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.     

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.     

Confirmation of QTL effects and evidence of genetic dominance of honeybee defensive behavior: results of colony and individual behavioral assays

The stinging and guarding components of the defensive behavior of European, Africanized, hybrid, and backcross honeybees (Apis mellifera L.) were compared and analyzed at both colony and individual levels. Hybrid and Africanized backcross colonies stung as many times as Africanized ones. European backcross colonies stung more than European bees but not as many times as Africanized or Africanized backcross colonies. The degree of dominance for the number of times that worker bees stung a leather patch was estimated to be 84.3%, 200.8%, and 145.8% for hybrid, backcross European, and backcross Africanized colonies, respectively. Additionally, both guards at the colony entrance and fast-stinging workers of one European backcross colony had a significantly higher frequency of an Africanized DNA marker allele, located near sting1, a QTL previously implicated in stinging behavior at the colony level. However, guards and fast-stinging bees from a backcross to the Africanized parental colony did not differ from control bees in their frequency for the Africanized and European markers, as would be expected if large genetic dominance effects for sting1 exist. These results support the hypothesis that genetic dominance influences the defensive behavior of honeybees and confirm the effect of sting1 on the defensiveness of individual worker bees.     

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.     

Effects of anger/hostility, defensiveness, gender, and family history of hypertension on cardiovascular reactivity

In this study, we examined the combined effects of anger/hostility and defensiveness on reactivity to three stressors (math, handgrip, cold pressor) and how these effects are moderated by gender and family history of hypertension. The subjects were 209 college students. Low compared with high Spielberger anger-out scores were associated with greater diastolic blood pressure (DBP) reactivity in low-but not high-defensive women with a negative family history. High compared with low hostility scores were associated with greater heart rate reactivity in low-defensive men with an opposite effect in high-defensive men. Gender, family history, and defensiveness each affected differential DBP reactivity to the three tasks. Combined study of biological factors and coping styles can improve our understanding of cardiovascular reactivity and disease.     

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).     

Alarm pheromone increases defensive and risk assessment behaviors in male rats

Previously, we reported that alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased Fos expression in the vomeronasal pathway and stress-related nuclei in pheromone-recipient rats. However, the alarm property of this pheromone in terms of behavior modification is still unclear. We recently found that this alarm pheromone could be trapped in water. Based on this finding, we developed an experimental paradigm to assess the effect of alarm pheromone on recipient behavior. Male Wistar rats were acclimatized for 5 min to an open field, where two pieces of filter paper soaked with 750 microl of either pheromone-containing water or vehicle water were attached to the wall. Then, a small "hiding box" was placed in one corner of the field and the behavioral responses of the subject rat were recorded for 10 subsequent minutes. Exposure to alarm pheromone significantly increased defensive and risk assessment behaviors and decreased exploratory and grooming behaviors compared to the vehicle control group, indicating the alarm property of the pheromone. In addition, the comparison with previous results suggests that the alarm pheromone released from the perianal region of the male rat increases anxiety in recipients, rather than evoking a stereotyped autonomic response.     

The volatility of an alarm pheromone in male rats

The volatility of an alarm pheromone in male rats. PHYSIOL BEHAV 00(0) 000-000, 2008. We previously reported that an alarm pheromone released from the perianal region of male rats is perceived by the vomeronasal organ and evokes stress-induced hyperthermia and defensive and risk assessment behavior. In addition, we recently reported that the alarm pheromone enhances the acoustic startle reflex (ASR). However, in contrast to our knowledge about such biological aspects of the pheromone, information concerning the physical character of the alarm pheromone is extremely limited. In this study, we investigated the volatility of the alarm pheromone using enhancement of the ASR as an index of the pheromone effect. The alarm pheromone enhanced the ASR when it was presented at a distance of 10 mm but not at 200 mm. In addition, the pheromone effect was observed even after the pheromone was trapped in the adsorbent (Tenax) and then extracted using purified water. These results suggest that the alarm pheromone is both volatile and water soluble.     

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.     

Enhancement of the acoustic startle reflex by an alarm pheromone in male rats

Recently, we reported that an alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased defensive and risk assessment behaviors in recipient male rats. Based on these results, we hypothesized that the primary effect of the alarm pheromone is to increase anxiety; however, there is still no clear evidence for this pheromone effect. Therefore, we examined this issue by assessing the effect of the alarm pheromone on the acoustic startle reflex (ASR), which is a useful index for studying negative emotions such as anxiety in rats. The alarm pheromone enhanced the ASR for 105-dB auditory stimuli, but not for those of 90 and 120 dB, when these three intensities of sound were used randomly. The same results were obtained when one of these three intensities was used repeatedly. In addition, pretreatment with diazepam (i.p.) at doses of 0.7 and 2.0 mg/kg suppressed the ASR of the pheromone recipients, whereas the lower dose (0.2 mg/kg) slightly attenuated the pheromone effect and the control injection (vehicle) had no effect. These results indicate that the alarm pheromone enhances the ASR by increasing anxiety in recipient rats, suggesting that the primary effect of the alarm pheromone is to increase the anxiety level.     

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.     

Single nucleotide polymorphisms as a prerequisite for autoantigens

It is still elusive why certain self proteins induce an autoimmune response. One immunological hypothesis is that only modified or altered self-proteins may become a target. Thus, we asked whether such alterations may actually be genetic polymorphisms that can be revealed by analyzing sequence variability in the known human autoantigens. Indeed, we found autoantigens to contain significantly more single nucleotide polymorphisms (SNP) than other human genes do. Our finding may offer an explanation for autoimmune responses through allogeneic exposure. Besides other contributing factors in autoimmunity, SNP may represent an essential prerequisite for the primary induction of an autoimmune response.     

Prenatal stress alters cardiovascular responses in adult rats

Environmental factors in early life are clearly established risk factors for cardiovascular disease in later life. Most studies have focused on nutritional programming and analysed basal cardiovascular parameters rather than responses. In the present study we have investigated whether prenatal stress has long-term effects on cardiovascular responses in adult offspring. Female pregnant Sprague-Dawley rats were subjected to stress three times daily from day 15 to day 21 of gestation. Litters from stressed and control females were cross-fostered at birth to control for mothering effects. When the offspring were 6 months old, blood pressure was measured in the conscious rats through implanted catheters at rest, during restraint stress and during recovery. Basal haemodynamic parameters were similar in the different groups but the pattern of cardiovascular responses during stress and recovery differed markedly between prenatally stressed (PS) and control animals. PS rats had higher and longer-lasting systolic arterial pressure elevations to restraint stress than control animals. They also showed elevated systolic and diastolic blood pressure values during the recovery phase. PS rats demonstrated a greater increase in blood pressure variability compared with control animals during exposure to restraint stress, and showed more prolonged heart rate responses to acute stress and delayed recovery than controls. There was no effect of prenatal stress on baroreflex regulation of heart rate. PS females showed a greater increase in systolic arterial pressure and blood pressure variability and delayed heart rate recovery following return to the home cage then did PS males. These findings demonstrate for the first time that prenatal stress can induce long-term, sex-related changes in the sensitivity of the cardiovascular system to subsequent stress.     

Genome-wide patterns of gene flow across a house mouse hybrid zone

Hybrid zones between closely related species or subspecies provide useful settings for studying the genetic architecture of speciation. Using markers distributed throughout the mouse genome, we use a hybrid zone between two recently diverged species of house mice (Mus musculus and Mus domesticus) as a natural mapping experiment to identify genomic regions that may be involved in reproductive isolation. Using cline analysis we document a nearly 50-fold variation in level of introgression among markers. Some markers have extremely narrow cline widths; these genomic regions may contribute to reproductive isolation. Biological processes associated with these narrow clines include physiological and immune responses to the environment as well as physiological and behavioral aspects of reproduction. Other autosomal markers exhibit asymmetrically broad clines, usually with high frequencies of M. domesticus alleles on the M. musculus side of the hybrid zone. These markers identify genome regions likely housing genes with alleles that are spreading from one species to the other. Biological processes associated with these wide clines include cell signaling, olfaction, and pheromone response. These processes play important roles in survival and reproduction, and associated genes are likely targets of selection. Patterns of linkage disequilibrium in the center of the hybrid zone suggest that isolation may be caused by multiple epistatic interactions between sets of genes. These data highlight the complex genetic architecture underlying speciation even at early stages of divergence and point to some of the biological processes that may govern this architecture.     

Effect of Fusarium verticillioides on maize-root-associated Burkholderia cenocepacia populations

Burkholderia cepacia complex (Bcc) bacteria are naturally present in the rhizosphere of several crop plants and have been found to antagonize a wide range of important plant pathogens. In this study, we evaluated the effect of the pathogenic fungus Fusarium verticillioides on Bcc populations recovered from the roots of Zea mays plants. Maize plants were cultivated under greenhouse conditions and bacterial colonies were randomly isolated from distinct root portions of Fusarium-treated and control plants. We obtained a total of 120 Bcc isolates which all belonged to the species Burkholderia cenocepacia, a species of the Bcc widely distributed in natural habitats such as the rhizosphere of several crop plants. Results obtained revealed that the presence of the plant pathogen F. verticillioides had an effect at the root colonization level of B. cenocepacia populations, since an increase in indigenous B. cenocepacia bacteria was found in the rhizospheres of maize plants grown in infested soil, compared to the rhizospheres of control plants. The analysis of diversity indices as well as the investigation of genetic polymorphism of B. cenocepacia strains, isolated from Fusarium-treated and control root portions, revealed greater genetic variability in the presence of F. verticillioides, especially in the terminal root system portion. Finally, all B. cenocepacia isolates were also tested for in vitro inhibition of F. verticillioides growth as a functional property. Our results revealed that all B. cenocepacia isolates were able to restrict in vitro fungal growth, suggesting that there was no relationship between genetic polymorphism and biocontrol traits.     

Pheromone response in fission yeast: modulation by pat1

Summary The pat1 gene, which encodes an essential protein kinase, was identified in temperature-sensitive mutants in which, at a nonpermissive temperature, copious sporulation occurred irrespective of mating type and ploidy. In addition, at a semi-permissive temperature, conjugation was uncoupled from nutritional control, although it still required both mating types. To further characterize this intermediate effect on conjugation, we studied pheromone production and response as influenced by a pat1 mutation. Using microscopic pheromone assay conducted under starvation conditions, we observed that the pat1-114 mutant allele exerted a strong sensitizing effect towards the complementary pheromone in both mating types even at the low temperature of 23°C. The production of the pheromone was affected as well, being strongest in the P cells. Presumably the pat1 protein kinase can be tuned in to various levels of activity and thereby is able to function as a universal inhibitor of sexual differentiation in S. pombe.     

Intergenerational transfers may have decoupled physiological and chronological age in a eusocial insect

Life-history theory generally predicts that there should be no selection for longevity beyond the limit of reproductive capacity. However, the capacity to increase fitness may not end when individuals reach a state of functional sterility. Recent studies show that intergenerational transfers of resources from post-reproductive parents can increase the offspring's fitness, and analytical theory shows that age-trajectories of transfers may shape the course of senescence in social organisms. In eusocial insects, female roles are partitioned so that one phenotype or "caste" reproduces while another is responsible for resource transfers: the reproductive "queens" are arrested in a continuous reproductive mode, while transfer-activities such as hygienic behaviors, guarding, foraging and further food processing ("nursing") that increases the nutritional value of provisions are conducted by sterile "workers". Worker honey bees normally perform these tasks in a sequence so that nursing inside the protected nest is conducted prior to more risky exterior hive activities such as guarding and foraging. However, foragers may revert to nurse-activity in response to demographic changes, and worker bees can also develop into a stress resistant survival form with a 10-fold increase in lifespan. This elastic division of parental functions is believed to increase colony fitness. Further, it generates a stage-dependent trajectory of senescence that is difficult to address with established theories of aging. In the following, we show how a recent theory that includes resource transfers can be used to elucidate patterns of senescence in eusocial, non-reproducing individuals like the honey bee worker.     

Side-specific olfactory conditioning leads to more specific odor representation between sides but not within sides in the honeybee antennal lobes

Honeybees can be trained to associate odorants to sucrose reward by conditioning the proboscis extension response. Using this paradigm, we have recently shown that bees can solve a side-specific task: they learn simultaneously to discriminate a reinforced odor A from a non-reinforced odor B at one antenna (A+B−) and the reversed problem at the other antenna (A−B+). Side-specific (A+B−/B+A−) conditioning is an interesting tool to measure neurophysiological changes due to olfactory learning because the same odorant is excitatory (CS+) on one brain side and inhibitory (CS−) on the opposite side. In the bee brain, the antennal lobe (AL) is the first olfactory relay where the olfactory memory is established. Using calcium imaging, we compared odor-evoked activity in the functional units, the glomeruli, of the two ALs, both in naive and conditioned individuals. Each odor evoked a different pattern of glomerular activity, which was symmetrical between sides and highly conserved among naive animals. In conditioned bees, response patterns were overall symmetrical but showed more active glomeruli and topical differences between sides. By representing odor vectors in a virtual olfactory space whose dimensions are the responses of 23 identified glomeruli, we found that distances between odor representations on each brain side were significantly higher in conditioned than in naive bees, but only for CS+ and CS−. However, the distance between CS+ and CS− representations was equal to that of naive individuals. Our work suggests that side-specific conditioning decorrelates odor representations between AL sides but not between CS+ and CS− within one AL.     

Intrapopulational variation of olfactory responses inDrosophila melanogaster

The existence of genetic variability for olfactory response within natural populations ofDrosophila melanogaster was studied in two newly collected natural populations from two very different habitats, the Sandiche population, from a very heterogeneous environment, and the Los Areneros population, from a very homogeneous one. Intrapopulational variability was estimated over approximately 50 isofemale lines derived from each population. Results confirm significant differences in olfactory response to ethyl alcohol and acetaldehyde in both populations and to ethyl acetate in the Sandiche population. The differences were due partially to common components of the olfactory responses to different chemical (nonspecifics) in the Sandiche population, but they were specific for the stimulus in the Los Areneros population.     

Effects of Cross Fostering on Open-Field Behavior, Acoustic Startle, Lipopolysaccharide-Induced Corticosterone Release, and Body Weight in Lewis and Fischer Rats

Lewis (LEW/N) and Fischer (F344/N) rats differ on a myriad of behavioral and physiological endpoints, some of which have been reported to be affected by maternal experience in outbred rats and other strains. To assess whether epigenetic factors contribute to the differential behavioral responses to stress and pro-inflammatory challenges in these strains, the effects of cross fostering on open-field, acoustic startle, and glucocorticoid reactivity to lipopolysaccharide (LPS) were examined in the present experiment. In the open-field test, although in-fostered female LEW/N and F344/N strains did not differ, female LEW/N rats displayed significantly greater activity than female F344/N rats in the cross-fostered condition. Differences between males of the two strains were increased by cross fostering, with the LEW/N strain displaying greater total activity. In acoustic startle, there was little strain difference between in-fostered or cross-fostered female rats. On the other hand, in-fostered male LEW/N rats had a significantly greater startle response than in-fostered male F344/N rats, an effect that was dramatically reduced by cross fostering. In-fostered female LEW/N rats displayed a blunted corticosterone response relative to in-fostered female F344/N rats, an effect that was reduced by cross fostering. Conversely, although there was no strain difference between male in-fostered rats, cross-fostered male F344/N rats displayed a significantly greater corticosterone response to LPS than cross-fostered male LEW/N rats. Finally, body weight differences between in-fostered LEW/N and F344/N rats were reduced by cross fostering. Together, these data illustrate that maternal factors play a role in the behavioral and physiological responses to stress between the two strains.