Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.     

Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5' untranslated region (UTR) of 869 bp, and the 3' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The Mr of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.     

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.     

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.     

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.     

意蜂工蜂酸性磷酸酶的纯化及其酶学特性

从意蜂Apis mellifera工蜂体内分离提纯酸性磷酸酶（ACPase, EC3.1.3.2）,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE-Sepharose FF离子交换层析及Sephadex G-200 凝胶过滤等纯化步骤,得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为77.24,酶液比活力为16.22 U/mg（对硝基苯磷酸二钠作底物）。利用凝胶过滤法测定酶的相对分子质量为135 kD,SDS-PAGE测定酶的亚基相对分子质量为63 .1 kD。酶的等电点为4.46和4.79。非还原/还原（NR/R）单向、双向SDS-PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示,酶分子中α-螺旋占13.84%,β-折叠占25.68%,无规则卷曲占56.34%。氨基酸组成分析结果表明, 酸性磷酸酶约含有507个氨基酸残基,富含门冬氨酸残基。     

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.     

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.     

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.     

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.     

Differential parallel processing of olfactory information in the honeybee, Apis mellifera L.

Two distinct neuronal pathways connect the first olfactory neuropil, the antennal lobe, with higher integration areas, such as the mushroom bodies, via antennal lobe projection neurons. Intracellular recordings were used to address the question whether neuroanatomical features affect odor-coding properties. We found that neurons in the median antennocerebral tract code odors by latency differences or specific inhibitory phases in combination with excitatory phases, have a more specific activity profile for different odors and convey the information with a delay. The neurons of the lateral antennocerebral tract code odors by spike rate differences, have a broader activity profile for different odors, and convey the information quickly. Thus, rather preliminary information about the olfactory stimulus first reaches the mushroom bodies and the lateral horn via neurons of the lateral antennocerebral tract and subsequently odor information becomes more specified by activities of neurons of the median antennocerebral tract. We conclude that this neuroanatomical feature is not related to the distinction between different odors, but rather reflects a dual coding of the same odor stimuli by two different neuronal strategies focusing different properties of the same stimulus.     

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。     

Hypertrehalosaemic peptides in the honeybee (Apis mellifera): purification,identification and function

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation.     

Modelling the subgenual organ of the honeybee, Apis mellifera

In a recent study on the honeybee (Apis mellifera), the subgenual organ was observed moving inside the leg during sinusoidal vibrations of the leg (Kilpinen and Storm 1997). The subgenual organ of the honeybee is suspended in a haemolymph channel in the tibia of each leg. When the leg accelerates, the inertia causes the haemolymph and the subgenual organ to lag behind the movement of the rest of the leg. To elucidate the biophysics of the subgenual organ system of the honeybee, two mathematical models to simulate the experimentally observed mechanical response are considered. The models are a classical mass-spring model and a newly developed tube model consisting of an open-ended, fluid-filled tube occluded by an elastic structure midway. Both models suggest that the subgenual organ included in the haemolymph channel resembles that of an overdamped system. In resembling the biophysics of the subgenual organ system in the honeybee, we consider the tube model to be the better of the two because it simulates a mechanical response which complies best with the experimental data, and the physical parameters in the model can be related to the␣constituent parts of the subgenual organ included in the haemolymph channel. Received: 25 July 1997 / Accepted in revised form: 8 December 1997     

Net reproductive rate of unmanaged honeybee colonies, (Apis mellifera L.)

Summary The net reproductive rate of unmanaged honeybee colonies has never been fully determined for honey bees in temperate climates. In this study, five overwintered colonies in Kansas, USA, were allowed to swarm naturally (Winston. 1980). These colonies and their swarms were studied over the winter (i.e. one generation). The net reproductive rateR ₀ was estimated to be 2.18. Afterswarms were found to contribute substantially (41.2%) to this net reproductive rate. The autumn and spring food reserves and brood areas of established colonies and colonies established from prime swarms and afterswarms are compared. Winter survival of afterswarms was related to autumn honey stores, and the brood areas of surviving afterswarms were smaller than those of prime swarms or established colonies.     

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.     

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。