Sex mosaics in the honeybee: how haplodiploidy makes possible the evolution of novel forms of reproduction in social Hymenoptera.

Hymenoptera are haplodiploid: females arise from fertilized, diploid eggs, while males arise from unfertilized, haploid eggs. The cytogenetic mechanisms underlying haplodiploidy enable remarkable phenomena including female cloning, male cloning and gynandromorphy (sex mosaics). We collected 11 newly emerged putative gynandromorph honeybees from a single colony, assessed the sex of various tissues morphologically and determined the genetic origin (maternal or paternal) of each tissue by genotyping. Ten bees were gynandromorphs with one to three distinct paternal origins. Remarkably, one bee carried no maternal alleles. This bee had female organs throughout, and arose from the fusion of two sperm nuclei. This is the first reported case in the Hymenoptera of sperm fusion resulting in a female, emphasizing the flexibility for social insect reproduction and potentially novel colony-level social structures.

Allocation of endogenous and dietary protein in the reconstitution of the gastrointestinal tract in migratory blackcaps at stopover sites.

During migratory flight, the mass of the gastrointestinal tract (GIT) and its associated organs in small birds decreases in size by as much as 40%, compared with the preflight condition because of the catabolism of protein. At stopover sites, birds need 2-3 days to rebuild their GIT so that they can restore body mass and fat reserves to continue migration. The source of protein used to rebuild the GIT may be exogenous (from food ingested) or endogenous (reallocated from other organs) or both. Because the relative contribution of these sources to rebuild the GIT of migratory birds is not yet known, we mimicked in-flight fasting and then re-feeding in two groups of blackcaps (Sylvia atricapilla), a Palearctic migratory passerine. The birds were fed a diet containing either 3% or 20% protein to simulate different refueling scenarios. During re-feeding, birds received known doses of (15)N-(l)-leucine before we measured the isotope concentrations in GIT and associated digestive organs and in locomotory muscles. We then quantified the extent to which blackcaps rebuilt their GIT with endogenous and/or dietary protein while refeeding after a fast. Our results indicate that blackcaps fed the low-protein diet incorporated less exogenous nitrogen into their tissues than birds fed the 20% protein diet. They also allocated relatively more exogenous protein to the GIT than to pectoral muscle than those birds re-fed with the high-protein diet. However, this compensation was not sufficient for birds eating the low-protein diet to rebuild their intestine at the same rate as the birds re-fed the high-protein diet. We concluded that blackcaps must choose stopover sites at which they can maximize protein intake to minimize the time it takes to rebuild their GIT and, thus, resume migration as soon as possible.

Reproductive plasticity and oogenesis in the queen honey bee (Apis mellifera).

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2000 eggs daily whereas workers are sterile in the queen's presence. Periodically queens stop laying: during swarming, when resources are scarce in winter, and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in the ovaries of honey bee queens that were caged inside and outside the colony. We find evidence that queens use a different combination of 'checkpoints' to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes likely use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that a key factor driving the termination of oogenesis in queens is nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but likely have adjusted physiological checkpoints to suit their highly-derived life history.

A Single Gene Causes Thelytokous Parthenogenesis, the Defining Feature of the Cape Honeybee Apis mellifera capensis.

In honeybees, the ability of workers to produce daughters asexually, i.e., thelytokous parthenogenesis, is restricted to a single subspecies inhabiting the Cape region of South Africa, Apis mellifera capensis. Thelytoky has unleashed new selective pressures and the evolution of traits such as social parasitism, invasiveness, and social cancer. Thelytoky arises from an abnormal meiosis that results in the fusion of two maternal pronuclei, restoring diploidy in newly laid eggs. The genetic basis underlying thelytoky is disputed. To resolve this controversy, we generated a backcross between thelytokous A. m. capensis and non-thelytokous A. m. scutellata from the neighboring population and looked for evidence of genetic markers that co-segregated with thelytokous reproduction in 49 backcross females. We found that markers associated with the gene GB45239 on chromosome 11, including non-synonymous variants, showed consistent co-segregation with thelytoky, whereas no other region did so. Alleles associated with thelytoky were present in all A. m. capensis genomes examined but were absent from all other honeybees worldwide including A. m. scutellata. GB45239 is derived in A. m. capensis and has a putative role in chromosome segregation. It is expressed in ovaries and is downregulated in thelytokous bees, likely because of polymorphisms in the promoter region. Our study reveals how mutations affecting the sequence and/or expression of a single gene can change the reproductive mode of a population.

Viable Triploid Honey Bees (Apis mellifera capensis) Are Reliably Produced in the Progeny of CO2 Narcotised Queens.

The haplodiploid system of sex determination of Hymenoptera acts as an exaptation for species to evolve novel forms of asexual reproduction including thelytoky (clonal offspring of the mother). During normal reproduction in Hymenoptera, three of the four products of meiosis that are present in newly-laid eggs are lost as polar bodies, while the remaining pronucleus either develops as a haploid male or fuses with a sperm nucleus to produce a diploid zygote. In contrast, in thelytokous reproduction, which is uncommon but taxonomically widespread, two of the four products of meiosis fuse, as if one acted as a sperm. Queenless workers of Apis mellifera capensis, a subspecies of honey bee from South Africa, routinely reproduce thelytokously. Unmated A. m. capensis queens can also be induced to lay thelytokously by narcosis with carbon dioxide, but mated queens are never thelytokous. We artificially inseminated A. m. capensis queens using CO2 narcosis. Up to 1/3 of offspring workers carried two maternal alleles and an allele of one father whereas no three-allele progeny were seen in control queens of the arrhenotokous (unfertilized eggs result in males) subspecies A. m. scutellata Flow cytometry of three-allele individuals revealed that they were triploid and arose from the fertilization of a thelytokous fusion nucleus. We then reared six queens from a narcotized A. m. capensis queen and determined the ploidy of the offspring queens based on microsatellites. One of the five daughters was triploid. Following artificial insemination, this queen produced unfertilized thelytokous diploid eggs at high frequency, and unfertilized triploid eggs at much lower frequency. If fertilized, thelytokous diploid eggs were non-viable, even though triploidy in itself does not impede normal development. In contrast, when the rarer triploid eggs were fertilized, a proportion developed into viable tetraploids. Our study highlights the extraordinary developmental flexibility of haplo-diploid systems.

During stopover, migrating blackcaps adjust behavior and intake of food depending on the content of protein in their diets.

During migration, birds undergo alternating periods of fasting and re-feeding that are associated with dynamic changes in body mass (m(b)) and in organ size, including that of the digestive tract. After arrival at a migratory stopover site, following a long flight, a bird must restore the tissues of its digestive tract before it can refuel. In the present study we examined how the availability of dietary protein influences refueling of migrating blackcaps (Sylvia atricapilla) during a migratory stopover. We tested the following predictions in blackcaps deprived of food and water for 1-2 days to induce stopover behavior: (1) birds provided with a low-protein diet will gain m(b), lean mass and fat mass, and increase in pectoral muscle size slower than do birds fed a high-protein diet; (2) since stopover time is shorter in spring, birds will gain m(b) and build up fat tissue and lean tissue faster than in autumn; and (3) if low dietary protein limits a bird's ability to gain m(b) and fat reserves, then birds that do not obtain enough protein will initiate migratory restlessness (Zugunruhe) earlier than will birds with adequate dietary protein. These predictions were tested by providing captured migrating blackcaps with semisynthetic isocaloric diets differing only in their protein content. Each day, we measured m(b), and food intake; also lean mass and fat mass were measured using dual energy X-ray absorptiometry. In addition, we monitored nocturnal activity with a video recording system. In both spring and autumn, birds fed diets containing either 3 or 20% protein increased in m(b), lean mass and fat mass at similar rates during the experiment. However, the group receiving 3% protein ate more than did the group receiving 20% protein. In support of our predictions, m(b), lean mass, fat mass, and intake of food all were higher in spring than in autumn. We also found that in spring all birds had higher levels of migratory restlessness, but birds fed 3% protein were less active at night than were birds fed 20% protein, possibly an adaptation conserving energy and protein. We conclude that protein requirements of migrating blackcaps during stopover are lower than expected, and that birds can compensate for low dietary protein by behavioral responses, i.e. hyperphagia and decreased migratory restlessness, that ensure rapid refueling.

Availability of water affects renewal of tissues in migratory blackcaps during stopover.

Migrating blackcaps (Sylvia atricapilla) were used to test the predictions that (1) the rebuilding of the digestive tract, as reflected by mass-specific consumption of food on the first 2-3 days of a stopover, is faster in birds with access to drinking water than in birds without, and (2) that adipose tissue and pectoral muscles grow faster and to a greater extent in birds with unlimited access to water. We simulated migratory stopover in two experiments. In Experiment I, each of 31 birds was randomly assigned to one of three experimental groups for 6 days. Along with mealworms (∼64% water) ad libitum, Group 1 received drinking water ad libitum; Group 2 had 0.5 h/day access to water; and Group 3 had no access to water. In Experiment II, 30 birds were offered a mixed diet for insectivorous birds (∼33% water) ad libitum for 6 days, while randomly assigned to two groups: (1) Water ad libitum-control; and (2) 30 min access to water twice a day. We measured lean mass and fat mass using dual energy X-ray absorptiometry, as well as body mass (m(b)), pectoral muscle index (PMI), and daily intake of food and water. Mean daily water intake was significantly different among the groups in both experiments. However, the availability of drinking water positively affected the rates of gain of lean and fat mass only in birds fed with the mixed, relatively dry diet. Furthermore, mass-specific daily food intake was affected by the availability of drinking water only in the mixed diet experiment, in which birds with unlimited access to drinking water reached an asymptote, 1 day earlier than birds in the water-restricted group. We suggest that in birds consuming diets with low water content, the lack of sufficient drinking water may result in slower rebuilding of the digestive tract, or may influence biochemical processes in the gut that result in slower growth of tissue. Although blackcaps obtained sufficient water from preformed and metabolic water to renew lost tissues when eating mealworms, given access to water, the birds drank prodigiously. Our results also suggest that if drinking water is unavailable to migrating blackcaps, their choices are restricted to water-rich foods, which may constrain their rate of feeding and thus the rate at which they deposit fat. Consequently, drinking water may have an important influence on birds' migratory strategies with respect to habitat selection, use of energy, and the saving of time.