Worker Progeny Research Articles

Indirect exposure to insect growth disruptors affects honey bee (Apis mellifera) reproductive behaviors and ovarian protein expression.

Pesticide exposure and queen loss are considered to be major causes of honey bee colony mortality, yet little is known regarding the effects of regularly encountered agrochemicals on honey bee reproduction. Here, we present the results of a two-generational study using specialized cages to expose queens to commonly used insect growth disrupting pesticides (IGDs) via their retinue of worker bees. Under IGD exposure, we tracked queen performance and worker responses to queens, then the performance of the exposed queens' offspring was assessed to identify patterns that may contribute to the long-term health and stability of a social insect colony. The positive control, novaluron, resulted in deformed larvae hatching from eggs laid by exposed queens, and methoxyfenozide, diflubenzuron, and novaluron caused a slight decrease in daily egg laying rates, but this was not reflected in the total egg production over the course of the experiment. Curiously, eggs laid by queens exposed to pyriproxyfen exhibited increased hatching rates, and those larvae developed into worker progeny with increased responsiveness to their queens. Additionally, pyriproxyfen and novaluron exposure affected the queen ovarian protein expression, with the overwhelming majority of differentially expressed proteins coming from the pyriproxyfen exposure. We discuss these results and the potential implications for honey bee reproduction and colony health.

Highly efficient site-specific integration of DNA fragments into the honeybee genome using CRISPR/Cas9.

Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal, and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9-mediated homology-directed repair. We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly, 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.

Reproductive conflicts in polyandrous and polygynous ant Formica sanguinea

The occurrence of multiple reproductives within an ant colony changes the balance between indirect fitness benefits and reproductive competition. We test whether the number of matings by an ant queen (polyandry) correlates negatively with the number of reproductive queens in the colony (polygyny), whether the patrilines and matrilines differ in their contribution to the sexual and worker progeny and whether there is an overall reproductive skew. For these aims, we genotyped both worker and sexual offspring from colonies of the ant Formica sanguinea in three populations. Most colonies were monogynous, but eight (11%) were polygynous with closely related queens. Most queens in the monogynous colonies (86%) had mated with multiple males. The effective paternity was lower than the actual number of mates, and the paternity skew was significant. Furthermore, in some monogynous colonies, the patrilines were differently represented in the worker pupae and sexual pupae produced at the same time. Likewise, the matrilines in polygynous colonies were differently present in worker pupae and male offspring. The effective number of matings by a queen was significantly lower in polygynous colonies (mean m(e) = 1.68) than in monogynous colonies (means 2.06-2.61). The results give support to the hypotheses that polyandry and polygyny are alternative breeding strategies and that reproductive competition can lead to different representation of patrilines and matrilines among the sexual and worker broods.

Reproductive constraint is a developmental mechanism that maintains social harmony in advanced ant societies

A hallmark of eusociality in ants is the reproductive division of labor between queens and workers. Yet, nothing is known about the molecular mechanisms underlying reproduction in this group. We therefore compared the developmental genetic capacity of queens and workers to reproduce in several eusocially advanced species from the two largest subfamilies of ants, the Myrmicinae and Formicinae. In flies, the asymmetric localization of maternally encoded determinants (mRNAs and proteins) during oogenesis establishes oocyte polarity and subsequently ensures proper embryonic development. Vasa and nanos, two key maternal determinants, are properly localized in the posterior of queen oocytes, but their localization is impaired in those of the workers. This mislocalization leads to severe embryonic defects in worker progeny, and therefore, represents a constraint on worker reproduction that we call 'reproductive constraint.' We show that reproductive constraint is phylogenetically widespread, and is at high levels in most species tested. Reproductive constraint can simultaneously reduce or eliminate the workers' ability to produce viable eggs for reproduction, while preserving their ability to produce trophic eggs for nutrition, and thus, may have been the basis for the evolutionary retention of worker ovaries in the majority of ants. We propose that high levels of reproductive constraint has most likely evolved as a consequence of selection at the colony level to reduce or eliminate any potential conflict over worker reproduction, therefore maintaining harmony and colony efficiency in advanced ant societies.

Microgeographic genetic structure and intraspecific parasitism in the antLeptothorax nylanderi

Summary1. Genetic colony structure of the small central European antLeptothorax nylanderiis affected strongly by ecological constraints such as nest site availability and intraspecific social parasitism.2. AlthoughL. nylanderiis generally monogynous and monandrous, more than a quarter of all nests collected in a dense population near Würzburg, Germany, contained several matrilines. As shown by microsatellite analysis, the average nest‐mate relatedness in these nests was 0.20. Genetically heterogeneous nests arise from nest take‐over by alien colonies or founding queens, a result of severe competition for nest sites.3. In summer, more than one‐third of all colonies inhabited several nest sites at a time. Polydomy appears to be rather limited, with two or three nests belonging to a single polydomous colony.4. Queens appear to dominate male production; only a small fraction (8%) of males was definitively not progeny of the queen present but might have been worker progeny or offspring of another queen.5. Strong evidence for heterozygote deficiency was found and a total of nine diploid males was discovered in two colonies. These findings suggest deviation from random mating through small, localised nuptial flights.

Microsatellite analysis of sperm admixture in honeybee.

Sperm usage was investigated in an instrumentally inseminated honeybee queen. Her progeny were examined in the first 3 months of the egg-laying period using a microsatellite marker. Frequencies of different subfamilies differed significantly from one month to another. However, there was no evidence for sperm displacement or sperm precedence of a specific male in the worker progeny. The variance of subfamily proportions decreased over time suggesting that sperm admixture in the spermatheca was incomplete at the beginning of the egg-laying period of the queen and improved progressively during the first months after mating.

Heritability in Honey Bees (Hymenoptera: Apidae) of Characteristics Associated with Resistance to Varroa jacobsoni(Mesostigmata: Varroidae)

This study uses sibling analysis to measure the heritability in honey bees, Apis mellifera L., of characteristics that have been associated with resistance to the mite, Varroa jacobsoni Oudemans. Twenty-eight uniform colonies of bees were established on 13 May in Baton Rouge, LA, each with 1 kg of mite-infested bees and a queen. The 28 unrelated queens in these colonies were divided into 7 groups of 4 based on the insemination of 4 queens with the same mixture of semen from 1 of 7 sire colonies. After worker progeny from these queens had replaced the initial bee populations, a colony was related as a full sister to the other 3 colonies in its sire group and unrelated to the other 24 colonies. Heritability ( h 2) was 1.24 for proportion of mites in brood, 0.65 for hygienic behavior, 0.89 for the duration of the capped period, 0.46 for suppression of mite reproduction, and 0.00 for physical damage to mites (measured by the presence of physically broken or dented mites on the bottom board). These results suggest that it should be possible to enhance the expression of 4 of these 5 characteristics with selective breeding of bees, thus reinforcing confidence in our ability to breed honey bees for resistance to V. jacobsoni.

Ployandry versus polygyny versus parasites

Although social insect colonies are most easily conceptualized as consisting of a single, once–mated queen and her worker progeny, the number of queens per colony and the number of times queens mate varies broadly in ants and other social insects. Various hypotheses have been suggested for the resulting range of breeding systems and social organizations, respectively; one set of hypotheses relating to both queen number and mate number at the same time is a need for genetic variation, especially in relation to disease resistance. We here carry out a comparative analysis using phylogenetic information and, contrary to one non–phylogenetic previous study, we find that polyandry and polygyny are not significantly associated. However, the level of relatedness within colonies, a quantity affected by both polyandry and polygyny, is significantly associated with parasite loads: species with colonies with low relatedness levels have lower parasite loads. Given that, under the variance–reduction principle, selection on queens for mating frequency ought to continue even in polygynous colonies, we suggest that while parasite loads indeed seem to correlate with intra–colony genetic variability, the relationship to polyandry and polygyny may be complex and requires considerably more experimental investigation.

Variation in worker response to honey bee (Apis mellifera L.) queen mandibular pheromone (Hymenoptera: Apidae)

Genetic and environmental influences on the worker honey bee retinue response to queen mandibular gland pheromone (QMP) were investigated. Worker progeny were reared from queens originating from four sources: Australia, New Zealand, and two locations in British Columbia, Canada (Simon Fraser University and Vancouver Island). Progeny from New Zealand queens responded significantly higher (P < 0.05) than progeny from Australia in a QMP retinue bioassay. Retinue response was not related to queen production of pheromone or colony environment, and the strain-dependent differences in retinue bioassay responses were maintained over a wide range of dosages. Selected high- and low-responding colonies were bioassayed over the course of 1 year. High-responding colonies contacted QMP lures more frequently than low-responding colonies (P < 0.05) throughout the year except in late summer. We conclude that there is a strong genetic component to QMP response by worker honey bees, as well as a seasonal effect on response.

Corbicular Size in Workers from Honeybee Lines Selected for High or Low Pollen Hoarding

SummaryMeasurements were made of corbicular areas (of a total of 1025 corbiculae) in 2 lines of honeybees (Apis mellifera) selected for high and low pollen hoarding respectively. The sample from each line comprised about 30 newly emerged workers from each of 9 queens representing 3 sublines. Analyses of variance revealed highly significant differences among the 18 queens (P<lt; 0·0001) and between the lines (P<lt; 0·0001). Mean corbicular area for the high pollen-hoarding line (1·909 ± 0·004 mm2) was greater than for the low hoarding line (1 874 ± 0·003 mm2). Of the 9 queens whose worker progeny had the largest corbiculae, 7 belonged to the high pollen-hoarding line, A Mann-Whitney U-test of rank indicated that the distribution of the queens fur the 2 lines differed significantly (P < 0·05). Assuming the difference to be genetic, as indicated from a previous heritability estimate, unintentional selection for worker corbicular area must have been performed during selection for divergent pollen-hoarding abi...

An Estimate of the Heritability of the Corbicular Area of the Honeybee

SummaryHeritability (h2) of corbicular area in worker honeybees (Apis mellifera) was estimated from the variance components of half-sib families. Corbicular area was measured on 30 worker progeny from each of 73 free-mated queens which were the daughters of 8 mother queens. Heritability was estimated to be 1·014 ± 0·195. This large value for h2 indicates that selection for larger corbicular area is possible and, depending upon the genetic correlation, may result in concomitant selection for improved honey production.

An Estimate of the Heritability of Worker Longevity or Length of Life in the Honeybee

SummaryHeritability of longevity or length of life of worker honeybees (Apis mellifera) was estimated from the variance components of half-sib families. Measurements were made of the life-spans of worker progeny from 27 free-mated queens which were the daughters of 6 queens. The heritability of length of life-span was estimated to be 0·196 ± 0·024. Selection for longer life, therefore, appears feasible and a correlated increase in colony honey production, depending upon the magnitude of the genetic correlation between these two traits, should also be possible.

Preservation of Honeybee Spermatozoa in Liquid Nitrogen

SummaryHoneybee (Apis mellifera) semen was stored for 359 days at −196°C in liquid nitrogen. Eleven semen diluents were tested. Use of a mixture of 40% semen and 60% Kiev diluent containing 10% dimethylsulphoxide, combined with freezing of samples at the rate of 3–4°/min produced relatively good post-thaw sperm motility. Fifteen queens were inseminated instrumentally with semen stored for 305–359 days. Thirteen survived and produced a brood area of 345 ± 60 cm2, of which 47·3 ± 6·42% were worker progeny from the frozen spermatozoa. Four queens were superseded by their daughters which mated naturally and built up strong colonies.

Early Death of Newly Emerged Worker Honeybees in Laboratory Test Cages

SummaryNewly emerged worker progeny of certain queens exhibited an early period of death after being put into laboratory test cages. Examination of early death in the progeny of 82 queens demonstrated that: it occurred during days 1–8 in the cage; 0–22% of the cage population died during this period; among progeny of the median queen, an average of 2–0% of the bees died; there were significant differences in the incidence of early death among the progeny of different queens (P<0·001); the incidence of early death appeared to be repeatable. Early death in the cage could be virtually eliminated by providing young laying queens.

Length of life and Dry Weight of Worker Honeybees Reared in Colonies with Different Worker-Larva Ratios

SummarySmall colonies consisting of a caged queen and young adult workers, ranging in number from 200 to 2800, were given 400 eggs to rear. Positive correlations were found between the numbers of workers and the adult life-span and dry weight of worker progeny they reared. Dry weight and life-span were also positively correlated. It is suggested that a nutritional factor is involved. Syrup, pollen and water were provided in a flight cage.

Survival of Honey Bee Spermatozoa in Liquid Nitrogen1

Worker progeny were produced by queen honey bees ( Apis mellifera L.) inseminated with semen that had been stored in liquid nitrogen. The cryoprotectant was DMSO (dimethyl sulfoxide). Queen bees inseminated with a semen mixture (60% semen, 30% saline, and 10% DMSO) that was stored in liquid nitrogen retained 41% as many spermatozoa in their spermathecae as control queens.

Measuring the Heritability of Characters of Honeybees

SummaryParent-offspring regression, intra-sire regression of offspring on dam, and sibling analysis, were examined for their usefulness in estimating heritabilities of characters of the honeybee (Apismellifera). These examinations indicate the allowances required for sibling workers with a coefficient of relationship of 0·75, and provide an approach to evaluate the usefulness of selecting breeding queens on the basis of their response, although the aim is improvement of response in worker progeny. Consideration is given to the consequences of a character being an attribute of individual workers, of groups of workers, or of queens.