Reproductive Potential Accelerates Preimaginal Development of Rebel Workers in Apis mellifera

Rebel workers develop from eggs laid by the previous queen, before it went swarming and left the colony orphaned, until the emergence of a new queen. In contrast to normal workers developing in the queen’s presence, rebels are set to reproduce and avoid rearing of successive bee generations. They have more ovarioles in their ovaries, as well as more developed mandibular glands and underdeveloped hypopharyngeal glands, just like the queen. We posited that rebels are not only similar to queens in some anatomical features, but also develop in a shorter time in comparison to normal workers. Therefore, the aim of this study was to compare preimaginal development duration in rebel and normal workers. The results show that rebels, i.e., workers with a higher reproductive potential, had a significantly shorter preimaginal development period (mean ± SD, 19.24 ± 0.07 days) than normal workers (22.29 ± 0.32 days). Our result confirmed that workers who develop in a queen-less colony undergo a shorter preimaginal development than those in a queen-right colony.

Reproductive Potential Impacts Body Maintenance Parameters and Global DNA Methylation in Honeybee Workers (Apis mellifera L.)

The widely accepted hypothesis in life history evolution about the trade-off between fecundity and longevity is not confirmed by long-living and highly fecund queens in eusocial insects. The fact that the queens and facultatively sterile workers usually arise from genetically identical eggs but differ in DNA methylation makes them a good model for studies on senescence, eusocial evolution, and epigenetics. Therefore, honeybees seem to be especially useful here because of long living rebel-workers (RW) with high reproductive potential recently described. Longevity, ovariole number, nosema tolerance, and global DNA methylation have been assayed in normal workers (NW) versus RW in hives and cages. RW always lived longer than NW and unexpectedly extended longevity of NW when they were together, similarly as the presence of a queen did. RW lived longer despite the fact that they had higher Nosema spore load; surprisingly they became infected more easily but tolerated the infection better. Global DNA methylation increased with age, being lower in RW than in NW. Therefore, RW are queen-like considering global DNA methylation and the link between fecundity, longevity, and body maintenance. Presented features of RW expands possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.

Humoral and Cellular Defense Mechanisms in Rebel Workers of Apis mellifera

The physiological state of an insect depends on efficiently functioning immune mechanisms such as cellular and humoral defenses. However, compounds participating in these mechanisms also regulate reproductive caste formation and are responsible for reproductive division of labor as well as for labor division in sterile workers. Divergent reaction of the same genotype yielding reproductive queens and worker castes led to shaping of the physiological and behavioral plasticity of sterile or reproductive workers. Rebels that can lay eggs while maintaining tasks inside and outside the colony exhibit both queen and worker traits. So, we expected that the phagocytic index, JH3 titer, and Vg concentration would be higher in rebels than in normal workers and would increase with their age. We also assumed that the numbers of oenocytes and their sizes would be greater in rebels than in normal workers. The rebels and the normal workers were collected at the age of 1, 7, 14, and 21 days, respectively. Hemolymph and fat bodies were collected for biochemical and morphological analyses. The high levels of JH, Vg, and the phagocytic index, as well as increased numbers and sizes of oenocytes in the fat body cells demonstrate the physiological and phenotypic adaptation of rebels to the eusocial life of honeybees.

Segmentation of the subcuticular fat body in Apis mellifera females with different reproductive potentials

Evolution has created different castes of females in eusocial haplodiploids. The difference between them lies in their functions and vulnerability but above all in their reproductive potentials. Honeybee queens are highly fertile. On the other hand, the workers are facultatively sterile. However, rebel workers, i.e. workers that develop in a queenless colony, reproduce more often than normal workers. As a result, the fat body of these bees, which apart from acting as the energy reserve, is also the site of numerous metabolic processes, had to specialize in different functions perfected over millions of years of eusocial evolution. Assuming that the variety of functions manifests itself in the pleomorphic structure of the fat body cells, we predicted that also different parts of the fat body, e.g. from different segments of the abdomen, contain different sets of cells. Such differences could be expected between queens, rebels and normal workers, i.e. females with dramatically different reproductive potentials. We confirmed all these expectations. Although all bees had the same types of cells, their proportion and segmental character corresponded with the caste reproductive potential and physiological characteristics shaped in the evolutionary process. The females with an increased reproductive potential were characterized by the presence of oenocytes in the third tergite and high concentrations of compounds responsible for energy reserves, like glucose, glycogen and triglycerides. Queens had very large trophocytes, especially in the third tergite. Only in workers did we observe intercellular spaces in all the segments of the fat body, as well as high protein concentrations—especially in the sternite. As expected, the rebels combined many features of the queens and normal workers, what with other findings can help understand the ways that led to the origin of different castes in females of eusocial Hymenoptera.

Honeybee workers with higher reproductive potential have a greater learning ability

Learning ability, which allows individuals to adjust their behaviour to changing environmental conditions, has a considerable positive impact on individual fitness. However, in addition to benefits, learning also incurs a cost, which means that investment in learning and maintaining learned skills can lead to trade-offs impacting other biological functions. Here, we tested whether a trade-off exists between learning skills and reproductive potential in honeybee workers. For this purpose, we compared learning ability between two groups of workers that differed in reproductive potential—normal and rebel workers. The results showed that workers with high reproductive potential (rebels), measured according to the number of ovarioles in the ovary, learned faster than normal workers with low reproductive potential. Moreover, by performing separate regression analyses within the rebel and non-rebel worker groups, we found that the reproductive potential of workers was positively correlated with their learning ability. The results show that in honeybees, there is no trade-off in resource allocation between two costly biological functions, learning and reproduction.

Do rebel workers in the honeybee Apis mellifera avoid worker policing?

A recent study showed that worker larvae fed in a queenless colony develop into another female polyphenic form—rebel workers. The rebel workers are more queen-like than normal workers because they have higher reproductive potential revealed by more ovarioles in their ovaries. However, it was unclear whether eggs laid by rebel workers avoided worker policing. Worker-laid eggs are normally eaten by other workers in a queenright colony. The aim of this study was to compare the survival of three classes of eggs, namely, those laid by normal workers, rebel workers, and the queen. All eggs were tested in queenright colonies. We expected that rebel workers would avoid policing by laying more queen-like eggs. Contrary to our expectations, eggs laid by rebel workers were eaten by other workers, as were eggs laid by normal workers, and only a few worker-laid eggs (both normal and rebel) survived for more than 3 h. Therefore, in a queenright colony, eggs laid by rebel workers do not avoid policing.