Revealing the Development Patterns of the Mandibular Glands of Apis mellifera carnica Based on Transcriptomics and Morphology.

The mandibular gland in worker bees synthesizes and secretes the organic acids present in royal jelly, and its development directly affects yield and quality. Therefore, we aimed to analyze the differences in morphology and gene expression in the mandibular glands of Apis mellifera carnica worker bees of different ages (3, 6, 9, 12, and 16 d). We dissected their mandibular glands and performed morphological and transcriptomic analyses to investigate the development of the mandibular gland and the molecular regulatory mechanisms involved in royal jelly secretion. Microscopy revealed that mandibular gland development is likely completed in the early stages. There were no significant differences in the structural morphology or organelles involved in the secretion of royal jelly at different ages. Transcriptomics revealed a total of 1554 differentially expressed genes, which were mainly involved in fat metabolism, lipid transport, and energy metabolism. The extracellular matrix-receptor interaction pathway was significantly enriched and contributed to the royal jelly secretion process. These results elucidate the genetic basis of the role of the mandibular gland in royal jelly secretion in A. mellifera and provide a reference for the genetic improvement of bees with high royal jelly production in the future.

Biotic and abiotic stresses on honeybee health.

Honeybees are the most critical pollinators providing key ecosystem services that underpin crop production and sustainable agriculture. Amidst a backdrop of rapid global change, this eusocial insect encounters a succession of stressors during nesting, foraging, and pollination. Ectoparasitic mites, together with vectored viruses, have been recognized as central biotic threats to honeybee health, while the spread of invasive giant hornets and small hive beetles also increasingly threatens colonies worldwide. Cocktails of agrochemicals, including acaricides used for mite treatment, and other pollutants of the environment have been widely documented to affect bee health in various ways. Additionally, expanding urbanization, climate change, and agricultural intensification often result in the destruction or fragmentation of flower-rich bee habitats. The anthropogenic pressures exerted by beekeeping management practices affect the natural selection and evolution of honeybees, and colony translocations facilitate alien species invasion and disease transmission. In this review, the multiple biotic and abiotic threats and their interactions that potentially undermine bee colony health are discussed, while taking into consideration the sensitivity, large foraging area, dense network among related nestmates, and social behaviors of honeybees.

Effects of local domestication warrant attention in honey bee population genetics.

Honey bees, Apis mellifera, have for millennia been managed and exploited by humans and introduced into most suitable regions worldwide. However, given the lack of records for many introduction events, treating A. mellifera populations as native would predictably bias genetic studies regarding origin and evolution. Here, we used the Dongbei bee, a well-documented population, introduced beyond the natural distribution range approximately 100 years ago, to elucidate the effects of local domestication on animal population genetic analyses. Strong domestication pressure was detected in this population, and the genetic divergence between Dongbei bee and its ancestral subspecies was found to have occurred at the lineage level. Results of phylogenetic and time divergence analyses could consequently be misinterpreted. Proposing new subspecies or lineages and performing analyses of origin should thus strive to eliminate anthropogenic effects. We highlight the need for definitions of landrace and breed in honey bee sciences and make preliminary suggestions.

Evolution of piggyBac Transposons in Apoidea.

In this study, we investigated the presence of piggyBac (PB) transposons in 44 bee genomes from the Apoidea order, which is a superfamily within the Hymenoptera, which includes a large number of bee species crucial for pollination. We annotated the PB transposons in these 44 bee genomes and examined their evolution profiles, including structural characteristics, distribution, diversity, activity, and abundance. The mined PB transposons were divided into three clades, with uneven distribution in each genus of PB transposons in Apoidea. The complete PB transposons we discovered are around 2.23-3.52 kb in length and encode transposases of approximately 580 aa, with terminal inverted repeats (TIRs) of about 14 bp and 4 bp (TTAA) target-site duplications. Long TIRs (200 bp, 201 bp, and 493 bp) were also detected in some species of bees. The DDD domains of the three transposon types were more conserved, while the other protein domains were less conserved. Generally, most PB transposons showed low abundance in the genomes of Apoidea. Divergent evolution dynamics of PB were observed in the genomes of Apoidea. PB transposons in some identified species were relatively young, whiles others were older and with some either active or inactive. In addition, multiple invasions of PB were also detected in some genomes of Apoidea. Our findings highlight the contribution of PB transposons to genomic variation in these species and suggest their potential as candidates for future gene transfer tools.

A key gene for the climatic adaptation of Apis cerana populations in China according to selective sweep analysis.

BACKGROUND: Apis cerana is widely distributed in China and, prior to the introduction of western honeybees, was the only bee species kept in China. During the long-term natural evolutionary process, many unique phenotypic variations have occurred among A. cerana populations in different geographical regions under varied climates. Understanding the molecular genetic basis and the effects of climate change on the adaptive evolution of A. cerana can promote A. cerana conservation in face of climate change and allow for the effective utilization of its genetic resources. RESULT: To investigate the genetic basis of phenotypic variations and the impact of climate change on adaptive evolution, A. cerana workers from 100 colonies located at similar geographical latitudes or longitudes were analyzed. Our results revealed an important relationship between climate types and the genetic variation of A. cerana in China, and a greater influence of latitude compared with longitude was observed. Upon selection and morphometry analyses combination for populations under different climate types, we identified a key gene RAPTOR, which was deeply involved in developmental processes and influenced the body size. CONCLUSION: The selection of RAPTOR at the genomic level during adaptive evolution could allow A. cerana to actively regulate its metabolism, thereby fine-tuning body sizes in response to harsh conditions caused by climate change, such as food shortages and extreme temperatures, which may partially elucidate the size differences of A. cerana populations. This study provides crucial support for the molecular genetic basis of the expansion and evolution of naturally distributed honeybee populations.

Atrazine exposure can dysregulate the immune system and increase the susceptibility against pathogens in honeybees in a dose-dependent manner.

Recently, concerns regarding the impact of agrochemical pesticides on non-target organisms have increased. The effect of atrazine, the second-most widely used herbicide in commercial farming globally, on honeybees remains poorly understood. Here, we evaluated how atrazine impacts the survival of honeybees and pollen and sucrose consumption, investigating the morphology and mRNA expression levels of midgut tissue, along with bacterial composition (relative abundance) and load (absolute abundance) in the whole gut. Atrazine did not affect mortality, but high exposure (37.3 mg/L) reduced pollen and sucrose consumption, resulting in peritrophic membrane dysplasia. Sodium channels and chitin synthesis were considered potential atrazine targets, with the expression of various genes related to lipid metabolism, detoxification, immunity, and chemosensory activity being inhibited after atrazine exposure. Importantly, 37.3 mg/L atrazine exposure substantially altered the composition and size of the gut microbial community, clearly reducing both the absolute and relative abundance of three core gram-positive taxa, Lactobacillus Firm-5, Lactobacillus Firm-4, and Bifidobacterium asteroides. With altered microbiome composition and a weakened immune system following atrazine exposure, honeybees became more susceptible to infection by the opportunistic pathogen Serratia marcescens. Thus, considering its scale of use, atrazine could negatively impact honeybee populations worldwide, which may adversely affect global food security.

Acute and chronic viruses mediated by an ectoparasite targeting different developmental stages of honeybee (Apis mellifera and Apis cerana) brood.

The health of the western honeybee, Apis mellifera, the most crucial pollinator, has been challenged globally over the past decades. An ectoparasitic mite, Varroa destructor, together with the viruses it vectored, is generally regarded as the vital pathogenic agent. Although the poor health status of A. mellifera compared to its eastern counterpart, Apis cerana, has been broadly identified, the underlying mechanism remains poorly understood and comparison between susceptible and resistant hosts will potentially ameliorate this predicament. Here, we investigated the impacts of two widespread viruses-deformed wing virus type A (DWV-A) and Israeli acute paralysis virus (IAPV), mediated by V. destructor mite, on the capped developing honeybee brood, in the absence of adult workers, of A. mellifera and A. cerana, with positive and negative controls. Our results demonstrated that the endogenous viruses imposed limited damage on the hosts even if the brood was wounded. In contrast, the exogenous viruses introduced by ectoparasites triggered variable mortality of the infested brood between host species. Intriguingly, death causes of both honeybee species presented a similar trend: the acute IAPV generally causes morbidity and mortality of late larvae, while the chronic DWV-A typically leads to brood mortality during and after pupation. Notably, the susceptible immature A. cerana individuals, supported by higher observed mortality and a lower virus tolerance, serve the interests of the colony and foster the overall survival of a resistant honeybee superorganism. These results improve our understanding of the interactions between viruses carried by ectoparasites and their developing hosts, and the novel insight of weak individuals fostering strong colonies may promote breeding efforts to mitigate the indefensible colony losses globally.

Transcriptome-Wide Characterization of piRNAs during the Developmental Process of European Honey-Bee Larval Guts.

piRNAs play pivotal roles in maintaining genome stability, regulating gene expression, and modulating development and immunity. However, there are few piRNA-associated studies on honey-bees, and the regulatory role of piRNAs in the development of bee guts is largely unknown. Here, the differential expression pattern of piRNAs during the developmental process of the European honey-bee (Apis mellifera) larval guts was analyzed, followed by investigation of the regulatory network and the potential function of differentially expressed piRNAs (DEpiRNAs) in regulating gut development. A total of 843 piRNAs were identified in the larval guts of A. mellifera; among these, 764 piRNAs were shared by 4- (Am4 group), 5- (Am5 group), and 6-day-old (Am6 group) larval guts, while 11, 67, and one, respectively, were unique. The first base of piRNAs in each group had a cytosine (C) bias. Additionally, 61 up-regulated and 17 down-regulated piRNAs were identified in the "Am4 vs. Am5" comparison group, further targeting 9, 983 genes, which were involved in 50 GO terms and 142 pathways, while two up-regulated and five down-regulated piRNAs were detected in the "Am5 vs. Am6" comparison group, further targeting 1, 936 genes, which were engaged in 41 functional terms and 101 pathways. piR-ame-742536 and piR-ame-856650 in the "Am4 vs. Am5" comparison group as well as piR-ame-592661 and piR-ame-31653 in the "Am5 vs. Am6" comparison group were found to link to the highest number of targets. Further analysis indicated that targets of DEpiRNAs in these two comparison groups putatively regulate seven development-associated signaling pathways, seven immune-associated pathways, and three energy metabolism pathways. Moreover, the expression trends of five randomly selected DEpiRNAs were verified based on stem-loop RT-PCR and RT-qPCR. These results were suggestive of the overall alteration of piRNAs during the larval developmental process and demonstrated that DEpiRNAs potentially modulate development-, immune-, and energy metabolism-associated pathways by regulating the expression of corresponding genes via target binding, further affecting the development of A. mellifera larval guts. Our data offer a novel insight into the development of bee larval guts and lay a basis for clarifying the underlying mechanisms.

Effect of carbendazim on honey bee health: Assessment of survival, pollen consumption, and gut microbiome composition.

Gut microbiota and nutrition play major roles in honey bee health. Recent reports have shown that pesticides can disrupt the gut microbiota and cause malnutrition in honey bees. Carbendazim is the most commonly used fungicide in China, but it is not clear whether carbendazim negatively affects the gut microbes and nutrient intake levels in honey bees. To address this research gap, we assessed the effects of carbendazim on the survival, pollen consumption, and sequenced 16 S rRNA gene to determine the bacterial composition in the midgut and hindgut. Our results suggest that carbendazim exposure does not cause acute death in honey bees even at high concentrations (5000 mg/L), which are extremely unlikely to exist under field conditions. Carbendazim does not disturb the microbiome composition in the gut of young worker bees during gut microbial colonization and adult worker bees with established gut communities in the mid and hindgut. However, carbendazim exposure significantly decreases pollen consumption in honey bees. Thus, exposure of bees to carbendazim can perturb their beneficial nutrition homeostasis, potentially reducing honey bee immunity and increasing their susceptibility to infection by pathogens, which influence effectiveness as pollinators, even colony health.

Dose-Dependent Effects of Royal Jelly on Estrogen- and Progesterone-Induced Mammary Gland Hyperplasia in Rats.

SCOPE: Royal jelly (RJ) has a wide range of biological functions, its effect on hyperplasia of the mammary gland (HMG) in mammals is unclear. This study aims to investigate the effect of RJ on HMG and the dose-response relationship of RJ in the treatment of HMG. METHODS AND RESULTS: HMG rats are induced by intramuscular injection of estrogen (E2) and progesterone, and are treated with different doses of RJ (100, 200, 400, and 800 mg kg-1 d-1 ). As a result, RJ improves the expansion of acinar and breast tissue ducts, particularly at 100 and 800 mg kg-1 d-1 . These two doses also inhibit serum E2 and prolactin (PRL) secretion and increase serum progesterone secretion and the expression of estrogen receptor (ER)-ß in the breast tissue. In addition, 800 mg kg-1 d-1 decrease and increase the mRNA expression of, respectively, hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary GnRH receptors (GnRH-R). The lowest dosage (100 mg kg-1 d-1 ) increases GnRH-R mRNA expression as well. However, the effects of 200 and 400 mg kg-1 d-1 RJ on the reproductive parameters of HMG are not significant, implying a dose-dependent effect. CONCLUSION: RJ regulates endocrine dyscrasia in HMG rats and improves the breast tissue structure, indicating its potential in the prevention and treatment on HMG.

Population genetics and host specificity of Varroa destructor mites infesting eastern and western honeybees.

In a globalized world, parasites are often brought in contact with new potential hosts. When parasites successfully shift host, severe diseases can emerge at a large cost to society. However, the evolutionary processes leading to successful shifts are rarely understood, hindering risk assessment, prevention, or mitigation of their effects. Here, we screened populations of Varroa destructor, an ectoparasitic mite of the honeybee genus Apis, to investigate their genetic structure and reproductive potential on new and original hosts. From the patterns identified, we deduce the factors that influenced the macro- and microevolutionary processes that led to the structure observed. Among the mite variants identified, we found two genetically similar populations that differed in their reproductive abilities and thus in their host specificity. These lineages could interbreed, which represents a threat due to the possible increased virulence of the parasite on its original host. However, interbreeding was unidirectional from the host-shifted to the nonshifted native mites and could thus lead to speciation of the former. The results improve our understanding of the processes affecting the population structure and evolution of this economically important mite genus and suggest that introgression between shifted and nonshifted lineages may endanger the original host. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-020-01322-7.

Metabolomic analysis of honey bees (Apis mellifera) response to carbendazim based on UPLC-MS.

Pesticides are one of the main causes of colony losses globally, posing a huge threat to the beekeeping industry. The fungicide carbendazim is commonly used on many crops worldwide, but the effects of fungicides on honey bees have received less attention than those of insecticides. Previous studies have shown that sublethal doses of carbendazim hinder growth and development and may destabilize and impede the development of honey bee colonies. The metabolome closely reflects brain activity at the functional level, allowing the effects of compounds such as fungicides to be investigated. Here, we established a model of carbendazim-treated honey bees, Apis mellifera, and used metabolomic approaches to better understand the effect of carbendazim on bee metabolic profiles. The results showed that 112 metabolites were significantly affected in carbendazim-treated bees compared to the control. Metabolites associated with energy and amino acid metabolism showed high abundance and were enriched for a wide range of pathways. In addition, the down-regulation of Aflatoxin B1exo-8,9-epoxide-GSH and glycerol diphosphate showed that carbenazim may affect the detoxification and immune system of honey bees. These results provide new insights into the interaction between fungicides and honey bees.

Diversity and Evolution of pogo and Tc1/mariner Transposons in the Apoidea Genomes.

Bees (Apoidea), the largest and most crucial radiation of pollinators, play a vital role in the ecosystem balance. Transposons are widely distributed in nature and are important drivers of species diversity. However, transposons are rarely reported in important pollinators such as bees. Here, we surveyed 37 bee genomesin Apoidea, annotated the pogo and Tc1/mariner transposons in the genome of each species, and performed a phylogenetic analysis and determined their overall distribution. The pogo and Tc1/mariner families showed high diversity and low abundance in the 37 species, and their proportion was significantly higher in solitary bees than in social bees. DD34D/mariner was found to be distributed in almost all species and was found in Apis mellifera, Apis mellifera carnica, Apis mellifera caucasia, and Apis mellifera mellifera, and Euglossa dilemma may still be active. Using horizontal transfer analysis, we found that DD29-30D/Tigger may have experienced horizontal transfer (HT) events. The current study displayed the evolution profiles (including diversity, activity, and abundance) of the pogo and Tc1/mariner transposons across 37 species of Apoidea. Our data revealed their contributions to the genomic variations across these species and facilitated in understanding of the genome evolution of this lineage.

A Survey of Common Pathogens of Apis spp. in Wild Non-Cave Honeybees in Southwest China.

Honeybees are crucial pollinators with significant ecologic value. The decline of wild honeybee populations has been recognized and documented during recent decades. However, the health status of wild non-cave Apis spp., including giant and dwarf honeybees, remains generally unknown. We investigated eight common viruses and five bacterial or fungal pathogens in four wild non-cave honeybee species at 11 locations in Southwest China. As a result, Melissococcus plutonius, the pathogenic agent of European foulbrood, was detected in all the species, and the sequences were identical to the pathogen in managed cave honeybees. Only one virus, black queen cell virus (BQCV), was positive in one dwarf species, Apis florea, in our study. The positive BQCV infected three A. florea colonies in Guangxi Province, with distinct sequences from this virus reported in cave honeybees or in the same host in the nearby Yunnan Province. Although our results indicated a low pathogenic level of common diseases in the wild non-cave Apis spp. in Southwest China, the conservation of these wild pollinators is of importance in light of the noticeable decline in populations and the irreplaceable position of pollination.

CXCL1 Clone Evolution Induced by the HDAC Inhibitor Belinostat Might Be a Favorable Prognostic Indicator in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer due to its lack of treatment options. Patients with TNBC frequently develop resistance to chemotherapy. As epigenetic-based antineoplastic drugs, histone deacetylase inhibitors (HDACis) have achieved particular efficacy in lymphoma but are less efficacious in solid tumors, and the resistance mechanism remains poorly understood. In this study, the GSE129944 microarray dataset from the Gene Expression Omnibus database was downloaded, and fold changes at the transcriptome level of a TNBC line (MDA-MB-231) after treatment with belinostat were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the critical biological processes. Construction and analysis of the protein-protein interaction (PPI) network were performed to screen candidate genes related to cancer prognosis. A total of 465 DEGs were identified, including 240 downregulated and 225 upregulated genes. The cytokine-cytokine receptor pathway was identified as being significantly changed. Furthermore, the expression of CXCL1 was implicated as a favorable factor in the overall survival of breast cancer patients. With in vitro approaches, we also showed that belinostat could induce the expression of CXCL1 in another 2 TNBC cell lines (BT-549 and HCC-1937). We speculate that belinostat-induced CXCL1 expression could be one of the results of the stress clone evolution of cells after HDACi treatment. These findings provide new insights into clone evolution during HDACi treatment, which might guide us to a novel perspective that various mutation-targeted treatments should be implemented during the whole treatment cycle.

Investigation of circular RNAs in an ectoparasitic mite Varroa destructor (Acarina: Varroidae) of the honey bee.

Circular RNAs (circRNAs) are a large class of non-protein-coding transcripts that are involved in a diverse spectrum of regulatory mechanisms across a broad range of biological processes. To date, however, few studies on circRNAs have investigated their role in the biology of invertebrate parasites. The ectoparasitic mite Varroa destructor is perceived as the principal biotic threat towards global honey bee health. This parasite cannot be sustainably controlled partially due to the lack of knowledge about its basic molecular biology. In this paper, we unveil the circRNA profile of V. destructor for the first time and report the sources, distribution, and features of the identified circRNAs. Exonic, intronic, exon-intron, and intergenic circRNAs were discovered and exon-intron circRNAs were the most abundant within the largest spliced length. Three hundred and eighty-six (8.3%) circRNAs were predicted to possess translational potential. Eleven circRNAs, derived from six parental genes, exhibited strong bonds with miRNAs as sponges, suggesting an efficient post-transcriptional regulation. GO term and KEGG pathway enrichment analyses of the parental genes of the identified circRNAs showed that these non-coding RNAs were mainly engaged in protein processing, signal transduction, and various metabolism processes. To our knowledge, this is the first catalog of a circRNA profile of parasitiformes species, which reveals the prevalence of circRNAs in the parasite and provides biological insights for future genetic studies on this ubiquitous parasitic mite.

Carbendazim exposure during the larval stage suppresses major royal jelly protein expression in nurse bees (Apis mellifera).

Studying the sublethal effects of agrochemical pesticides on nontarget honeybees (Apis mellifera) is important for agricultural development. Carbendazim is a widely used broad-spectrum fungicide that inhibits mitotic microtubule formation and cell division. However, the impact of carbendazim on bee health and development has not been fully elucidated. Here, using proteomics approaches, we assessed in vitro the changes in the expression of functional proteins in the head of newly emerged adults following treatment with field concentration of carbendazim during the larval stage. Treatment with carbendazim severely altered 266 protein expression patterns in the heads of adults and 218 of them showed downregulation after carbendazim exposure. Notably, major royal jelly proteins, a crucial multifunctional protein family with irreplaceable function in sustaining the development of colonies, were significantly suppressed in carbendazim-treated bees. This result was verified in both head and hypopharyngeal gland of nurse bees. Moreover, visual and olfactory loss, immune functions, muscular activity, social behavior, neural and brain development, protein synthesis and modification, and metabolism-related proteins were likely inhibited by carbendazim treatment. Together, these results suggest that carbendazim is an environmental risk factor that likely weakens bee colonies, partially due to reduced expression of major royal jelly proteins, which may be potential causes of colony collapse disorder.

Genome-Wide Identification of Long Non-coding RNAs in the Gravid Ectoparasite Varroa destructor.

Long non-coding RNAs (lncRNAs) emerge as critical regulators across a wide variety of biological functions in living organisms. However, to date, no systematic characterization of lncRNAs has been investigated in the ectoparasitic mite Varroa destructor, the most severe biotic threat to honey bees worldwide. Here, we performed an initial genome-wide identification of lncRNAs in V. destructor via high-throughput sequencing technology and reported, for the first time, the transcriptomic landscape of lncRNAs in the devastating parasite. By means of a lncRNA identification pipeline, 6,645 novel lncRNA transcripts, encoded by 3,897 gene loci, were identified, including 2,066 sense lncRNAs, 2,772 lincRNAs, and 1,807 lncNATs. Compared with protein-coding mRNAs, V. destructor lncRNAs are shorter in terms of full length, as well as of the ORF length, contain less exons, and express at lower level. GO term and KEGG pathway enrichment analyses of the lncRNA target genes demonstrated that these predicted lncRNAs may be potentially responsible for the regulatory functions of cellular and biological progresses in the reproductive phase of V. destructor. To our knowledge, this is the first catalog of lncRNA profile in the parasitiformes species, providing a valuable resource for genetic and genomic studies. Understanding the characteristics and features of lncRNAs in V. destructor would promote sustainable parasite control.

Epigenetic Modifications May Regulate the Activation of the Hypopharyngeal Gland of Honeybees (Apis Mellifera) During Winter.

DNA methylation is an epigenetic modification primarily responsible for individual phenotypic variation. This modification has been reported to play an important role in caste, brain plasticity, and body development in honeybees (Apis mellifera). Here, we report the DNA methylation profile of honeybee hypopharyngeal glands, from atrophy in winter to arousal in the following spring, through the use of whole-genome bisulfite sequencing. Consistent with previous studies in other Apis species, we found low methylation levels of the hypopharyngeal gland genome that were mostly of the CG type. Notably, we observed a strong preference for CpG methylation, which was localized in promoters and exon regions. This result further indicated that, in honeybees, DNA methylation may regulate gene expression by mediating alternative splicing, in addition to silencing gene in the promoter regions. After assessment by correlation analysis, we identified seven candidate proteins encoded by differentially methylated genes, including aristaless-related homeobox, forkhead box protein O, headcase, alpha-amylase, neural-cadherin, epidermal growth factor receptor, and aquaporin, which are reported to be involved in cell growth, proliferation, and differentiation. Hypomethylation followed by upregulated expression of these candidates suggested that DNA methylation may play significant roles in the activation of hypopharyngeal glands in overwintering honeybees. Overall, this study elucidates epigenetic modification differences in honeybee hypopharyngeal glands by comparing an inactive winter state to an aroused state in the following spring, which could provide further insight into the evolution of insect sociality and regulatory plasticity.