Improving genetic transformation rates in honeybees.

Functional genetic studies in honeybees have been limited by transformation tools that lead to a high rate of transposon integration into the germline of the queens. A high transformation rate is required to reduce screening efforts because each treated queen needs to be maintained in a separate honeybee colony. Here, we report on further improvement of the transformation rate in honeybees by using a combination of different procedures. We employed a hyperactive transposase protein (hyPBaseapis), we tripled the amount of injected transposase mRNAs and we injected embryos into the first third (anterior part) of the embryo. These three improvements together doubled the transformation rate from 19% to 44%. We propose that the hyperactive transposase (hyPBaseapis) and the other steps used may also help to improve the transformation rates in other species in which screening and crossing procedures are laborious.

Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism.

Social evolution in honey bees has produced strong queen-worker dimorphism for plastic traits that depend on larval nutrition. The honey bee developmental programme includes both larval components that determine plastic growth responses to larval nutrition and nurse components that regulate larval nutrition. We studied how these two components contribute to variation in worker and queen body size and ovary size for two pairs of honey bee lineages that show similar differences in worker body-ovary size allometry but have diverged over different evolutionary timescales. Our results indicate that the lineages have diverged for both nurse and larval developmental components, that rapid changes in worker body-ovary size allometry may disrupt queen development and that queen-worker dimorphism arises mainly from discrete nurse-provided nutritional environments, not from a developmental switch that converts variable nutritional environments into discrete phenotypes. Both larval and nurse components have likely contributed to the evolution of queen-worker dimorphism.

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.

Use of Wolfe graft for the treatment of mucous cysts.

Many surgical procedures have been described for the treatment of mucous cysts. We report a case series of a surgical technique that excises the cyst along with overlying skin and reconstructs the defect using a Wolfe graft harvested from the wrist crease. This technique can be applied to cysts in all locations, even those adjacent to the nail. The procedure has been performed on 51 mucous cysts with satisfactory results, a very low recurrence rate (4%) and negligible complications.

High recombination frequency creates genotypic diversity in colonies of the leaf-cutting ant Acromyrmex echinatior.

Honeybees are known to have genetically diverse colonies because queens mate with many males and the recombination rate is extremely high. Genetic diversity among social insect workers has been hypothesized to improve general performance of large and complex colonies, but this idea has not been tested in other social insects. Here, we present a linkage map and an estimate of the recombination rate for Acromyrmex echinatior, a leaf-cutting ant that resembles the honeybee in having multiple mating of queens and colonies of approximately the same size. A map of 145 AFLP markers in 22 linkage groups yielded a total recombinational size of 2076 cM and an inferred recombination rate of 161 kb cM(-1) (or 6.2 cM Mb(-1)). This estimate is lower than in the honeybee but, as far as the mapping criteria can be compared, higher than in any other insect mapped so far. Earlier studies on A. echinatior have demonstrated that variation in division of labour and pathogen resistance has a genetic component and that genotypic diversity among workers may thus give colonies of this leaf-cutting ant a functional advantage. The present result is therefore consistent with the hypothesis that complex social life can select for an increased recombination rate through effects on genotypic diversity and colony performance.

Surgical delay in the management of dog bite injuries in children, does it increase the risk of infection?

Dog bites are contaminated wounds and convention dictates that any surgical treatment should be performed at the earliest opportunity. This practice, however, may be in direct conflict with NCEPOD guidelines to reduce night time operating. We audited our treatment of dog bite injuries in children over a 5-year period (1998-2002) to determine whether delaying surgery to conform to operating guidelines increased the incidence of infection. A retrospective case note study identified 118 patients admitted to Sheffield Children's Hospital with dog bite injuries. Details of their injury, treatment and outcome were recorded. There were 70 males and 48 females and an average age of 6.6 years (range 1-15 years). The most common type of injury was a single laceration (32%) and the most frequent site of injury was the face (59%). The majority of wounds (68%) were cleaned and dressed before theatre and 89% were closed directly after minimal debridement. All patients received prophylactic antibiotics. Of the 114 patients that required surgery, 57 (50%) were operated on within 12 h of their injury and 57 (50%) after 12 h. There were three cases of infection in the early group (<12 h) and none in the delayed group (>12 h). We suggest that delaying the surgical treatment of dog bites in children to avoid operating at night does not increase the infection rate.

Locomotion and the pollen hoarding behavioral syndrome of the honeybee (Apis mellifera L.).

Honeybees selected for the colony level phenotype of storing large quantities of pollen (pollen hoarding) in the nest exhibit greater walking activity than those selected against pollen hoarding. In this study, we use a simple walking assay to demonstrate that walking activity increases with the proportion of high pollen-hoarding alleles in pure and backcrossed strains of bees (high-strain bees > offspring generated from a high backcross > offspring generated from a low backcross > low-strain bees). The trait is heritable but is not associated with markers linked to three quantitative trait loci (QTL) mapped for their effects on pollen hoarding with demonstrated pleiotropic effects on pollen and nectar foraging and learning behavior. However, locomotion in non-selected bees is correlated with responsiveness to sucrose, a trait that correlates with foraging and learning behavior. We propose that pollen-hoarding behavior involves a syndrome of behavioral traits with complex genetic and regulatory architectures that span sensory sensitivity, foraging behavior, and learning. We propose that locomotor activity is the component of this syndrome and reflects the early maturation of the bees that become pollen foragers.

Paternal effects on the defensive behavior of honeybees.

The defensive behavior of 52 hybrid honeybee (Apis mellifera L.) colonies from four sets of crosses was studied and compared with that of European and Africanized bee colonies. Colonies containing F(1) hybrid workers were obtained through reciprocal crosses between European and Africanized bees. The total number of stings deposited by workers in a moving leather patch in 1 min was recorded. In each of the four sets of crosses, bees from hybrid colonies of Africanized paternity left more stings in leather patches than bees from hybrid colonies of European paternity. Results strongly suggest paternal effects of African origin increasing the defensive behavior of hybrid colonies. Although some degree of dominance was observed for high-defensive behavior in one of the four sets of crosses involving European paternity, most of the dominance effects reported in the literature appear to be the result of paternal effects. Several hypotheses to explain this phenomenon, as well as the implications of these effects on the fitness and breeding of honeybees are discussed.

Pleiotropy, epistasis and new QTL: the genetic architecture of honey bee foraging behavior.

The regulation of division of labor in social insects, particularly in the honey bee (Apis mellifera L.), has received considerable attention from a number of biological subdisciplines, including quantitative and behavioral genetics, because of the high complexity of the behavioral traits involved. The foraging choices of honey bee workers can be accurately quantified, and previous studies have made the foraging behavior of honey bees one of the best studied naturally occurring behavioral phenotypes. Three quantitative trait loci (QTL) have been identified that influence a set of foraging variables, including the concentration of nectar collected and the amount of pollen and nectar brought back to the hive. This study extends previous genetic investigations and represents the most comprehensive investigation of the genetic architecture of these foraging variables. We examined the effects of markers for the three established QTL and for one further candidate gene (Amfor), in two reciprocal backcross populations. These populations were also used to carry out two new QTL mapping studies, with over 400 Amplified Fragment Length Polymorphism (AFLP) markers in each. We detected a variety of effects of the genetic markers for the established QTL and the candidate gene, which were mostly epistatic in nature. A few new QTL could be detected with a variety of mapping techniques. Our results add complexity to the genetic architecture of the foraging behavior of the honey bee. Specifically, we support the hypotheses that pln1, pln2, pln3, and Amfor are involved in the regulation of foraging behavior in the honey bee and add some new factors that deserve further study in the future.

Spindle cell haemangioendothelioma in an arteriovenous fistula of the ring finger after blunt trauma.

We present a case of traumatic arteriovenous fistula of the palm and ring finger, which posed management dilemmas and eventually necessitated ray amputation. Subsequent histology revealed a spindle cell haemangioendothelioma that had developed within the fistula. We report the clinical features and management of this patient.

Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.).

The responsiveness of bees to sucrose is an important indicator of honey bee foraging decisions. Correlated with sucrose responsiveness is forage choice behavior, age of first foraging, and conditioned learning response. Pheromones and hormones are significant components in social insect systems associated with the regulation of colony-level and individual foraging behavior. Bees were treated to different exposure regimes of queen and brood pheromones and their sucrose responsiveness measured. Bees reared with queen or brood pheromone were less responsive than controls. Our results suggest responsiveness to sucrose is a physiologically, neuronally mediated response. Orally administered octopamine significantly reduced sucrose response thresholds. Change in response to octopamine was on a time scale of minutes. The greatest separation between octopamine treated and control bees occurred 30 min after feeding. There was no significant sucrose response difference to doses ranging from 0.2 mug to 20 mug of octopamine. Topically applied methoprene significantly increased sucrose responsiveness. Handling method significantly affected sucrose responsiveness. Bees that were anesthetized by chilling or CO(2) treatment were significantly more responsive than control bees 30 min after handling. Sixty minutes after handling there were no significant treatment differences. We concluded that putative stress effects of handling were blocked by anesthetic.

PKA and PKC content in the honey bee central brain differs in genotypic strains with distinct foraging behavior.

Selection of honey bees for pollen storage resulted in high and low pollen-hoarding strains differing in foraging behavior traits including resource choice and quality, load size, sucrose responsiveness, age of foraging initiation, and learning performance. To determine how these genotypic differences correlate with changes at the level of proteins involved in neuronal function, we measured the content of protein kinase A, protein kinase C, and synapsin in the brains of high- and low-strain bees. In the central brain protein kinase A and protein kinase C levels were greater in high-strain bees and increased from emergence to 5 days in both strains. By 15 days, high-strain bees retained significantly higher levels of protein kinase C than low-strain bees, but overall protein kinase C content decreased in both strains. Synapsin levels increased from emergence to 5 days but did not differ between the two strains. In contrast to the protein kinase A content in the central brain, the basal protein kinase A activity did not differ between the strains or between the two age groups. This provides first evidence that the two genetic strains of honey bees show characteristic differences in the regulation of protein expression that may contribute to the behavioral differences between them.

New genomic resources for the honey bee(Apis mellifera L): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2 empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99 probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/)

The genetic basis of the interspecific differences in wing size in Nasonia (Hymenoptera; Pteromalidae): major quantitative trait loci and epistasis.

There is a 2.5-fold difference in male wing size between two haplodiploid insect species, Nasonia vitripennis and N. giraulti. The haploidy of males facilitated a full genomic screen for quantitative trait loci (QTL) affecting wing size and the detection of epistatic interactions. A QTL analysis of the interspecific wing-size difference revealed QTL with major effects and epistatic interactions among loci affecting the trait. We analyzed 178 hybrid males and initially found two major QTL for wing length, one for wing width, three for a normalized wing-size variable, and five for wing seta density. One QTL for wing width explains 38.1% of the phenotypic variance, and the same QTL explains 22% of the phenotypic variance in normalized wing size. This corresponds to a region previously introgressed from N. giraulti into N. vitripennis that accounts for 44% of the normalized wing-size difference between the species. Significant epistatic interactions were also found that affect wing size and density of setae on the wing. Screening for pairwise epistatic interactions between loci on different linkage groups revealed four additional loci for wing length and four loci for normalized wing size that were not detected in the original QTL analysis. We propose that the evolution of smaller wings in N. vitripennis males is primarily the result of major mutations at few genomic regions and involves epistatic interactions among some loci.

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database.

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).

A linkage analysis of sex determination in Bombus terrestris (L.) (Hymenoptera: Apidae).

We constructed a linkage map of Bombus terrestris (Hymenoptera, Apidae) phase unknown. The map contains 79 markers (six microsatellite and 73 RAPD markers) in 21 linkage groups and spans over 953.1 cM. The minimal recombinational size of the B. terrestris genome was estimated to be 1073 cM. Using flow cytometry, the physical size of the haploid genome of B. terrestris was calculated to be 274 Mb. This is the second linkage map for a social insect species. Bombus terrestris has on average five times less recombinational events per kb than the honey bee Apis mellifera. Male haploidy, chromosome size, and eusociality can now be excluded as reasons for the high recombination frequency of Apis mellifera. Finally, the sex determination locus of B. terrestris was placed on the map using bulked segregant analysis.

The effects of genotype, foraging role, and sucrose responsiveness on the tactile learning performance of honey bees (Apis mellifera L.).

We analyzed sucrose responsiveness and associative tactile learning in two genetic strains of honey bees under laboratory conditions. These strains differ in their foraging behavior. Bees of the "high" strain preferentially collect pollen. "Low"-strain bees mainly forage for nectar. Responsiveness to different sucrose concentrations and tactile learning were examined using the proboscis extension reflex. Acquisition, extinction of conditioned responses, and responses to an alternative tactile stimulus were tested. High-strain bees are more responsive to sucrose than low-strain bees. Regardless of genotype, pollen foragers are more responsive to sucrose than nectar foragers. In bees of both strains we find the same relationship between responsiveness to sucrose and acquisition. Bees responding to low sucrose concentrations show more often the conditioned response during acquisition than those responding only to higher sucrose concentrations. Extinction of conditioned responses depends on the response probability during acquisition. Discrimination between the two tactile stimuli is affected by genotype but not by responsiveness to sucrose. High-strain bees discriminate better than low-strain bees. Our experiments thus establish links between division of labor, responsiveness to sucrose, and associative learning in honey bees.

Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): influence of genotype, feeding, and foraging experience.

The perception of sugar is important to honey bees for making foraging decisions. We measured bees' perception by determining what concentration of sucrose touched to the antennae elicited the proboscis extension response (response threshold). A low response threshold (extension at low concentration) suggests a high perceptual value of sucrose. and vice versa. Perception of sucrose solutions differed between two artificially selected genotypic strains and was modulated by the bees' recent feeding experiences. Bees offered 10%, 30%, or 50% sucrose solutions in small cages overnight, and in large flight-cages or free-flying in the field for several days, had subsequent response thresholds positively correlated to the concentration offered. Empty bees, whether they were nectar, water or pollen foragers, dancers or non-dancers, had a significantly lower threshold than loaded bees. Crop volume affected response thresholds directly and independently of sucrose concentration. We interpret these findings as multiple mechanisms that operate in different time scales, modulating perception of sucrose. Changes occurred in the time scale of evolutionary processes as demonstrated by genotypic differences. Changes with foraging experience occur in hours or minutes while effects of crop filling are instantaneous.