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify -600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development （Cactus, Relish, dorsal, Ank2, baz）, Varroa hygiene （NorpA2, Zasp, LanA, gasp, impl3） and Varroa resistance （Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch） functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.

Genomics and proteomics of Apis mellifera filamentous virus isolated from honeybees in China

Apis mellifera filamentous virus(Am FV)is a large DNA virus that is endemic in honeybee colonies.The genome sequence of the Am FV Swiss isolate(Am FV CH–C05)has been reported,but so far very few molecular studies have been conducted on this virus.In this study,we isolated and purified Am FV(Am FV CN)from Chinese honeybee(Apis mellifera)colonies and elucidated its genomics and proteomics.Electron microscopy showed ovoid purified virions with dimensions of 300–500×210–285 nm,wrapping a 3165×40 nm filamentous nucleocapsid in three figure-eight loops.Unlike Am FV CH–C05,which was reported to have a circular genome,our data suggest that Am FV CN has a linear genome of approximately 493 kb.A total of 197 ORFs were identified,among which36 putative genes including 18 baculoviral homologs were annotated.The overall nucleotide similarity between the CN and CH–C05 isolates was 96.9%.Several ORFs were newly annotated in Am FV CN,including homologs of per os infectivity factor 4(PIF4)and a putative integrase.Phylogenomic analysis placed Am FVs on a separate branch within the newly proposed virus class Naldaviricetes.Proteomic analysis revealed 47 Am FV virionassociated proteins,of which 14 had over 50%sequence coverage,suggesting that they are likely to be main structural proteins.In addition,all six of the annotated PIFs(PIF-0–5)were identified by proteomics,suggesting that they may function as entry factors in Am FV infection.This study provides fundamental information regarding the molecular biology of Am FV.

Distribution and variability of deformed wing virus of honeybees （Apis mellifera） in the Middle East and North Africa

Three hundred and eleven honeybee samples from 12 countries in the Middle East and North Africa （MENA） （Jordan, Lebanon, Syria, Iraq, Egypt, Libya, Tunisia, Algeria, Morocco, Yemen, Palestine, and Sudan） were analyzed for the presence of deformed wing virus （DWV）. The prevalence of DWV throughout the MENA region was pervasive, but variable. The highest prevalence was found in Lebanon and Syria, with prevalence dropping in Palestine, Jordan, and Egypt before increasing slightly moving westwards to Algeria and Morocco Phylogenetic analysis of a 194 nucleotide section of the DWV Lp gene did not identify any significant phylogenetic resolution among the samples, although the sequences did show consistent regional clustering, including an interesting geographic gradient from Morocco through North Africa to Jordan and Syria. The sequences revealed several clear variability hotspots in the deduced amino acid sequence, which furthermore showed some patterns of regional identity. Furthermore, the sequence variants from the Middle East and North Africa appear more numerous and diverse than those from Europe.

Methionine as a methyl donor regulates caste differentiation in the European honey bee(Apis mellifera)

Nutrition contributes to honey bee caste differentiation,but the role of individual nutrients is still unclear.Most essential amino acid contents,except that of methionine(Met),are greater in royal jelly than worker jelly.After〜3.5 i the Met content in the latter was slightly greater than in the former.Met is the major raw material used in the synthesis of S-adenosyl-L-methionine,an active methyl donor for DNA methylation,which is an epigenetic driver of caste differentiation.Here,we tested whether Met regulates caste differentiation in honey bees by determining its effects on the caste development of bees receiving four diets:the basic,basic+0.2%Met,basic+0.2%Met+20 mg/kg 5-azacytidine,and basic+20 mg/kg 5-azacytidine.The presence of Met decreased the adult bee body length and the numbers of ovarioles,indicating that Met may direct the development of female larvae toward worker bees.The upregulated expression of SAMS,Dnmtl,and Dnmt3 caused by Met exposure in 4-d-old larvae indicated that the worker-inductive effects of Met may occur through the promotion of DNA methylation.We investigated the co-effects of Met and glucose on bee development,and found that the effects of an increased glucose level on the number of ovarioles and body length did not strengthen the worker-inductive effects caused by Met.Our results contribute to caste development theory and suggest that Met-as a methyl donor一plays a regulatory,but not decisive,role in caste differentiation.

Comparative Analyses of Proteome Complement Between Worker Bee Larvae of High Royal Jelly Producing Bees (A. m. ligustica) and Carniolian Bees (A. m. carnica)

This study is to compare the protein composition of the high royal jelly producing bee （A. m. ligustica） with that of Carniolian bee （A. m. carnica） during their worker larval developmental stage. The experiment was carried out by two- dimensional gel electrophoresis. The results showed that significant higher numbers of total proteins （283） were detected in larvae of high royal jelly producing bees （Jelly bee） than those of Carniolian bees （152） on 2-d-old larvae. Among them, 110 proteins were presented on both strains of bee larvae, whereas 173 proteins were specific to larvae of Jelly bees, and 42 proteins were exclusive to Carniolian larvae. However, on the 4th d, a significant higher number of total proteins （290） were detected in larvae of Jelly bees than those of Carniolian bees （240）, 163 proteins resolved to both bee larvae, and 127 proteins were specific to Jelly bees and 77 proteins to Camiolian bees. Until the 6th d, also a significant higher number of total proteins （236） were detected in larvae of Jelly bees than those of Carniolian bees （180）, 132 proteins were constantly expressed in two bee larvae, whereas 104 and 48 proteins are unique to Jelly bee and Carniolian bee larvae, respectively. We tentatively concluded that the metabolic rate and gene expression of Jelly bees larvae is higher than those of Carniolian bees based proteins detected as total proteins and proteins specific to each stage of two strains of bee larvae. Proteins constantly expressed on 3 stages of larval development with some significant differences between two bee strains, and proteins unique to each stage expressed differences in term of quality and quantity, indicating that larval development needed house keeping and specific proteins to regulate its growth at different development phage, but the expression mold is different between two strains of larval development.

Influence of visual targets and landmarks on honey bee foraging and waggle dancing

Animals use diverse sensory stimuli to navigate their environment and to recognize rewarding food sources.Honey bees use visual atributes of the targeted food source,such as its color,shape,size,direction and distance from the hive,and the landmarks around it to navigate during foraging.They transmit the location information of the food source to other bees if it is highly rewarding.To investigate the relative importance of these attributes,we trained bees to feeders in two different experiments.In the first experiment,we asked whether bees prefer to land on(a)a similar feeder at a different distance on the same heading or on(b)a visually distinct feeder located at the exact same location.We found that,within a short foraging range,bees relied heavily on the color and the shape of the food source and to a lesser extent on its distance from the hive.In the second experiment,we asked if moving the main landmark or the feeder(visual target)influenced recruitment dancing for the feeder.We found that foragers took longer to land and danced fewer circuits when the location of the food source,or a major landmark associated with it,changed.These results demonstrate that prominent visual atributes of food sources and landmarks are evidently more reliable than distance information and that foraging bees heavily utilize these visual cues at the later stages of their journey.

Do introduced honeybees affect seed set and seed quality in a plant adapted for bird pollination?

Aims Worldwide,evidence suggests that exotic pollinators can disrupt plant mating patterns.However,few studies have determined if pol-lination by the honeybee Apis mellifera(the world’s most widely introduced pollinator)reduces offspring quality when compared with pollination by native birds.The Australian Proteaceae provides an excellent opportunity to test the impact of honeybees in pol-lination systems that are adapted to birds and non-flying mammals.Methods We compared the frequency of flower visitation and foraging behav-iour of birds and insects within seven populations of Banksia erici-folia.Banksia ericifolia is hermaphroditic and has large nectar-rich,orange inflorescences typical of bird and mammal pollinated spe-cies.For a subset of the study populations,we compared the qual-ity of seed produced via an exclusion treatment(that only allowed invertebrates to access flowers)with an open-pollination treatment(potentially visited by mammals,birds and invertebrates),by meas-uring seed weight,germination rates(T50),percent germination,seedling height after 14 days since the emergence of the cotyledon and time to emergence of the cotyledon.Important Findings Apis mellifera was the only apparent insect pollinator and the most frequent flower visitor,while the open treatment inflorescences were also frequently visited by avian pollinators,primarily hone-yeater species.The foraging behaviour of honeybees and honeyeat-ers showed striking differences that potentially affect patterns of pollen transfer.Honeybees made significantly greater proportions of within cf.among plant movements and only 30%(n=48)of honeybees foraged for pollen(nectar foragers carried no pollen)whilst all birds were observed to contact both stigmas and anthers when foraging for nectar.Despite these fundamental differences in behaviour,there was little effect of treatment on seed set or qual-ity.Our data show that while honeybees appear to alter patterns of pollen transfer within B.ericifolia populations,they do not impact reproductive rates or performance of early life-stages.

Spatial distribution of Vespa velutina individuals hunting at domestic honeybee hives： heterogeneity at a local scale

Since its recent introduction into Europe, the yellow-legged hornet, Vespa velutina, has become a major predator of the domestic honeybee, Apis mellifera, but little is known about its hunting behavior. We studied V. velutina hunting behavior by a capture- mark-recapture procedure in an experimental apiary. A total of 360 hornets were captured and tagged, and we determined： （i） the number of hornets visiting the apiary and the changes in time, （ii） the average number of individual visits per half-day and the time elapsed between consecutive recaptures, and （iii） the individual and global distribution of the hornets in the apiary. More than 50% of the marked hornets were recaptured at least once, this increased to 74% in considering the first marked individuals. We estimated 350 hornets visiting the patch daily with at least 1 visit per half-day. The number of marked hornets decreased over time while the number of unmarked ones increased, suggesting a turnover of individuals. The reduction of the delay between consecutive visits indicates that hornets became more efficient over time. Most of the hornets （88%） were recaptured in front of different hives but, overall, the global distribution was aggregative. Hornets were mainly recaptured in front of 1 hive which was neither the smallest nor the biggest colony, suggesting that the major cue used by hornets is not the amount of food. We hypothesize that the defensive behavior of the honeybee colony could explain our results which may be promising to further studies.

Changing climate patterns risk the spread of Varroa destructor infestation of African honey bees in Tanzania

Background:Climate change creates opportune conditions that favour the spread of pests and diseases outside their known active range.Modelling climate change scenarios is oftentimes useful tool to assess the climate analogues to unveil the potential risk of spreading suitability conditions for pests and diseases and hence allows development of appropriate responses to address the impending challenge.In the current study,we modelled the impact of climate change on the distribution of Varroa destructor,a parasitic mite that attacks all life forms of honey bees and remains a significant threat to their survival and productivity of bee products in Tanzania and elsewhere.Methods:The data about the presence of V.destructor were collected in eight regions of Tanzania selected in consideration of several factors including potentials for beekeeping activities,elevation(highlands vs.lowlands)and differences in climatic conditions.A total of 19 bioclimatic datasets covering the entire country were used for developing climate scenarios of mid-century 2055 and late-century 2085 for both rcp4.5 and rcp8.5.We thereafter modelled the current and future risk distribution of V.destructor using MaxEnt.Results:The results indicated a model performance of AUC=0.85,with mean diurnal range in temperature(Bio2,43.9%),mean temperature(Bio1,20.6%)and mean annual rainfall(Bio12,11.7%)as the important variables.Future risk projections indicated mixed responses of the potential risk of spreads of V.destructor,exhibiting both decrease and increases in the mid-century 2055 and late-century 2085 on different sites.Overall,there is a general decline of highly suitable areas of V.destructor in mid-and late-century across all scenarios(rcp4.5 and rcp8.5).The moderately suitable areas indicated a mixed response in mid-century with decline(under rcp4.5)and increase(under rcp8.5)and consistent increase in late century.The marginally suitable areas show a decline in mid-century and increase in late-century.Our results suggest that the climate change will continue to significantly affect the distribution and risks spread of V.destructor in Tanzania.The suitability range of V.destructor will shift where highly suitable areas will be diminishing to the advantage of the honey bees’populations,but increase of moderately suitable sites indicates an expansion to new areas.The late century projections show the increased risks due to surge in the moderate and marginal suitability which means expansion in the areas where V.destructor will operate Conclusion:The current and predicted areas of habitat suitability for V.destructor’s host provides information useful for beekeeping stakeholders in Tanzania to consider the impending risks and allow adequate interventions to address challenges facing honey bees and the beekeeping industry.We recommend further studies on understanding the severity of V.destructor in health and stability of the honey bees in Tanzania.This will provide a better picture on how the country will need to monitor and reduce the risks associated with the increase of V.destructor activities as triggered by climate change.The loss of honey bees’colonies and its subsequent impact in bees’products production and pollination effect have both ecological and economic implications that need to have prioritization by the stakeholders in the country to address the challenge of spreading V.destructor.