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%.     

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.     

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.     

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.     

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.     

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.     

An ionotropic GABA receptor in cultured mushroom body Kenyon cells of the honeybee and its modulation by intracellular calcium

GABAergic inhibitory transmission is very abundant within the insect brain. We, therefore, studied the functional properties of the ionotropic GABA receptor of honeybee mushroom body Kenyon cells in vitro. GABA applications elicit rapidly activating and desensitizing currents, which are concentration-dependent between 10 and 500 μM. The mean peak amplitude induced by 500 μM GABA at a holding potential of −110 mV is −1.55 ± 0.23 nA (SEM, n = 29). The GABA-induced current is mediated by Cl⁻ ions because (1) the reversal potential of the GABA-induced current of −40.6 mV is very close to the calculated Nernst potential of chloride (−44.8 mV). (2) With equimolar chloride concentrations the reversal potential shifted to about 0 mV. GABA or muscimol are equally efficient channel agonists, whereas CACA is a partial agonist. Picrotoxin or philanthotoxin (100 μM) completely and reversibly block the GABA-induced current, bicuculline (100 μM) has no effect. Elevating the intracellular Ca²⁺ concentration increases the GABA current amplitude. This modualtory effect is blocked by the kinase blocker K 252a, but not by blockers of CaMkinaseII (KN-93), PKC (bisindolylmaleimide) or PKA (KT 5720). We conclude that Kenyon cells express functional GABA receptors whose properties support an inhibitory role of GABAergic transmission.     

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.     

Hygropreference and brood care in the honeybee (Apis mellifera)

Terrestrial organisms need to limit evaporation from their bodies in order to maintain a homeostatic water balance. Owing to a large surface to volume ratio, arthropods are particularly susceptible to desiccation and have evolved behavioural and physiological mechanisms to conserve water. In social insects, water balance is also affected by the interactions between nestmates and by the architecture of the nest. For honeybees, humidity is particularly important for the brood because it affects the hatching success of eggs and because, unlike ants, honeybees cannot relocate their brood to parts of the nest with more favourable humidity. To advance the understanding of the water economy in honeybee nests, we investigated whether workers exhibit a hygropreference when exposed to a gradient of 24-90% relative humidity (RH) and whether the expression of this preference and their behaviour is affected by the presence of brood. The results show that young honeybee workers in the absence of brood exhibit a weak hygropreference for approximately 75% RH. When brood is present the expression of this preference is further weakened, suggesting that workers tend to the brood by distributing evenly in the gradient. In addition, fanning behaviour is shown to be triggered by an increase in humidity above the preferred level but not by a decrease. Our results suggest that humidity in honeybee colonies is actively controlled by workers.     

Biosynthesis of the scFv Antibody to Human Ferritin in Plant and Bacterial Producers

A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures. As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility. Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny. Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His₆ immobilized on a metal-affinity carrier.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

Community response to transgenic plant release: using mathematical theory to predict effects of transgenic plants

Predicting the potential effects of introductions of plants on the structure of plant communities has been elusive. I suggest that mathematical models of resource competition might be useful for identifying categories of plants that either are unlikely to alter community structure or that have the potential for altering community structure. Assuming that the transgenic plant will escape and establish viable populations in nontarget habitats, this theory suggests that species that have a high minimum resource requirement are unlikely to alter community structure. The theory is elaborated to evaluate the potential effects on community structure of transgenic plants with resistance to primary consumers. Results indicate that the greatest reduction in the minimum resource requirement caused by resistance will occur when consumers are consuming enough plant biomass that the plant can no longer grow. If resistance to such a consumer were incorporated into a plant, it could lower the minimum resource requirement sufficiently that a transgenic plant would be able to alter community structure substantially. Examples of introductions of exotic plants, plant pathogens, and insect herbivores are given to support the conceptual basis of the theory. Not all transgenic plants with resistance, however, have the potential to alter community structure. Resistance to primary consumers that strongly reduce the biomass producing ability of a plant will probably be able to alter community structure, whereas resistance that reduces most other types of yield loss is less likely to alter community structure. The theory should be elaborated to incorporate more-realistic assumptions, such as those regarding reproduction, dormancy, and dispersal of the transgenic plants, and provide more detailed characterization of the potential hazard of transgenic plants to plant communities.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Production of recombinant proteins by microbes and higher organisms

Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S–S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.