Acetylcholine and choline in honey bee (Apis mellifera) worker brood food are seasonal and age-dependent.

Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April-September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020-2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.

Rare finding of a symptomatic epidermoid cyst of the diaphragm - a case report.

Cystic lesions of the diaphragm are rare and accordingly present a diagnostic challenge. Specific radiological features with which to clinch a diagnosis may be elusive. Herein we present the case of a patient who presented with symptoms attributable to a cyst in the left upper abdomen, irritating the diaphragm. Surgery was considered appropriate for diagnostic and symptomatic purposes. Final histology demonstrated an epidermoid cyst. Resolution of symptoms was reported after surgery. Diaphragmatic epidermoid cysts appear to be a rare entity with only three prior cases reported in the literature. Given the rarity of this lesion and the lack of unique features by which they can be characterized, accurately diagnosing epidermoid cysts of the diaphragm is likely to remain difficult without surgery, although they are presumed to have a benign behaviour.

Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee.

Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities.

Are Honey Bees at Risk from Microplastics?

Microplastics (MPs) are ubiquitous and persistent pollutants, and have been detected in a wide variety of media, from soils to aquatic systems. MPs, consisting primarily of polyethylene, polypropylene, and polyacrylamide polymers, have recently been found in 12% of samples of honey collected in Ecuador. Recently, MPs have also been identified in honey bees collected from apiaries in Copenhagen, Denmark, as well as nearby semiurban and rural areas. Given these documented exposures, assessment of their effects is critical for understanding the risks of MP exposure to honey bees. Exposure to polystyrene (PS)-MPs decreased diversity of the honey bee gut microbiota, followed by changes in gene expression related to oxidative damage, detoxification, and immunity. As a result, the aim of this perspective was to investigate whether wide-spread prevalence of MPs might have unintended negative effects on health and fitness of honey bees, as well as to draw the scientific community's attention to the possible risks of MPs to the fitness of honey bees. Several research questions must be answered before MPs can be considered a potential threat to bees.

Honey bee behaviours within the hive: Insights from long-term video analysis.

The combined behaviours of individuals within insect societies determine the survival and development of the colony. For the western honey bee (Apis mellifera), individual behaviours include nest building, foraging, storing and ripening food, nursing the brood, temperature regulation, hygiene and defence. However, the various behaviours inside the colony, especially within the cells, are hidden from sight, and until recently, were primarily described through texts and line drawings, which lack the dynamics of moving images. In this study, we provide a comprehensive source of online video material that offers a view of honey bee behaviour within comb cells, thereby providing a new mode of observation for the scientific community and the general public. We analysed long-term video recordings from longitudinally truncated cells, which allowed us to see sideways into the cells in the middle of a colony. Our qualitative study provides insight into worker behaviours, including the use of wax scales and existing nest material to remodel combs, storing pollen and nectar in cells, brood care and thermoregulation, and hygienic practices, such as cannibalism, grooming and surface cleaning. We reveal unique processes that have not been previously published, such as the rare mouth-to-mouth feeding by nurses to larvae as well as thermoregulation within cells containing the developing brood. With our unique video method, we are able to bring the processes of a fully functioning social insect colony into classrooms and homes, facilitating ecological awareness in modern times. We provide new details and images that will help scientists test their hypotheses on social behaviours. In addition, we encourage the non-commercial use of our material to educate beekeepers, the media and the public and, in turn, call attention to the general decline of insect biomass and diversity.

The complete mitochondrial genome of Apis mellifera jemenitica (Insecta: Hymenoptera: Apidae), the Arabian honey bee.

The mitochondrial genome of a worker Apis mellifera jemenitica was 16,623 bp. It consisted of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and a control region. Phylogenetic analyses suggest a close relationship between A. m. jemenitica, A. m. lamarckii and A. m. syriaca.

The mitochondrial genome of the Maltese honey bee, Apis mellifera ruttneri (Insecta: Hymenoptera: Apidae).

The mitochondrial genome of Apis mellifera ruttneri consisted of 13 protein-coding genes, two rRNAs, 22 tRNAs, an AT-rich control region, and was 16,577 bp long. The phylogenetic analyses suggested that A. m. ruttneri was closely related to two North African subspecies: A. m. sahariensis and A. m. intermissa.

Chronic within-hive video recordings detect altered nursing behaviour and retarded larval development of neonicotinoid treated honey bees.

Risk evaluations for agricultural chemicals are necessary to preserve healthy populations of honey bee colonies. Field studies on whole colonies are limited in behavioural research, while results from lab studies allow only restricted conclusions on whole colony impacts. Methods for automated long-term investigations of behaviours within comb cells, such as brood care, were hitherto missing. In the present study, we demonstrate an innovative video method that enables within-cell analysis in honey bee (Apis mellifera) observation hives to detect chronic sublethal neonicotinoid effects of clothianidin (1 and 10 ppb) and thiacloprid (200 ppb) on worker behaviour and development. In May and June, colonies which were fed 10 ppb clothianidin and 200 ppb thiacloprid in syrup over three weeks showed reduced feeding visits and duration throughout various larval development days (LDDs). On LDD 6 (capping day) total feeding duration did not differ between treatments. Behavioural adaptation was exhibited by nurses in the treatment groups in response to retarded larval development by increasing the overall feeding timespan. Using our machine learning algorithm, we demonstrate a novel method for detecting behaviours in an intact hive that can be applied in a versatile manner to conduct impact analyses of chemicals, pests and other stressors.

Acetylcholine and Its Receptors in Honeybees: Involvement in Development and Impairments by Neonicotinoids.

Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.

The complete mitochondrial genome of Apis mellifera unicolor (Insecta: Hymenoptera: Apidae), the Malagasy honey bee.

The complete mitochondrial genome of the endemic Malagasy honey bee Apis mellifera unicolor is 16,373 bp and comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The mitochondrial genome closely resembles mitogenomes of other published Apis mellifera subspecies, and the phylogenetic analysis suggests that A. m. unicolor is distinct from other African (A) lineage honey bees but is most closely related to the honey bees from southern African: A. m. scutellata and A. m. capensis.

The mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica (Insecta: Hymenoptera: Apidae).

Sequencing the mitochondrial genome of the Carniolan honey bee, Apis mellifera carnica, revealed 16,358 bp, consisting of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. Phylogenetic analysis supported a close relationship to another south-eastern European (C-lineage) honey bee, A. m. ligustica.

The mitochondrial genome of Apis mellifera simensis (Hymenoptera: Apidae), an Ethiopian honey bee.

The complete mitochondrial genome of Apis mellifera simensis was 16,523 bp long. The 13 protein-coding genes, two rRNAs, and 22 tRNAs resembled other Apis mitogenomes. The location of this Apis subspecies in our phylogenetic tree supported the hypothesis that this subspecies is distinct, and is most closely related to A. m. scutellata and A. m. monticola.

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the West African honey bee Apis mellifera adansonii consisted of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. It was 16,466 bp and consisted of 84.7% AT nucleotides. This subspecies had a similar mitogenome to those of other southern African honey bees, namely A. m. scutellata, A. m. capensis, and A. m. monticola.

The mitochondrial genome of the Spanish honey bee, Apis mellifera iberiensis (Insecta: Hymenoptera: Apidae), from Portugal.

The Spanish honey bee Apis mellifera iberiensis, had a mitochondrial genome of 16,560 bp. It consisted of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and an AT-rich control region. The sample was from Portugal and its mitogenome resembled those of the African (A)-lineage honey bee subspecies. It was most closely related to other North African honey bees, namely A. m. sahariensis and A. m. intermissa.

Neural Organization of A3 Mushroom Body Extrinsic Neurons in the Honeybee Brain.

In the insect brain, the mushroom body is a higher order brain area that is key to memory formation and sensory processing. Mushroom body (MB) extrinsic neurons leaving the output region of the MB, the lobes and the peduncle, are thought to be especially important in these processes. In the honeybee brain, a distinct class of MB extrinsic neurons, A3 neurons, are implicated in playing a role in learning. Their MB arborisations are either restricted to the lobes and the peduncle, here called A3 lobe connecting neurons, or they provide feedback information from the lobes to the input region of the MB, the calyces, here called A3 feedback neurons. In this study, we analyzed the morphology of individual A3 lobe connecting and feedback neurons using confocal imaging. A3 feedback neurons were previously assumed to innervate each lip compartment homogenously. We demonstrate here that A3 feedback neurons do not innervate whole subcompartments, but rather innervate zones of varying sizes in the MB lip, collar, and basal ring. We describe for the first time the anatomical details of A3 lobe connecting neurons and show that their connection pattern in the lobes resemble those of A3 feedback cells. Previous studies showed that A3 feedback neurons mostly connect zones of the vertical lobe that receive input from Kenyon cells of distinct calycal subcompartments with the corresponding subcompartments of the calyces. We can show that this also applies to the neck of the peduncle and the medial lobe, where both types of A3 neurons arborize only in corresponding zones in the calycal subcompartments. Some A3 lobe connecting neurons however connect multiple vertical lobe areas. Contrarily, in the medial lobe, the A3 neurons only innervate one division. We found evidence for both input and output areas in the vertical lobe. Thus, A3 neurons are more diverse than previously thought. The understanding of their detailed anatomy might enable us to derive circuit models for learning and memory and test physiological data.

Performance of honey bee colonies under a long-lasting dietary exposure to sublethal concentrations of the neonicotinoid insecticide thiacloprid.

BACKGROUND: Substantial honey bee colony losses have occurred periodically in the last decades. The drivers for these losses are not fully understood. The influence of pests and pathogens are beyond dispute, but in addition, chronic exposure to sublethal concentrations of pesticides has been suggested to affect the performance of honey bee colonies. This study aims to elucidate the potential effects of a chronic exposure to sublethal concentrations (one realistic worst-case concentration) of the neonicotinoid thiacloprid to honey bee colonies in a three year replicated colony feeding study. RESULTS: Thiacloprid did not significantly affect the colony strength. No differences between treatment and control were observed for the mortality of bees, the infestation with the parasitic mite Varroa destructor and the infection levels of viruses. No colony losses occurred during the overwintering seasons. Furthermore, thiacloprid did not influence the constitutive expression of the immunity-related hymenoptaecin gene. However, upregulation of hymenoptaecin expression as a response to bacterial challenge was less pronounced in exposed bees than in control bees. CONCLUSION: Under field conditions, bee colonies are not adversely affected by a long-lasting exposure to sublethal concentrations of thiacloprid. No indications were found that field-realistic and higher doses exerted a biologically significant effect on colony performance. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The complete mitochondrial genome and phylogenetic placement of Apis nigrocincta Smith (Insecta: Hymenoptera: Apidae), an Asian, cavity-nesting honey bee.

The complete mitochondrial genome of Apis nigrocincta was sequenced. The mitochondrial genome is a circular molecule of 15,855 bp, including 37 classical eukaryotic mitochondrial regions and an A + T-rich region. Gene directions and arrangements are similar to those of other Apis mitogenomes. Most genes initiated with ATT, though ATG and ATA were also used as start codons. Twelve of 13 protein-coding genes terminated with TAA, though ND2 terminated with TAG. Four PCG genes, eight tRNAs and both rRNAs were encoded on the heavy strand while all others were encoded on the light strand (9 PCGs and 14 tRNAs). Overall, the GC content composed 15.6% of the mitogenome. All of the 22 tRNA genes, ranging from 66 to 114 bp, have a typical cloverleaf structure. A phylogenetic tree showed that A. nigrocincta clustered closest to A. cerana. The complete mitogenome of A. nigrocincta provides essential information on the biogeography and evolution of this Asian honey bee species.

The complete mitochondrial genome of Apis mellifera meda (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the western honey bee subspecies Apis mellifera meda was sequenced. This mitochondrial genome is 16,248 bp in length, with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene direction and arrangement are similar to those of other Apis mitogenomes. All genes initiate with ATT (six genes), ATG (four genes), ATA (two genes), and ATC (one gene) start codons and terminate with a TAA stop codon. Four genes are encoded on the heavy and nine on the light strands, respectively. All of the 22 tRNA genes, ranging from 66 to 78 bp, have a typical cloverleaf structure. The complete mitogenome of A.m. meda provides information on the biogeography and evolution of A. mellifera subspecies.

The complete mitochondrial genome of the Egyptian honey bee, Apis mellifera lamarckii (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of the western honey bee subspecies Apis mellifera lamarckii was sequenced. This mitochondrial genome is 16,589 bp in length with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene directions and arrangements are similar to those of other Apis mitogenomes. Seven genes begin with ATT, four with ATG, and two with ATA (none with ATC) and all genes terminate with TAA. Four genes are encoded on the heavy strand and nine are encoded on light strand. All of the 22 tRNA genes, ranging from 66 to 80 bp, have a typical cloverleaf structure. A phylogenetic tree showed that A.m. lamarckii clusters with other A. mellifera subspecies, as expected.

The complete mitochondrial genome of Apis nuluensis Tingek, an Asian honey bee (Insecta: Hymenoptera: Apidae).

The complete mitochondrial genome of Apis nuluensis Tingek was sequenced. The mitochondrial genome was 15,843 bp in length, with 37 classical eukaryotic mitochondrial genes and an A + T-rich region. Gene directions and arrangements were similar to those of other Apis mitogenomes. Most genes initiate with ATT (though ATG and ATC also were used) and all genes terminated with TAA. Nine genes were encoded on the light strand while four were encoded on the heavy strand. All 22 tRNA genes have a typical cloverleaf structure. The most likely phylogenetic tree showed A. nuluensis clustering with A. cerana. The complete mitogenome of A. nuluensis completes the sequencing of all mitogenomes of the currently accepted species of Apis.