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The neuropeptide Pigment-Dispersing Factor (PDF) plays a prominent role in the circadian clock of many insects including honey bees. In the honey bee brain, PDF is expressed in about 15 clock neurons per hemisphere that lie between the central brain and the optic lobes. As in other insects, the bee PDF neurons form wide arborizations in the brain; but certain differences are evident. For example, they arborize only sparsely in the accessory medulla (AME), which serves as important communication center of the circadian clock in cockroaches and flies. Furthermore, all bee PDF neurons cluster together, which makes it impossible to distinguish individual projections. Here, we investigated the developing bee PDF network and found that the first three PDF neurons arise in the 3rd larval instar and form a dense network of varicose fibers at the base of the developing medulla that strongly resembles the AME of hemimetabolous insects. In addition, they send faint fibers toward the lateral superior protocerebrum. In last larval instar, PDF cells with larger somata appear and send fibers toward the distal medulla and the medial protocerebrum. In the dorsal part of the medulla serpentine layer, a small PDF knot evolves from which PDF fibers extend ventrally. This knot disappears during metamorphosis and the varicose arborizations in the putative AME become fainter. Instead a new strongly stained PDF fiber hub appears in front of the lobula. Simultaneously, the number of PDF neurons increases and the PDF neuronal network in the brain gets continuously more complex. This article is protected by copyright. All rights reserved.

The benefits of honey bee dance communication for colony performance in different resource environments are still not well understood. Here, we test the hypothesis that directional dance communication enables honey bee colonies to maintain a diverse pollen diet, especially in landscapes with low resource diversity. To test this hypothesis, we placed 24 Apis mellifera L. colonies with either intact or experimentally disrupted dance communication in eight agricultural landscapes that differed in the diversity of flowering plants and in the dominance of mass-flowering crops. Pollen from incoming foragers was collected and identified via DNA metabarcoding. Disrupting dance communication affected the way the diversity of honey bee pollen diets was impacted by the dominance of mass-flowering crops in available flower resources (p = .04). With increasing dominance of mass-flowering crops in resource environments, foragers of colonies with intact communication foraged on an increasing proportion of available plant genera (p = .01). This was not the case for colonies with disrupted dance communication (p = .5). We conclude that the honey bee dance communication benefits pollen foraging on diverse plant resources and thereby contributes to high quality nutrition in environments with low-resource diversity.

Summary 1. To sustain the vital ecosystem service of pollination, new methodical developments are needed for research on the underlying factors of globally observed bee losses. In particular, robust laboratory methods for assessing adverse effects on honey bee brood are required. In addition, from a statistical point of view, the shared origin of test individuals must be considered when analysing ecotoxicological data. 2. To improve honey bee in vitro rearing, we adopted a nongrafting method to collect honey bee larvae without direct manipulation. Linear mixed effects model to evaluate LD50 ,l arvae survival and prepupae weights integrated the colony background of larvae as a random factor into the statistical analyses. The novel rearing approach and appropriate statistical tools for data analyses are illustrated in an in vitro case study on acute oral dimethoate toxicity. 3. We recommend our honey bee larvae collection approach for in vitro larvae-rearing applications, because of (i) a mere 3% background mortality upon the prepupae stage, (ii) a high quantitative capacity and (iii) because of robustness of performance which are great benefits for standardization. 4. The analyses indicate clear adverse effects of dimethoate by a significant survival reduction and prepupae weight reduction. For second instars, the acute 48-h LD50 was 1AE67 lg dimethoate per larva. 5. We conclude that both our larvae collection method and the applied statistical approaches will help to improve the quality of environmental risk assessment studies on honey bees, to secure honey bee pollination and to sustain biodiversity.

Determining the extent and causes of barriers to gene flow is essential for understanding sympatric speciation, but the practical difficulties of quantifying reproductive isolation remain an obstacle to analysing this process. Social parasites are common in eusocial insects and tend to be close phylogenetic relatives of their hosts (= Emery’s rule). Sympatric speciation caused by reproductive isolation between host and parasite is a possible evolutionary pathway. Socially parasitic workers of the Cape honeybee, Apis mellifera capensis, produce female clonal offspring parthenogenetically and invade colonies of the neighbouring subspecies A. m. scutellata. In the host colony, socially parasitic workers can become pseudoqueens, an intermediate caste with queenlike pheromone secretion. Here, we show that over an area of approximately 275.000 km 2 , all parasitic workers bear the genetic signature of a clone founded by a single ancestral worker genotype. Any gene flow from the host to the parasite is impossible because honeybee workers cannot mate. Gene flow from the parasite to the host is possible, as parasitic larvae can develop into queens. However, we show that despite sympatric coexistence for more than a decade, gene flow between host and social parasite (Fst = 0.32) and hybridizations (0.71%) are rare, resulting in reproductive isolation. Our data suggest a new barrier to gene flow in sympatry, which is not based on assortative matings but on thelytoky and reproductive division of labour in eusocial insects, thereby suggesting a new potential pathway to Emery’s rule.