Lipophorin of the larval honeybee, Apis mellifera L

Most insects have a major lipoprotein species in the blood (hemolymph) that serves to transport fat from the midgut to the storage depots in fat body cells and from the fat body to peripheral tissues. The generic name lipophorin is used for this lipoprotein. In larvae of the honeybee, Apis mellifera, a lipophorin has been found with properties that correlate well with those of the only other lipophorin reported for an immature insect, that of the tobacco hornworm, Manduca sexta. The honeybee lipophorin (Mr = 530,000) has a density of 1.13 g/ml, contains approximately 41% lipid and 59% protein, and contains two apoproteins, apoLp-I, Mr = 250,000 and apoLp-II, Mr = 80,000, both of which are glycosylated. The lipids consist predominantly of polar lipids, of which phospholipids and diacylglycerols represent 60% of the total. When the intact lipophorin is treated with trypsin, apoLp-I is rapidly proteolyzed, while apoLp-II is resistant, indicating a difference in exposure of the two apoproteins to the aqueous environment. Honeybee apoLp-II cross-reacts with antibodies to M. sexta apoLp-II, but not to anti-M. sexta apoLp-I. No cross-reactivity of honeybee apoLp-I to anti-M. sexta apoLp-I was observed.     

Local honeybee (Apis mellifera mellifera L.) populations in the Urals

The COI-COII intergenic region of mitochondrial DNA (mtDNA) was studied in local honeybee (Apis mellifera mellifera) L. populations from the Middle and Southern Urals. Analysis of bee colonies in these regions revealed apiaries enriched in families descending from A. m. mellifera in the maternal lineage. These results confirm the suggestion of preservation of A. m. mellifera refuges in the Urals and provide grounds for work on the preservation of the gene pool of this bee variety, valuable for all Russia.     

Disulfide pairing and secondary structure of ASP1, an olfactory-binding protein from honeybee (Apis mellifera L).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory-binding proteins (CBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion-spray mass spectrometry analysis of cyanylation-induced cleavage products of partially reduced protein with Tris(2-carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all-alpha protein in accordance with NMR preliminary data, but unlike lipocalin-like vertebrate OBPs.     

Physiology of protein digestion in the midgut of the honeybee (Apis mellifera L.)

Proteolytic activity in the midguts of pupae and imagos of worker honeybees was determined over a 1-yr period. The bees were of defined ages and the size of the hypopharyngeal glands was used as a parameter of their functional status. The activities of trypsin-like, chymotrypsin-like enzymes and the total caseinolytic activity were investigated; they did not depend on Ca²⁺ and showed optimal turnover at pH values above 7. Proteolytic activity is limited in pupae and newly emerged bees, then increases rapidly in the first hours of the imago stage. Proteolytic activity and the relation between trypsin- and chymotrypsin-like activity vary with age, season and functional status of the insects. Nurse bees show the greatest proteolytic activity. Age-dependent distributions of enzymatic activities in the endo- and ecto-peritrophic space indicate that the peritrophic membrane establishes compartments within the midgut lumen.     

Molecular characterization of hemoglobin from the honeybee Apis mellifera

Due to the prevailing importance of the tracheal system for insect respiration, hemoglobins had been considered rare exceptions in this arthropod subphylum. Here we report the identification, cloning and expression analysis of a true hemoglobin gene in the honeybee Apis mellifera (Hymenoptera). The deduced amino acid sequence covers 171 residues (19.5kDa) and harbors all globin-typical features, including the proximal and the distal histidines. The protein has no signal peptide for transmembrane transport and was predicted to localize in the cytoplasm. The honeybee hemoglobin gene shows an ancient structure, with introns in positions B12.2 and G7.0, while most other insect globins have divergent intron positions. In situ hybridization studies showed that hemoglobin expression in the honeybee is mainly associated with the tracheal system. We also observe hemoglobin expression in the Malpighi tubes and testis. We further demonstrated that hemoglobins occur in other insect orders (Hemiptera, Coleoptera, Lepidoptera), suggesting that such genes belong to the standard repertoire of an insect genome. Phylogenetic analyses show that globins evolved along with the accepted insect systematics, with a remarkable diversification within the Diptera. Although insect hemoglobins may be in fact involved in oxygen metabolism, it remains uncertain whether they carry out a myoglobin-like function in oxygen storage and delivery.     

意大利蜂营养腺的形态结构研究

研究了意大利蜂分泌蜂王浆的主要腺体──营养腺的形态结构及腺体随工蜂日龄增加而变化的规律。结果表明,工蜂羽化后,在各个日龄段随其职能分工的不同,营养腺形态结构也有相应的变化。从羽化出房的幼峰到哺育蜂到采集蜂,其营养腺要经历发育─→饱满发达─→衰退萎缩的过程。分泌细胞中的粗面内质网变化更为明显。     

A microsatellite-based linkage map of the honeybee, Apis mellifera L

A linkage map for the honeybee (Apis mellifera) was constructed mainly from the progeny of two hybrid queens (A. m. ligustica x A. m. mellifera). A total of 541 loci were mapped; 474 were microsatellite loci; a few were additional bands produced during PCRs, one of the two rDNA loci (using ITS), the MDH locus, and three sex-linked markers (Q and FB loci and one RAPD band). Twenty-four linkage groups were estimated of which 5 were minute (between 7.1 and 22.8 cM) and 19 were major groups (>76.5 cM). The number of major linkage groups exceeded by three the number of chromosomes of the complement (n = 16). The sum of the lengths of all linkage groups amounts to 4061 cM to which must be added at least 320 cM to link groups in excess, making a total of at least 4381 cM. The length of the largest linkage group I was 630 cM. The average density of markers was 7.5 cM and the average resolution was about one marker every 300 kb. For most of the large groups, the centromeric region was determined genetically, as described in (accompanying article in this issue), using half-tetrad analysis of thelytokous parthenogens in which diploid restoration occurs through central fusion. Several cases of segregation distortion that appreared to result from deleterious recessives were discovered. A low positive interference was also detected.     

Male fitness of honeybee colonies (Apis mellifera L.)

Honeybees (Apis mellifera L.) have an extreme polyandrous mating system. Worker offspring of 19 naturally mated queens was genotyped with DNA microsatellites, to estimate male reproductive success of 16 drone producing colonies. This allowed for estimating the male mating success on both the colony level and the level of individual drones. The experiment was conducted in a closed population on an isolated island to exclude interferences of drones from unknown colonies. Although all colonies had produced similar numbers of drones, differences among the colonies in male mating success exceeded one order of magnitude. These differences were enhanced by the siring success of individual drones within the offspring of mated queens. The siring success of individual drones was correlated with the mating frequency at the colony level. Thus more successful colonies not only produced drones with a higher chance of mating, but also with a significantly higher proportion of offspring sired than drones from less successful colonies. Although the life cycle of honeybee colonies is very female centred, the male reproductive success appears to be a major driver of natural selection in honeybees.     

Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L

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

The mating behaviour of the honeybee (Apis mellifera L.)

An account is given of the mating responses of free-flying drones towards a queen, and of the pheromones controlling these responses.     

Molecular cloning and characterization of a lipase from the honeybee Apis mellifera

Lipids perform essential and important functions, such as serving as an energy source for growth, development, reproduction, flight, starvation, and diapause in insects. Lipases are key enzymes involving in lipid metabolism and have been reported in several insect species. However, the molecular characterization of the pancreatic triacylglycerol lipase-like genes in honeybees has not been elucidated. Here, we describe the cDNA cloning and characterization of lipase from the honeybee Apis mellifera (Am-Lipase). Am-Lipase consists of 321 amino acids, including a Ser-Glu-His catalytic triad and a consensus active site motif GXSXG. Recombinant Am-Lipase protein degrades triglycerides, preferentially catalyzing the hydrolysis of long-chain fatty acids. Furthermore, the expression pattern of Am-Lipase seems to be a fat body-specific lipase, shows higher expression in forager bees, and is decreased under starvation conditions. Thus, our results suggest that Am-Lipase plays a role in the utilization of lipids stored in the fat body for lipid metabolism.     

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.     

茚虫威对意大利蜜蜂毒性研究

通过单次饲喂高浓度茚虫威药液以及连续饲喂亚致死浓度茚虫威药液进行染毒,记录和分析急性慢性经口毒性试验中,意大利蜜蜂的中毒症状和死亡情况;测定慢性毒性试验中意大利蜜蜂每天药液消耗量,并计算危害商值进行初步风险评价。结果表明,98%茚虫威原药对意大利蜜蜂的半致死剂量48 h-LD_(50)为0.399μg a.i./bee,每日半数致死剂量240 h-LDD_(50)为0.0233μg a.i./bee/day,其中慢性毒性试验中1.66 mg a.i./L处理组的意大利蜜蜂死亡率在144 h之后呈现显著性的增加,240 h达到70%,且该处理组意大利蜜蜂的摄食量为17.6μL,少于对照组和其他处理组。此外,茚虫威根据目前推荐使用剂量计算出的危害商值基本大于50。茚虫威对意大利蜜蜂急性毒性(48 h)是高毒,存在较高的风险;亚致死浓度在144 h后会对意大利蜜蜂造成较大的威胁。     

Genetic determination of honeybee (Apis mellifera L.) foraging preferences

Progeny from different honeybee queens that were reared in, and foraged from, the same colony sometimes differed in their floral preferences, confirming that these are to some extent innately determined.     

The fine structure of the tarsal glands of the honeybee Apis mellifera L. (Hymenoptera)

Summary Tarsal glands are located in the 6th tarsomere of adult honeybee queens, workers and drones. Their structural features are not cast or sex specific. The glandular epithelium is lined by a thin endocuticular layer. A cuticular pocket is formed from a postimaginal delamination of the cuticle secreted by the glandular epithelium. The apical plasma membrane of the glandular cells shows numerous cristae and microvilli lining large crypts that communicate with the subcuticular space. Pinocytotic vesicles, multivesicular bodies and residual dense bodies are present in the apical part of the glandular cells. The RER is well developed in perinuclear and basal parts of the glandular cells, but the Golgi apparatus is a discrete organelle without secretory granules. No exocytotic secretory structures were observed. To reach the glandular pocket, the non-proteinaceous secretory product must pass across the subcuticular space, the cuticular intima, the space between the intima and the cuticular wall, and the cuticular wall of the glandular pocket.     

Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.     

Differential protein expression in honeybee (Apis mellifera L.) larvae: underlying caste differentiation

Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level.     

Lateralization of olfaction in the honeybee Apis mellifera

Lateralization of function is a well-known phenomenon in humans. The two hemispheres of the human brain are functionally specialized such that certain cognitive skills, such as language or musical ability, conspecific recognition, and even emotional responses, are mediated by one hemisphere more than the other [1, 2]. Studies over the past 30 years suggest that lateralization occurs in other vertebrate species as well [3-11]. In general, lateralization is observed in different sensory modalities in humans as well as vertebrates, and there are interesting parallels (reviewed in [12]). However, little is known about functional asymmetry in invertebrates [13, 14] and there is only one investigation in insects [15]. Here we show, for the first time, that the honeybee Apis mellifera displays a clear laterality in responding to learned odors. By training honeybees on two different versions of the well-known proboscis extension reflex (PER) paradigm [16, 17], we demonstrate that bees respond to odors better when they are trained through their right antenna. To our knowledge, this is the first demonstration of asymmetrical learning performance in an insect.