Vitellogenin expression in the ovaries of adult honeybee workers provides insights into the evolution of reproductive and social traits.

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin-signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non-reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co-opted to regulate behavioural differences between queens and workers.

Extreme polyandry aids the establishment of invasive populations of a social insect.

Although monandry is believed to have facilitated the evolution of eusociality, many highly eusocial insects have since evolved extreme polyandry. The transition to extreme polyandry was likely driven by the benefits of within-colony genetic variance to task specialization and/or disease resistance, but the extent to which it confers secondary benefits, once evolved, is unclear. Here we investigate the consequences of extreme polyandry on the invasive potential of the Asian honey bee, Apis cerana. In honey bees and other Hymenoptera, small newly founded invasive populations must overcome the genetic constraint of their sex determination system that requires heterozygosity at a sex-determining locus to produce viable females. We find A. cerana queens in an invasive population mate with an average of 27 males (range 16-42) that would result in the founding queen/s carrying 75% of their source population's sex alleles in stored sperm. This mating frequency is similar to native-range Chinese A. cerana (mean 29 males, range 19-46). Simulations reveal that extreme polyandry reduces the risk, relative to monandry or moderate polyandry, that colonies produce a high incidence of inviable brood in populations that have experienced a founder event, that is, when sex allele diversity is low and/or allele frequencies are unequal. Thus, extreme polyandry aids the invasiveness of A. cerana in two ways: (1) by increasing the sex locus allelic richness carried to new populations with each founder, thereby increasing sex locus heterozygosity; and (2) by reducing the population variance in colony fitness following a founder event.

Queen pheromone regulates programmed cell death in the honey bee worker ovary.

In social insect colonies the presence of a queen, secreting her pheromones, is a key environmental cue for regulating the reproductive state of workers. However, until recently the proximate molecular mechanisms underlying facultative worker sterility were unidentified. Studies into worker oogenesis in the honey bee (Apis mellifera) have indicated that programmed cell death is central to the regulation of oogenesis. Here we investigate how queen pheromone, age of the worker and ovary state affect both programmed cell death and cell number in worker ovaries. We describe a novel method to simultaneously measure programmed cell death (caspase activity) and live cell number (estimated from the amount of adenosine triphosphate) in an insect tissue. Workers exposed to queen pheromone have higher levels of caspase activity in the ovary than those not exposed. Our results suggest that queen pheromone triggers programmed cell death at the mid-oogenesis checkpoint causing the abortion of worker oocytes and reproductive inhibition of the worker caste. Nonetheless, high caspase activity is present in activated ovaries from workers not exposed to queen pheromone. This caspase activity is most likely to be from the nurse cells undergoing programmed cell death, in late oogenesis, for normal oocyte development. Our study shows that the social environment of an organism can influence programmed cell death within a tissue.

Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana.

Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species.

Inheritance of thelytoky in the honey bee Apis mellifera capensis.

Asexual reproduction via thelytokous parthenogenesis is widespread in the Hymenoptera, but its genetic underpinnings have been described only twice. In the wasp Lysiphlebus fabarum and the Cape honey bee Apis mellifera capensis the origin of thelytoky have each been traced to a single recessive locus. In the Cape honey bee it has been argued that thelytoky (th) controls the thelytoky phenotype and that a deletion of 9 bp in the flanking intron downstream of exon 5 (tae) of the gemini gene switches parthenogenesis from arrhenotoky to thelytoky. To further explore the mode of inheritance of thelytoky, we generated reciprocal backcrosses between thelytokous A. m. capensis and the arrhenotokous A. m. scutellata. Ten genetic markers were used to identify 108 thelytokously produced offspring and 225 arrhenotokously produced offspring from 14 colonies. Patterns of appearance of thelytokous parthenogenesis were inconsistent with a single locus, either th or tae, controlling thelytoky. We further show that the 9 bp deletion is present in the arrhenotokous A. m. scutellata population in South Africa, in A. m. intermissa in Morocco and in Africanized bees from Brazil and Texas, USA, where thelytoky has not been reported. Thus the 9 p deletion cannot be the cause of thelytoky. Further, we found two novel tae alleles. One contains the previously described 9 bp deletion and an additional deletion of 7 bp nearby. The second carries a single base insertion with respect to the wild type. Our data are consistent with the putative th locus increasing reproductive capacity.

Why acquiesce? Worker reproductive parasitism in the Eastern honeybee (Apis cerana).

Most societies are vulnerable to rogue individuals that pursue their own interests at the expense of the collective entity. Societies often protect themselves from selfish behaviour by 'policing', thereby enforcing the interests of the collective over those of individuals. In insect societies, for example, selfish workers can activate their ovaries and lay eggs, exploiting the collective brood rearing system for individual benefit. Policing, usually in the form of oophagy of worker-laid eggs, controls selfish behaviour. Importantly, once an effective system of policing has evolved, the incentive for personal reproduction is lost, and 'reproductive acquiescence' in which ovary activation is rare or absent is predicted to evolve. Studies of social Hymenoptera have largely supported the prediction of worker 'acquiescence'; workers of most species where policing is well developed have inactive ovaries. However, the eastern honeybee Apis cerana appears to be an exception. A. cerana colonies are characterized by highly efficient policing, yet about 5% of workers have active ovaries, even when a queen is present. This suggests that the evolution of acquiescence is incomplete in A. cerana. We regularly sampled male eggs and pupae from four A. cerana colonies. Workers had high levels of ovary activation overall (11.7%), and 3.8% of assignable male eggs and 1.1% of assignable male pupae were worker-laid. We conclude that workers with active ovaries lay their eggs, but these rarely survive to pupation because of intense policing. We then used our findings as well as previously published data on A. cerana and A. mellifera to redo the meta-analysis on which reproductive acquiescence theory is based. Including data on both species did not affect the relationship between effectiveness of policing and levels of worker reproduction. Their inclusion did, however, seriously weaken the relationship between relatedness among workers and levels of worker reproduction. Our work thus suggests that relatedness among workers does not affect the probability that workers will attempt to reproduce, but that it is coercion by peers that limits worker reproduction.

Variable virulence among isolates of Ascosphaera apis: testing the parasite-pathogen hypothesis for the evolution of polyandry in social insects.

The queens of many eusocial insect species are polyandrous. The evolution of polyandry from ancestral monoandry is intriguing because polyandry undermines the kin-selected benefits of high intracolonial relatedness that are understood to have been central to the evolution of eusociality. An accumulating body of evidence suggests that polyandry evolved from monoandry in part because genetically diverse colonies better resist infection by pathogens. However, a core assumption of the "parasite-pathogen hypothesis", that there is variation in virulence among strains of pathogens, remains largely untested in vivo. Here, we demonstrate variation in virulence among isolates of Ascosphaera apis, the causative organism of chalkbrood disease in its honey bee (Apis mellifera) host. More importantly, we show a pathogen-host genotypic interaction for resistance and pathogenicity. Our findings therefore support the parasite-parasite hypothesis as a factor in the evolution of polyandry among eusocial insects.

Biogenic amine receptor gene expression in the ovarian tissue of the honey bee Apis mellifera.

In the honey bee Apis mellifera loss of the queen from a colony induces increased levels of the biogenic amine dopamine in the brain of workers, and this elevation is correlated with ovary activation. In the present study we use real-time quantitative PCR to investigate expression of five biogenic amine receptor genes. We show that biogenic amine receptors are expressed in ovarian tissue, and that their expression is strongly influenced by the presence or absence of a queen in the colony. In contrast to the brain, where all three dopamine receptors are expressed, only two dopamine receptors are expressed in the ovaries, and their expression is strongly correlated with the reproductive status of workers. We conclude that biogenic amine receptors are expressed in the ovaries and are likely to be directly influential in the regulation of worker sterility in honey bees.

Expression of genes related to reproduction and pollen foraging in honey bees (Apis mellifera) narcotized with carbon dioxide.

It has been proposed that a honey bee (Apis mellifera) worker's preference for foraging for pollen or nectar is modulated by a gene network that was originally involved in regulating the reproductive cycles of an ancestral solitary species. We used carbon dioxide to induce narcosis in queens and workers. This treatment is known to initiate oogenesis in queens, reduce oogenesis in queenless workers and to change worker foraging preference. We then assessed changes in gene expression of genes suspected to be involved in either foraging behaviour or reproduction. We show that some genes change expression in the opposite direction between castes in response to treatment. Our results therefore support the hypothesis that reproductive and foraging traits are causally related in the honey bee.

Hybridization between European and africanized honey bees in the neotropical yucatan peninsula.

A population genetic analysis of honey bees of the Mexican neotropical Yucatan peninsula shows that the range expansion of Africanized bees there has involved extensive introgressive hybridization with European bees. Yucatan honey bee populations now include many colonies with intermediate morphologies. Genotypes of mitochondria have disassociated from historically correlated Africanized or European morphology, producing diverse phenotypic associations. This suggests that the size of resident European populations may be important in explaining previously reported asymmetrical hybridization. Evidence of natural hybridization is encouraging for the use of genetic management to mitigate the effects of Africanized bees in the United States.

Mitochondrial sequencing reveals five separate origins of 'black' Apis mellifera (Hymenoptera: Apidae) in eastern Australian commercial colonies.

Establishment of a closed population honey bee, Apis mellifera L. (Hymenoptera: Apidae), breeding program based on 'black' strains has been proposed for eastern Australia. Long-term success of such a program requires a high level of genetic variance. To determine the likely extent of genetic variation available, 50 colonies from 11 different commercial apiaries were sequenced in the mitochondrial cytochrome oxidase I and II intergenic region. Five distinct and novel mitotypes were identified. No colonies were found with the A. mellifera mellifera mitotype, which is often associated with undesirable feral strains. One group of mitotypes was consistent with a caucasica origin, two with carnica, and two with ligustica. The results suggest that there is sufficient genetic diversity to support a breeding program provided all these five sources were pooled.

Genome-wide analysis of genes related to ovary activation in worker honey bees.

A defining characteristic of eusocial animals is their division of labour into reproductive and nonreproductive specialists. Here, we used a microarray study to identify genes associated with functional sterility in the worker honey bee Apis mellifera. We contrasted gene expression in workers from a functionally sterile wild-type strain with that in a mutant (anarchist) strain selected for high rates of ovary activation. We identified a small set of genes from the brain (n = 7) and from the abdomen (n = 5) that are correlated in their expression with early stages of ovary activation. Sterile wild-type workers up-regulated two unknown genes and a homologue of Drosophila CG6004. By contrast, reproductive anarchist workers up-regulated genes for the yolk protein vitellogenin, venom peptides and a member of the AdoHycase superfamily, among others. The differentially expressed genes identified are likely to be involved in early differentiation into sterile and reproductive worker phenotypes and may therefore form part of the gene networks associated with the regulation of honey bee worker sterility. Our study may have lacked sufficient power to detect all but a minority of biologically relevant changes taking place; however, the differential expression of vitellogenin and a putative AdoHycase suggests that our screen has captured core reproductive genes and that ovary activation may involve an epigenetic mechanism.

Multiple paternity or multiple queens: two routes to greater intracolonial genetic diversity in the eusocial Hymenoptera.

Understanding the evolution of multiple mating by females (polyandry) is an important question in behavioural ecology. Most leading explanations for polyandry by social insect queens are based upon a postulated fitness benefit from increased intracolonial genetic diversity, which also arises when colonies are headed by multiple queens (polygyny). An indirect test of the genetic diversity hypotheses is therefore provided by the relationship between polyandry and polygyny across species, which should be negative if the genetic diversity hypotheses are correct. Here, we conduct a powerful comparative investigation of the relationship between polyandry and polygyny for 241 species of eusocial Hymenoptera (ants, bees and wasps). We find a clear and significant negative relationship between polyandry and polygyny after controlling for phylogeny. These results strongly suggest that fitness benefits resulting from increased intracolonial genetic diversity have played an important role in the evolution of polyandry, and possibly polygyny, in social insects.

Factors affecting the dynamics of the honeybee (Apis mellifera) hybrid zone of South Africa.

Hybrid zones are found wherever two populations distinguishable on the basis of heritable characters overlap spatially and temporally and hybridization occurs. If hybrids have lower fitness than the parental types a tension zone may emerge, in which there is a barrier to gene flow between the two parental populations. Here we discuss a hybrid zone between two honeybee subspecies, Apis mellifera capensis and A. m. scutellata and argue that this zone is an example of a tension zone. This tension zone is particularly interesting because A. m. capensis can be a lethal social parasite of A. m. scutellata. However, despite its parasitic potential, A. m. capensis appears to be unable to increase its natural range unassisted. We propose three interlinked mechanisms that could maintain the South African honeybee hybrid zone: (1) low fitness of intercrossed and genetically mixed colonies arising from inadequate regulation of worker reproduction; (2) higher reproductive success of A. m. scutellata via both high dispersal rates into the hybrid zone and increased competitiveness of males, countered by (3) the parasitic nature of A. m. capensis.

Effects of procedure, upright equilibrium time, sex and BMI on the precision of body fluid measurements using bioelectrical impedance analysis.

BACKGROUND/OBJECTIVES: Extensive work has addressed the validity of bioimpedance (BIA) measurements and the effect of posture on fluid homeostasis. However, limited research has investigated effects of subject preparation. This study aimed to determine the precision of total body water (TBW) and extracellular water (ECW) measurements using a stand-on multifrequency BIA (MFBIA seca mBCA 514/515), in three pre-test procedures: supine, sitting, and following walking, with specific reference to the influence of sex and body mass index (BMI). SUBJECTS/METHODS: Fifty three healthy, ambulatory men (n=26, age:32.5±9.4 years) and women (n=27, age:35.2±10.3 years) received repeat MFBIA measurements (six measurements from 0 to 15 min). Agreement and precision were evaluated for each condition and paired time points. RESULTS: Significant TBW sex differences from supine posture were observed for walking (females) and sitting (males) postures. For BMI (⩽24.9 kg m-2) significant TBW differences from supine were observed for both sitting and walking and significant ECW differences from sitting were also observed with both supine and walking. There was no significant effect of sex or BMI (⩾25.0 kg m-2) on ECW measures. Irrespective of sex or BMI, there was close agreement in TBW and ECW precision over the three protocols. CONCLUSIONS: Practitioners can have confidence in the precision of TBW and ECW measurements within a 15 min time period and pre-testing conditions (supine, sitting or walking) in healthy subjects, though must be cautious in assessments when pre-test postures change. Further research to examine the impact of pre-testing procedures on stand-on MFBIA BIA measurements, including subjects with fluid disturbance, is warranted.

Differences in bone density, body composition, physical activity, and diet between child gymnasts and untrained children 7-8 years of age.

Strategies that enhance the acquisition of bone mass may be protective against osteoporosis. BMD was compared in 20 artistic gymnasts (10 boys; 10 girls) and 20 untrained children ages 7-8 years. Higher regional values of BMD were observed in female gymnasts than untrained girls. If retained to adulthood, this higher BMD may protect skeletal integrity in later life. Strategies that enhance the acquisition of bone mass in children may assist with the prevention of osteoporosis. This study explored the effects of regular high-impact and weight-bearing activity before the age of 7 years on total and regional bone mineral density (BMD). Twenty artistic gymnasts (10 boys and 10 girls) and 20 untrained children, 7-8 years of age, were recruited. The untrained children were matched to gymnasts by sex, height, weight, and age. Female gymnasts trained 8-10 h per week and had trained regularly for 3-4 years. Male gymnasts trained 4-6 h per week and had trained for 1-2 years. Measurements of bone mineral density were made using DXA for total body BMD (TBBMD); lumbar spine, both areal (aSBMD) and volumetric (vSBMD); total spine; pelvis; arms; and legs. Significant mean differences (8-10%) in aSBMD, vSBMD, arm BMD, and TBBMD were observed between female gymnasts and untrained girls (p < 0.05: aSBMD, vSBMD, and TBBMD body mass (BM); p < 0.01: arm BMD). A nonsignificant trend toward a higher TBBMD/BM and arm BMD was observed in male gymnasts compared with untrained boys. Trends toward a higher BMD within the pelvis, legs, and total spine were also observed in gymnasts. There were no differences in total and regional BMD between untrained boys and untrained girls. The results suggest that gymnastics training before the age of 7 years enhances the acquisition of bone mass at selected skeletal sites. The magnitude of this enhancement seems to be linked to the cumulative volume of such training. If retained during adolescence and young adulthood, a surfeit of bone acquired through high-impact and weight-bearing activity in early childhood may protect skeletal integrity in later life.

Influence of changes in peritoneal fluid on body-composition measurements by dual-energy X-ray absorptiometry in patients receiving continuous ambulatory peritoneal dialysis.

The effect of the presence of intraabdominal fluid on measurement of body composition by dual-energy X-ray absorptiometry (DXA) was determined by scanning 14 patients who were receiving continuous ambulatory peritoneal dialysis (CAPD) before and after the drainage of peritoneal dialysate, resulting in a mean (+/- SD) weight loss of 1.81 +/- 0.34 kg. DXA showed changes in whole-body soft tissue, which were correctly localized to the trunk region on regional analysis. Changes in DXA estimates of mean total lean tissue, 1.78 +/- 0.94 kg, and trunk lean tissue, 1.87 +/- 0.84 kg, were very similar to the actual change in body weight. However, the DXA estimate of change in total soft tissue, 2.11 +/- 0.44 kg, was significantly greater than the change in body weight by 0.3 kg (P < 0.005); the difference was accounted for by a tendency for a reduction in the estimate of body fat affecting the trunk region after drainage of dialysate. DXA was also less good at detecting changes in individual subjects. Estimates of total and regional bone mineral content and bone mineral density were not affected by the drainage of the dialysis fluid.

Comparison of body composition methods in overweight and obese children.

The objective of the present study was to investigate the accuracy of percent body fat (%fat) estimates from dual-energy X-ray absorptiometry, air-displacement plethysmography (ADP), and total body water (TBW) against a criterion four-compartment (4C) model in overweight and obese children. A volunteer sample of 30 children (18 male and 12 female), age of (mean +/- SD) 14.10 +/- 1.83 yr, body mass index of 31.6 +/- 5.5 kg/m, and %fat (4C model) of 41.2 +/- 8.2%, was assessed. Body density measurements were converted to %fat estimates by using the general equation of Siri (ADPSiri) (Siri WE. Techniques for Measuring Body Composition. 1961) and the age- and gender-specific constants of Lohman (ADPLoh) (Lohman TG. Exercise and Sport Sciences Reviews. 1986). TBW measurements were converted to %fat estimates by assuming that water accounts for 73% of fat-free mass (TBW73) and by utilizing the age- and gender-specific water contents of Lohman (TBWLoh). All estimates of %fat were highly correlated with those of the 4C model (r > or = 0.95, P < 0.001; SE < or = 2.14). For %fat, the total error and mean difference +/- 95% limits of agreement compared with the 4C model were 2.50, 1.8 +/- 3.5 (ADPSiri); 1.82, -0.04 +/- 3.6 (ADPLoh); 2.86, -2.0 +/- 4.1 (TBW73); 1.90, -0.3 +/- 3.8 (TBWLoh); and 2.74, 1.9 +/- 4.0 DXA (dual-energy X-ray absorptiometry), respectively. In conclusion, in overweight and obese children, ADPLoh and TBWLoh were the most accurate methods of measuring %fat compared with a 4C model. However, all methods under consideration produced similar limits of agreement.

Segmental bioelectrical impedance in patients with chronic renal failure.

We studied changes in hydration by whole body and segmental (arm, leg and trunk)bioelectrical impedance analysis (BIN in patients with chronic renal failure (CRF) undergoing haemodialysis and continuous ambulatory peritoneal dialysis (CAPD). Mean (SD) fluid removal by haemodialysis of 1.38 (0.81) kg was overestimated by whole body BIA at 1.83 (1.13) I, P < 0.005. Peritoneal fluid drained from the CAPD patients of 1.88 (0.36) kg was underestimated by whole body BIA at 0.59 (0.35) I, P < 0.0001. Resistance and reactance significantly increased for the whole body and all segments (except trunk reactance) after haemodialysis. Drainage of CAPD fluid resulted in smaller increases in trunk resistance and whole body resistance. The increase in trunk resistance was less in CAPD than haemodialysis patients, even though the volume of fluid drained from the peritoneum in CAPD patients exceeded that removed from the whole body during haemodialysis. We conclude that whole body BIA does not estimate changes in body fluid with sufficient accuracy to be of use in clinical practice. Segmental impedance may be a potentially useful method for investigation of regional changes in body fluid, though is insensitive to changes within the peritoneal cavity.