Genotype,Task Specialization,and Nest Environment Influence the Stinging Response Thresholds of Individual Africanized and European Honeybees to Electrical Stimulation

This study was conducted to analyze the stinging response thresholds of individual European and Africanized worker honeybees (Apis mellifera L.) to electrical stimulation. Newly emerged workers were identified, and either were placed into an incubator, into their natal colonies, or cross-fostered in common colonies of European or Africanized ancestry. Nest and guard bees of each type were collected and exposed to an electric stimulus of 0.5 mA, and the time they took to sting a leather substrate was recorded. Africanized bees consistently had significant lower thresholds of defensive response than European bees across all of the environments tested. Guards were faster to sting than nest bees only for the Africanized genotype, suggesting that alleles of African origin have pleiotropic effects on guarding and stinging. This is the first study that shows that single individuals specialized in guarding also may have a lower response threshold for stinging. Environmental effects were also evident. In all cases, bees responded faster to the electrical stimulation after being kept in environments other than their natal nest. Moreover, significant genotype by environment and genotype by task specialization interactions were found. Our results fit a model of division of labor based on differences in response thresholds to stimuli among workers of different genotypes and task groups that result in non-additive effects on colony behavior. Edited by Yong-Kyu Kim.     

Quantitative trait loci that influence the expression of guarding and stinging behaviors of individual honey bees

This study was conducted to test for the effect of three stinging behaviors QTLs (sting-1, sting-2 and sting-3) on the expression of guarding and stinging behavior of individual honey bees, and to determine if results of defensive behavior QTLs found in studies with Africanized honey bees could be extended to other populations of bees. Samples of guards, stingers, foragers and nurse bees were taken from two backcross colonies derived from a defensive colony and a gentle colony. The genotype of each bee for both types of colonies was determined for two sequence tagged site (STS) markers linked to sting-1 and for another two STSs, one linked to sting-2 and one linked to sting-3. Results showed that sting-1 had an effect on the expression of both stinging and guarding behaviors, sting-2 and sting-3 influenced the expression of guarding behavior. These results indicate that division of labor is influenced by specific QTLs. Results also show that QTLs mapped in a population of Africanized honey bees using colony level phenotypes also influenced the expression of guarding and stinging behavior of individual bees of other populations.     

A genetic analysis of the stinging and guarding behaviors of the honey bee

In order to identify genes that are influencing defensive behaviors, we have taken a new approach by dissecting colony-level defensive behavior into individual behavioral measurements using two families containing backcross workers from matings involving European and Africanized bees. We removed the social context from stinging behavior by using a laboratory assay to measure the stinging response of individual bees. A mild shock was given to bees using a constant-current stimulator. The time it took bees to sting in response to this stimulus was recorded. In addition, bees that were seen performing guard behaviors at the hive entrance were collected. We performed QTL mapping in two backcross families with SNP probes within genes and identified two new QTL regions for stinging behavior and another QTL region for guarding behavior. We also identified several candidate genes involved in neural signaling, neural development and muscle development that may be influencing stinging and guarding behaviors. The lack of overlap between these regions and previous defensive behavior QTL underscores the complexity of this behavior and increases our understanding of its genetic architecture.     

Biochemical variability of venoms from individual European and Africanized honeybees (Apis mellifera).

To study biochemical differences between venom from individual honeybees, venom sacs from 103 European (EU) bees and 92 Africanized bees representing 12 different colonies were dissected, and the dry weight (DW) of venom from each bee was determined. Venom from each of these bees was studied with isoelectric focusing and functional assays for phospholipase A2 and melittin. Phospholipase concentrations in individual EU bee venoms varied between 1.8% and 27.4% (wt/wt). The melittin concentration in EU bee venom varied less and, on the average, was found to be much lower than previously reported. There was an eightfold to ninefold difference between lowest and highest venom sac DW contents, suggesting the possibility of highly variable venom delivery from bee stings. One EU bee contained greater than 300 micrograms of venom, three times the recommended maintenance dose for venom immunotherapy. Isoelectric focusing also demonstrated large differences between individual bees, with respect to major and minor components of their venoms. Africanized bees contained significantly less venom but more phospholipase than did EU bees. Bee venoms from different colonies differed in their DW content and in their concentrations of phospholipase and melittin. The results are relevant to the uncertainty of responses from sting challenges and field stings in allergic patients and massive stinging attacks on normal subjects.     

The behavioral genetics of colony defense in honeybees: Genetic variability for guarding behavior

Guard honeybees stand at the entrance of colonies and facilitate the exclusion of nonnestmates from the colony. In this study, we examined the hypothesis that genetic variability among individuals in colonies might explain variability in guarding activity. To do this, we cross-fostered honey bees between colonies with high-defensive responses and colonies with low-defensive responses in alarm pheromone tests. Individuals from high-defensive colonies were more likely to guard in their own colonies (controls) than cross-fostered bees from low-defensive colonies. Cross-fostered high-defensive bees also were more likely to guard in low-defense colonies. These results support the hypothesis that interindividual differences in guarding behavior are at least partially under genetic control. A positive correlation between number of guards and response to alarm pheromone demonstrates a link between behaviorally separated components of the overall defensive response.This work was supported by NSF Grant BNS 8605604.     

Genetic study of the aggressiveness of two subspecies ofApis mellifera in Bazil. IV. Number of stings in the gloves of the observer

Data are analyzed on an aspect of aggressiveness in workers from colonies of Africanized bees (Apis mellifera adansonii), Italian bees (Apis mellifera ligustica), their F ₁ hybrids, and backcrosses of the F ₁ to the parental stocks (Rothenbuhler method). The segregation values (3:1) in the backcrosses to the Africanized stock and nonsegregation in the backcrosses to the Italian stock suggest the existence of two pairs of genes (F₁/F₁;F₂/F₂ in the Italian bees and f₁/f₁;f₂/f₂ in the Africanized bees) which control a character defined as the number of stings in the gloves of the observer.This work was carried out in the Department of Genetics, Faculdade de Medicina de Ribeirão Preto, and in the Laboratory of Biology, Faculdade de Filosofia, Ciências, e Letras de Araraquara, with the financial assistance of Fundação de Amparo à Pesquisa do Estado de São Paulo. This paper is part of the author's doctoral dissertation.     

Olfactory stimulation of Africanized honey bee (Hymenoptera: Apidae) attacks by insect repellents

Three common insect repellents (N,N-diethyl-m-toluamide [DEET], Pyranha, and Repel X) were tested to determine whether they affected Africanized honey bee attack behavior. Eight Africanized honey bee (Apis mellifera L.) colonies were exposed in an alternating series to the test repellents or blank controls delivered in a stream of air directed toward the colony entrances. The response generated by the repellents and the controls was measured as the number of attacking honey bees recorded with an electronic temper tester. Neither a citronella-based repellent (Pyranha) nor DEET had any effect on colony behavior; however, Repel X consistently caused a greater attack response after exposure.     

The ontogeny of kin discrimination cues in the honey bee,Apis mellifera

Cues used in the discrimination of relatives from nonrelatives by the honey bee reflect both genetic and environmental differences between groups. Discrimination is behaviorally expressed by acceptance into or agonistic rejection from the social group. We examine the development of these cues in field colonies and in controlled laboratory settings. Newly emerged worker honey bees are accepted by honey bee social groups at a high frequency. When bees are kept in a controlled laboratory environment for 5 days, acceptability into laboratory groups is determined largely by relatedness. Cues indicating relatedness develop in the laboratory within 12 h after the adult bee emerges. Bees older than 12 h are not accepted by field colonies regardless of relatedness. Bees maintained in a hive until 5 days after emergence are not accepted by related or unrelated laboratory groups (this is termed the hive effect). Bees maintained in hives for times as short as 5 h acquired the hive effect. In a cross-fostering experiment, the hive effect completely masked genetic differences.This work was supported by NSF Grants BNS 82-16787 and BNS 86-05604.     

Genetic differences in learning behavior in honeybees (Apis mellifera capensis)

Workers from colonies of Cape honeybees show marked phenotypic differences in performance in proboscis extension reflex (PER) conditioning. Analysis of these differences using parthenogenetic offspring groups permitted the estimation of genotypic values and revealed a high degree of genetic variability that is evident among related as well as unrelated bees. The results obtained from related groups are of particular importance, since they demonstrated the existence of strong genetic variability among individuals of the same colony. Quantitative analysis yielded high estimates of additive genetic effects and low estimates of dominance effects. Selection of individual workers resulted in an explicit increase in genetic variance of the next generation (G1). However, selection of bees from the parthenogenetic G1 generation, which was done to obtain parthenogenetic G2 offspring, did not lead to further improvement in selection. This observation suggests that recombination of linked genes underlying proboscis extension reflex was neglible during selection in parthenogenetic groups. Taken together with further behavioral analysis (Brandes and Menzel, 1990; Brandes et al., 1988), results from these quantitative genetic experiments suggest that additive genetic factors contribute significantly to variability among individuals for associative learning.This work was supported by a fellowship from the Deutsche Forschnungsgemmeinschaft (Br 827/1, Br 827/2).     

Unusual Reactions to Hymenoptera Stings: What Should we Keep in Mind?

This review includes a variety of extremely rare and unusual hymenoptera sting (HS) circumstances with regard to sting localization, geographic region, massivity of multiple stings, and particularly related to clinical symptoms. Such reactions occur in a temporal relationship to HS (s), differ from typical allergic symptomatology, and sometimes need follow-up during many months. With respect to pathogenesis, the major mechanisms involved are toxic, autoimmune, and other delayed immunological ones. While delayed inflammatory symptoms of the nervous system are considered as delayed hypersensitization or autoimmune entities, generalized rhabdomyolysis and consecutive acute kidney injury is considered a toxic reaction, mostly induced by massive envenomation to wasps or “Africanized” bees. Hemorrhagic episodes of targeted organ (s) could be additional potential risk for acute kidney injury, while the bee venom-induced hemorrhage is proposed to be a nonimmune-mediated anaphylactic symptom. The hemodynamic involvement of vital organs and systems with hypoxia and hypovolemia together with simultaneous immunoglobulin E (IgE) sensitization are considered potential indications for venom immunotherapy. In contrast, patients who have experienced various complications with unknown or nonallergic mechanisms should be informed about the importance of epinephrine’s use and additional measures on future sting avoidance. In conclusion, although unusual reactions are extremely rare, it is important to keep them in mind.     

Einsatz der automatischen Bildanalyse beim Screening von leistungsfähigen Selektanten Turimycin-bildender Streptomyces-Stämme

The growth of the surface colonies on solid media is studied with the image analyzing system QUANTIMET 720 M controlled by a PDP 11 computer. For the bacterial strain Streptomyces hygroscopicus JA 6599 a new differential equation describing the kinetic behaviour of the area of the colony is suggested It is shown that this time dependence of area describes more realistically the experiment than the function resulting from PIRT'S model. Two new constants arise from the growing curve, α describes the profile of density of the colony and k₀ is correlated to the growing constant of the colony. Both parameters can be correlated to the amount of antibiotics produced and, therefore, it is possible to use this information for the selection procedure of the colonies. A uniform criterion for selection was found for each colony of two mutants of the turimycin-producing strain Streptomyces hygroscopicus JA 6599, which reads: The quality of the selection criterion of surface colonies with higher productivity is dependent on the mean value of the growing parameters of all colonies under investigation Limitations of the selection method arise from the need for great uniformity of environmental conditions, growth medium and a homogeneous spore suspension, which have to consist of a large amount of single spores in order to assure a reproducible growing curve of the colonies. For the last condition a special technique is pointed out.     

Transformation of Cochliobolus lunatus with pUT 720 changes the steroid hydroxylating ability of the fungus

Summary The filamentous fungus Cochliobolus lunatus, a known 11-hydroxylator of steroids, was transformed to bleomycin resistance using the heterologous plasmid pUT 720. This plasmid contains the Sh ble gene expressed under the control of the Aspergillus nidulans gpd and trpC expression signals. The bleomycin-resistant colonies appeared with a frequency of six per g of DNA. All colonies were real transformants and no abortive growth was observed. In all transformants tested the plasmid molecules became stably integrated into the genome of the host, and one of the plasmid molecules integrated in a site-specific manner. Transformants retained the ability to hydroxylate the steroid ring, but the hydroxy group was inserted at the 15 position.     

Insect sting allergy in children: what is the real cost of the disease?

The impact of insect sting allergies on the quality of life of 118 children and their parents is assessed using attitudinal and psychometric questionnaires. Children, ranging in age from 7-15 years, manifested more anxiety in the clinical setting (state anxiety) than usual (trait anxiety), whereas for parents the trend was reversed. Most children believed that they could control being stung, and restrictions imposed by two-thirds of the parents assisted in preventing stinging episodes. Parents perceived their child's academic achievement, social abilities and extracurricular involvement as superior to that of their peers and closest aged siblings.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

High frequency recombination and the expression of genes cloned on chimeric yeast plasmids: Identification of a fragment of 2-μm circle essential for transformation

Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-m circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-m circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-m circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per g in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-m circle transform LL20 at a reduced frequency (6,000–16,000 colonies per g) and YF233 at extremely low frequencies (1–5 colonies per g). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-m circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-m circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-m circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-m circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-m circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.     

Effect of chromosome loss on the leavening ability of Saccharomyces cerevisiae in dough

Summary To investigate the leavening ability of yeast in dough, chromosome loss was induced by benomyl treatment in YOY1037, a diploid between a baking strain and a laboratory strain, and its effect on the leavening ability was studied. When benomyl-treated cells were spread on plates with a dye indicator for ploidy, about 20% of the visible colonies were stained dark blue or dark purple; the rest stained pale blue, similar to the diploid YOY1037. Strains showing the MAT phenotype, and non-galactose fermenting strains, apparently having lost particular chromosomes, were observed only in those with darkcoloured colonies. Strains with dark-coloured colonies showed a wider range of leavening ability than did those with pale-coloured colonies.     

Parasexual cycle and genetic analysis following protoplast fusion in Nectria haematococca

Summary Protoplasts of multiauxotrophic strains of Nectria haematococca (the perfect form of Fusarium solani) were fused and grown on different selective media. Following protoplast fusion, rapidly growing heterokaryons were formed at high frequency (about 1%). By collecting uninucleate microconidia from these heterokaryons, it was possible to isolate a few colonies with new combinations of the parental markers. On some selective media, non-heterokaryotic fusion products, easily distinguishable from heterokaryons by their growth characteristics, were detected at low frequency. These colonies were either stable haploid recombinants or unstable hybrids of unknown ploidy. Genetic analysis of hybrids in which six factors were heterozygous provided evidence for nuclear fusion events. These hybrid colonies spontaneously segregated a large number of haploid recombinants, allowing the analysis of linkage relationships between markers. The detection of a mitotic linkage between two markers.which appeared unlinked following meiosis, demonstrates that mitotic mapping may be an important complement to meiotic analysis for mapping the chromosomes of Nectria haematococca.     

Cloning,sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene

Summary The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5 and 3 gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragments has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The idenity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae ghd-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.     

Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance

Summary The phytopathogenic fungus Septoria nodorum has been transformed using a plasmid (pAN7-1) containing the Escherichia coli hygromycin phosphotransferase gene (hph). Large, stable hygromycin-resistant transformant colonies appeared at frequencies between 2 and 25 per g DNA when wheat-adapted and barley-adapted wild type strains were used as recipients. These transformants grew at hygromycin concentrations up to ten times that which inhibits the wild types. A second type of colony also developed on transformation plates. These appeared at higher frequencies, grew less vigorously and could not be subcultured in the presence of hygromycin. They are believed to be abortive transformants. Southern hybridization analyses indicated that transformation takes place via the integration of plasmid DNA into the fungal chromosomal DNA. Multiple integrations occur producing tandemly iterated arrays of plasmid molecules. Some transformants arose as heterokaryons. These could be resolved by propagation through a single spore and transformants purified in this way remained mitotically stable. All of 1,025 transformants tested were unchanged in pathogenicity. Reisolates from leaves retained their hygromycin-resistance, indicating that transformants remain stable during growth in plant tissue. Cotransformation of an unselected plasmid (p3SR2) carrying the Aspergillus nidulans amdS gene occurred at a high frequency.     

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

Summary Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 ( haploid), P-540 (a/ diploid), and P-544 (a/ diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10^-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a// genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an / genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.