Identification of two piwi genes and their expression profile in honeybee,Apis mellifera

Piwi genes play an important role in regulating spermatogenesis and oogenesis because they participate in the biogenesis of piRNAs, a new class of noncoding RNAs. However, these genes are not well understood in most insects. To understand the function of piwi genes in honeybee reproduction, we amplified two full‐length piwi‐like genes, Am‐aub and Am‐ago3. Both the cloned Am‐aub and Am‐ago3 genes contained typical PAZ and PIWI domains and active catalytic motifs “Asp‐Asp‐Asp/His/Glu/Lys,” suggesting that the two piwi‐like genes possessed slicer activity. We examined the expression levels of Am‐aub and Am‐ago3 in workers, queens, drones, and female larvae by quantitative PCR. Am‐aub was more abundant than Am‐ago3 in all the tested samples. Both Am‐aub and Am‐ago3 were highly expressed in drones but not in workers and queens. The significant finding was that the larval food stream influenced the expression of Piwi genes in adult honeybees. This helps to understand the nutritional control of reproductive status in honeybees at the molecular level. © 2010 Wiley Periodicals, Inc.     

Origin and rearing season of honeybee queens affect some of their physiological and reproductive characteristics

Queen honeybees of Apis mellifera ligustica and Apis mellifera syriaca were raised to investigate physiological and reproductive characteristics and to determine the most suitable time for queen rearing under semi‐arid conditions in Jordan. The queen rearing season as well as the origin of the queens affected the queens’ weight, acceptance, preoviposition period, volume of the spermatheca, and quantity and quality of sperm in the spermatheca. Italian bees were heavier than Syrian bees at emergence. The introduced queen acceptance rate appeared to be a genetic influence of the queen: A. m. ligustica virgin queens were accepted at a higher rate than were A. m. syriaca queens. There were large seasonal variations in the acceptance rate. Experimental bee colonies accepted their virgin queens during spring with good honey flows at a higher rate compared to the other rearing periods. The greatest mating success was achieved in May and the smallest was during July and August. The preoviposition period was shorter in the Syrian than in the Italian queens, and was longer during summer for both honeybee subspecies. The volume of the spermatheca was smaller in Syrian bees and the spermatheca had lower numbers of spermatozoa compared with Italian bees. Thus, under semi‐arid Mediterranean region conditions, it is highly recommended to raise virgin queens in the spring months only to obtain their highest quality.     

Foraging pauses and their meaning as an economic strategy in the honeybee Apis mellifera L.

When conditioned honeybees collect sucrose solution delivered at a range of low-profit flow rates for the hive, they increase the pause length between successive visits. If sucrose solution was delivered continuously, it accumulated at the food source in an amount proportional to the pause length and the flow rate of nectar. When the flow rate of sucrose solution was further decreased but kept constant throughout the day, a threshold level was attained in which oscillations in the length of the pauses were observed. The relationship between the amount of accumulated nectar and subsequent pause length at this threshold level can be depicted by means of a power function. The best fit allowed the calculation of the values of parameters that quantitatively describe the control system regulating foraging activity. The importance of foraging pauses as a strategy to cope with changing nectar availability is discussed. Accepted: 7 January 1998     

Genetically modified organisms and risks of their introduction

The major goal of this review is to assess food risks of the introduction of genetically modified (GM) crops. The author analyzes the properties of the several classes of target proteins used in the transgenic constructions and discusses the problems that arise due to the pleiotropic action of transgenic proteins, the horizontal transfer of the transgenic constructions, primarily in bacteria, and their instability. Particular consideration is given to elevated risks of using the GM plant varieties for producing pharmaceutical preparations, due to the probability of uncontrolled cross-pollination between the GM plants and the plants grown for foodstuff production. The author emphasizes the requirement for assessing in detail all hypothetic risks in each particular case of cultivating GM varieties; as a control, such assessment must involve a comprehensive comparison with the conventional parental forms.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 115–128.Original Russian Text Copyright © 2005 by Kulikov.     

Amino acids and osmolarity in honeybee drone haemolymph

Summary In the haemolymph of honeybee drones, concentrations of free amino acids were higher than in worker haemolymph, with different relative proportions of individual amino acids. The overall concentration of free amino acids reached its highest level at the 5th day after adult drone emergence, and after the 9^th day only minor changes in the concentration and distribution of free amino acids were observed. This coincides with the age when drones reach sexual maturity and change their feeding behaviour. Levels of essential free amino acids were high during the first 3 days of life and thereafter decreased. Osmolarity was lowest at emergence (334 ± 41 mOsm), increased until the age of 3 days (423 ± 32mOsm) and then stayed generally constant until the 16^th day of life. Only 25-day-old drones had significantly higher osmolarity (532 ± 38 mOsm). The overall change in osmolarity during a drone's lifetime was about 40%.     

Activity of salivary glands in secreting honey‐elaborating enzymes in two subspecies of honeybee (Apis mellifera L)

The activity of invertase, glucose oxidase and amylase in the cephalic (post‐cerebral) and thoracic salivary glands is determined in Egyptian and Carniolan honeybees (Apis mellifera L). For this purpose, three ages of worker bees are selected for enzyme assays. The results show that the three target enzymes are detected in the two glands during the three worker ages, except invertase, which cannot be detected in the cephalic gland of newly emerged bees of both subspecies. In both glands, the secretion of invertase is highest, followed by amylase and then glucose oxidase. In Carniolan bees, invertase secretion of the cephalic and thoracic glands increases gradually with age. In Egyptian bees, invertase increases with age only in the cephalic gland, whereas, in the thoracic gland, the highest secretion activity is detected in 10–15‐day‐old bees. The highest amounts of glucose oxidase and amylase in the cephalic gland are detected in newly emerged individuals of both Egyptian and Carniolan bees. In the thoracic gland, however, the highest activity of both enzymes is recorded only in newly emerged Egyptian bees. The results are discussed in the light of bee management and biological aspects of the two subspecies.     

Preliminary investigations into possible resistance to oxytetracycline in Melissococcus plutonius,a pathogen of honeybee larvae

AIMS: To investigate the occurrence of oxytetracycline (OTC) resistance in Melissococcus plutonius, which causes European foulbrood in honeybee colonies. METHODS AND RESULTS: Strains of M. plutonius were isolated from diseased colonies in England and Wales and tested for resistance to OTC. The minimum inhibitory concentration (MIC) of OTC was also determined for selected isolates. No resistance to the antibiotic was found in any isolate and the average MIC was found to be 3.9 microg ml-1. Melissococcus plutonius was found to be susceptible to both chlortetracycline and tetracycline. CONCLUSIONS: No resistance to OTC was found in M. plutonius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that OTC can continue to be used to treat European foulbrood and that resistance may not explain why some treatments fail.     

Adaptation of microglomerular complexes in the honeybee mushroom body lip to manipulations of behavioral maturation and sensory experience

Worker honeybees proceed through a sequence of tasks, passing from hive and guard duties to foraging activities. The underlying neuronal changes accompanying and possibly mediating these behavioral transitions are not well understood. We studied changes in the microglomerular organization of the mushroom bodies, a brain region involved in sensory integration, learning, and memory, during adult maturation. We visualized the MB lips' microglomerular organization by applying double labeling of presynaptic projection neuron boutons and postsynaptic Kenyon cell spines, which form microglomerular complexes. Their number and density, as well as the bouton volume, were measured using 3D-based techniques. Our results show that the number of microglomerular complexes and the bouton volumes increased during maturation, independent of environmental conditions. In contrast, manipulations of behavior and sensory experience caused a decrease in the number of microglomerular complexes, but an increase in bouton volume. This may indicate an outgrowth of synaptic connections within the MB lips during honeybee maturation. Moreover, manipulations of behavioral and sensory experience led to adaptive changes, which indicate that the microglomerular organization of the MB lips is not static and determined by maturation, but rather that their organization is plastic, enabling the brain to retain its synaptic efficacy.     

气味结合蛋白OBPs在意大利蜜蜂头部表达模式的研究

昆虫气味结合蛋白(OBPs)是脂溶性化学物质的载体,参与了嗅觉识别和化学传输等生理过程.本研究利用MEME在线软件对意大利蜜蜂Apis mellifera ligustica OBPs基因家族21条氨基酸序列进行Motif预测分析;采用RT-PCR初步筛选出在意大利蜜蜂头部表达量较高的OBPs基因,然后利用qPCR技术对筛选出的基因进一步分析其在不同日龄内勤蜂头部的表达情况.结果 表明,在预测获得的6个motif中,motif1为21个OBPs所共有,具有极高的保守性,推测该序列元件可能是意大利蜜蜂OBPs与底物结合的区域;表达谱分析结果表明,13个OBPs基因的凝胶电泳条带明显(即在头部的表达量较高),且大多数基因在4日龄时表达量达到最高值,推测这类蛋白可能参与了幼虫信息素和糖类物质的识别过程.本研究结果丰富了蜜蜂OBPs表达特性的研究数据,同时也为探讨昆虫OBPs广泛的生理功能奠定了基础.     

Characterization and comparison of recombinant honeybee chymotrypsin-like protease (HCLPase) expressed in Escherichia coli and insect cells

We previously found a novel chymotrypsin-like protease in honeybee, designated as HCLPase. The recombinant enzyme expressed in insect cells was produced and compared to that in Escherichia coli. Both enzymes showed equivalent molecular size and specificity. However, HCLPase produced in insect cells showed higher specific activity. The C-terminal cleavage sites of HCLPase were phenylalanine, leucine, and tyrosine residues.     

Genetic architecture of ovary size and asymmetry in European honeybee workers

The molecular basis of complex traits is increasingly understood but a remaining challenge is to identify their co-regulation and inter-dependence. Pollen hoarding (pln) in honeybees is a complex trait associated with a well-characterized suite of linked behavioral and physiological traits. In European honeybee stocks bidirectionally selected for pln, worker (sterile helper) ovary size is pleiotropically affected by quantitative trait loci that were initially identified for their effect on foraging behavior. To gain a better understanding of the genetic architecture of worker ovary size in this model system, we analyzed a series of crosses between the selected strains. The crossing results were heterogeneous and suggested non-additive effects. Three significant and three suggestive quantitative trait loci of relatively large effect sizes were found in two reciprocal backcrosses. These loci are not located in genome regions of known effects on foraging behavior but contain several interesting candidate genes that may specifically affect worker-ovary size. Thus, the genetic architecture of this life history syndrome may be comprised of pleiotropic, central regulators that influence several linked traits and other genetic factors that may be downstream and trait specific.     

MtDNA COI‐COII marker and drone congregation area: An efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile‐de‐France, France. CANIF's honeybee colonies were intensively studied over a 3‐year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI‐COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.     

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences

Honeybee workers express a pronounced age‐dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age‐ and task‐controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse‐forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1368–1384, 2015     

Netted crop covers reduce honeybee foraging activity and colony strength in a mass flowering crop

The widespread use of protective covers in horticulture represents a novel landscape‐level change, presenting the challenges for crop pollination. Honeybees (Apis mellifera L) are pollinators of many crops, but their behavior can be affected by conditions under covers. To determine how netting crop covers can affect honeybee foraging dynamics, colony health, and pollination services, we assessed the performance of 52 nucleus honeybee colonies in five covered and six uncovered kiwifruit orchards. Colony strength was estimated pre‐ and postintroduction, and the foraging of individual bees (including pollen, nectar, and naïve foragers) was monitored in a subset of the hives fitted with RFID readers. Simultaneously, we evaluated pollination effectiveness by measuring flower visitation rates and the number of seeds produced after single honeybee visits. Honeybee colonies under cover exhibited both an acute loss of foragers and changes in the behavior of successful foragers. Under cover, bees were roughly three times less likely to return after their first trip outside the hive. Consequently, the number of adult bees in hives declined at a faster rate in these orchards, with colonies losing on average 1,057 ± 274 of their bees in under two weeks. Bees that did forage under cover completed fewer trips provisioning their colony, failing to reenter after a few short‐duration trips. These effects are likely to have implications for colony health and productivity. We also found that bee density (bees/thousand flowers) and visitation rates to flowers were lower under cover; however, we did not detect a resultant change in pollination. Our findings highlight the need for environment‐specific management techniques for pollinators. Improving honeybee orientation under covers and increasing our understanding of the effects of covers on bee nutrition and brood rearing should be primary objectives for maintaining colonies and potentially improving pollination in these systems.     

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.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

转基因植物环境监测进展

近20年来,转基因植物的商业化应用规模越来越大,而转基因生物安全问题依然是转基因植物产业进一步发展的最主要制约因素。转基因植物在商业化应用之前虽然预先进行了风险评估,但是,包括环境监测在内的风险管理措施是确保转基因植物安全应用的必要手段。在转基因作物大规模应用近20年之后,其在靶标生物抗性、对生物多样性的影响、基因漂移、在生态系统中的长期存留等方面产生的环境风险已经渐渐显现出来,表明风险评估无法为转基因植物应用提供足够的安全保障,还必须通过开展系统而长期的环境监测,明确转基因植物在生产应用后的实际环境影响。联合国环境规划署和欧盟等已经制定了转基因植物环境监测的法规和技术指南,一些国家实施了系统的转基因植物环境监测。对转基因植物所产生的环境风险以及环境监测应包括的内容进行了综述。     

Perizin, an acaricide to combat the mite Varroa jacobsoni: its distribution in and influence on the honeybee Apis mellifera

Abstract. The distribution of coumaphos (the active component of perizin), fed to individual honeybees, in the honey stomach, haemolymph, midgut and rectum was studied over time. Concurrently, we investigated changes occurring in the haemolymph volume due to the ingestion of perizin, and we examined the influence of a Nosema apis infection on the survival of bees that had been fed perizin. The maximum amount of coumaphos in the haemolymph was found 4h after ingestion, but it was only 2–3% of the total amount recovered. After 15 min 55% of the total amount of the coumaphos recovered was in the honey stomach and available for distribution within the colony by trophallaxis, while 45% had already passed the proventriculus. Ultimately the coumaphos accumulated in the rectum. The volume of the haemolymph significantly increased in bees which were fed perizin compared with bees which were fed syrup and with non-fed bees. The lethal dose of coumaphos to 3-day-old bees was three times higher than the lethal dose for 18- and 1-day-old bees. The number of Nosema apis spores in the alimentary canal was not correlated with the survival of the bees that were fed perizin. It is concluded that coumaphos can act as a systemic agent and can be distributed to other individuals in a colony through trophallaxis, but these effects are limited to a maximum period of 12h after ingestion.