The Dominating Role of Genetic Background in Shaping Gut Microbiota of Honeybee Queen Over Environmental Factors

A balanced, diverse gut microbiota is vital for animal health. The microbial population is shaped by multiple factors including genetic background and environment, but other determinants remain controversial. Numerous studies suggest that the dominant factor is genetic background while others emphasize the environmental factors. Here, we bred asexual hybridization queens (AHQs) of honeybees through nutritional crossbreeding (laid in Apis mellifera colony but bred in Apis cerana colony), sequenced their gut microbiome, and compared it with normally bred sister queens to determine the primary factor shaping the gut microbiota. Our results showed that the dominant genera in the gut microbiota of AHQs were Brevundimonas, Bombella, and Lactobacillus, and its microbial community was more related to A. mellifera queens. The AHQs had a moderate number of different bacterial species and diversity, but total bacterial numbers were low. There were more significant taxa identified in the comparison between AHQ and A. cerana queen according to LEfSe analysis results. The only genetic-specific taxon we figured out was Brevundimonas. The growth of core bacterial abundance showed different characteristics among different queen groups in the first week after emerging. Collectively, this study suggested that the genetic background played a more dominant role than environmental factors in shaping the gut microbiota of honeybee queen and the microbiota of midgut was more sensitive than that of rectum to this impact.

Successful Indoor Mass Storage of Honeybee Queens (Apis mellifera) during Winter

The production of young, mated honeybee queens (Apis mellifera) is essential to replace dead queens or to start new colonies after wintering. Mass storage of mated honeybee queens during winter and their use the following spring is an interesting strategy that could help fulfill this need. In this study, we investigated the survival, fertility, and fecundity of young, mated queens stored massively in queenless colonies from September to April (eight months). The queens were kept in environmentally controlled rooms at temperatures above and below cluster formation. The results show that indoor mass storage of mated queens can be achieved with success when queen banks are stored above cluster temperature. Significantly higher survival of queens was measured when wintering queen banks at 16 °C. Surviving queens wintered at different temperatures above or below cluster formation had similar fertility (sperm viability) and fecundity (egg laying and viable worker population). This study shows the potential of indoor overwintering of honeybee queen banks. The technique we describe could be applied on a commercial scale by beekeepers and queen breeders.

Honeybee Queen mandibular pheromone induces starvation in Drosophila melanogaster, implying a role for nutrition signalling in the evolution of eusociality.

Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant or queen. Reproductive dominance is maintained through behavioural dominance in some species as well as production of queen pheromones in others, or a mixture of both. Queen mandibular pheromone (QMP) is produced by honeybee (Apis mellifera) queens and has been characterised chemically. How QMP acts to repress worker reproduction, and how it has evolved this activity, remains less well understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods which have not co-evolved with QMP, are never exposed to QMP in nature, and are up to 530 million years diverged from the honeybee. Here we show that, in Drosophila melanogaster, QMP treatment mimics nutrient limiting conditions, leading to disrupted reproduction. Exposure to QMP induces an increase in food consumption, consistent with that observed in D. melanogaster in response to starvation conditions. This response induces the activation of two checkpoints within the ovary that inhibit oogenesis. The first is the 2a/b ovarian checkpoint in the germarium, which reduces the flow of presumptive oocytes. A stage 9 ovarian checkpoint is also activated, causing degradation of oocytes. The magnitude of activation of both checkpoints is indistinguishable between QMP treated and starved individuals. As QMP seems to trigger a starvation response in an insect highly diverged from honeybees, we propose that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction, a key step in the evolution of eusociality.

Non-Destructive Genotyping of Honeybee Queens to Support Selection and Breeding

In traditional bee breeding, the honeybee queen is chosen for breeding based on the performance of the colony produced by its mother. However, we cannot be entirely certain that a specific queen will produce offspring with desirable traits until we observe the young queen’s new colony. Collecting the queen’s genetic material enables quick and reliable determination of the relevant information. We sampled exuviae, feces, and wingtips for DNA extraction to avoid fatally injuring the queen when using tissue samples. Quantity and purity of extracted DNA were measured. Two mitochondrial markers were used to determine the lineage affiliation and exclude possible contamination of DNA extracts with non-honeybee DNA. dCAPS (derived Cleaved Amplified Polymorphic Sequences) markers allowed detection of single nucleotide polymorphisms (SNPs) in nuclear DNA regions presumably associated with Varroa sensitive hygiene and set the example of successful development of genotyping protocol from non-destructive DNA sources. One of the logical future steps in honeybee breeding is introducing genomic selection and non-destructive sampling methods of genetic material may be the prerequisite for successful genotyping. Our results demonstrate that the extraction of DNA from feces and exuviae can be introduced into practice. The advantage of these two sources over wingtips is reducing the time window for processing the samples, thus enabling genotyping directly after the queen’s emergence.

Absence of Nepotism in Waggle Communication of Honeybees (Apis mellifera)

The polyandrous mating behavior of the honeybee queen increases the genetic variability among her worker offspring and the workers of particular subfamilies tend to have a genetic predisposition for tasks preference. In this study, we intended to understand whether there is nepotism in dance communication of honeybees during natural conditions. Microsatellite DNA analyses revealed a total of fourteen and twelve subfamilies in two colonies. The subfamily composition of the dancer and the followers did not deviate from random. The majority of the subfamilies did not show kin recognition in dance-recruit communication in honeybee colonies, but some subfamilies showed significant nepotism for workers to follow their super-sister dancer. Because it seems unlikely that honeybee would change the tendency to follow dancers due to the degree of relatedness, we conclude that honeybees randomly follow a dancer in order to e benefit colony gain and development.

Cross-activity of honeybee queen mandibular pheromone in bumblebees provides evidence for sensory exploitation

The evolutionary origin of queen pheromones (QPs), which regulate reproductive division of labor in insect societies, has been explained by two evolutionary scenarios: the sender-precursor hypothesis and the sensory exploitation hypothesis. These scenarios differ in terms of whether the signaling system was built on preadaptations on the part of either the sender queens or the receiver workers. While some social insect QPs—such as cuticular hydrocarbons—were likely derived from ancestral fertility cues and evolved according to the former theory, the honeybee’s queen mandibular pheromone (QMP) has been suggested to act directly on preexisting gene-regulatory networks linked with reproduction. This is evidenced by the fact that QMP has been shown to also inhibit ovary activation in fruit flies, thereby implying exploitation of conserved physiological pathways. To verify whether QMP has similar effects on more closely related eusocial species, we here tested for QMP cross-activity in the bumblebee Bombus terrestris. Interestingly, we found that the non-native QMP blend significantly inhibited egg laying in both worker and queen bumblebees and caused accompanying shifts in ovary activation. The native bumblebee QP pentacosane, by contrast, only inhibited the reproduction of the workers. Overall, these findings support the hypothesis that honeybee QMP likely evolved via a route of sensory exploitation. We argue that such exploitation could allow social insect queens to produce compounds that manipulate the workers to remain sterile, but that a major hurdle would be that the queens themselves would have to be immune to such compounds.