Seasonal dynamics of the microbiota and nutritional composition in bee bread from Apis cerana and Apis mellifera colonies.

Bee bread is a product of honeybees, which collect and ferment pollen, that contains highly nutritious and easily digestible active substances. However, its nutritional composition varies significantly with fermentation strains and seasonal changes. To unveil the patterns of microbial community and nutritional component changes in bee bread across seasons, we employed high-throughput techniques to assess the diversity of bacteria and fungi in bee bread. The results indicated that the compositions of bacteria and fungi in bee bread undergo significant seasonal variation, with noticeable changes in the microbial diversity of bee bread from different bee species. Subsequently, metabolomic analysis revealed high activity of glycerophospholipid metabolism in bee bread. Furthermore, our analysis identifaied noteworthy differences in nutritional components, including pH values, sugar content, and free amino acid levels, in bee bread across different seasons.

Gut microbiota-driven regulation of queen bee ovarian metabolism.

With the global prevalence of Varroa mites, more and more beekeepers resort to confining the queen bee in a queen cage to control mite infestation or to breed superior and robust queen bees. However, the impact of such practices on the queen bee remains largely unknown. Therefore, we subjected the queen bees to a 21-day egg-laying restriction treatment (from the egg stage to the emergence of adult worker bees) and analyzed the queen bees' ovarian metabolites and gut microbiota after 21 days, aiming to assess the queen bees' quality and assist beekeepers in better hive management. Our findings revealed a significant reduction in the relative expression levels of Vg and Hex110 genes in the ovaries of egg laying-restricted queen bees compared to unrestricted egg-laying queens. The diversity of gut microbiota in the queen bee exhibited a notable decrease, accompanied by corresponding changes in the core bacteria of the microbial community, the relative abundance of Lactobacillus and Bifidobacterium increased from 22.34% to 53.14% (P = 0.01) and from 0.053% to 0.580% (P = 0.04), respectively. The relative abundance of Bombella decreased from 25.85% to 1.720% (P = 0.002). Following egg-laying restriction, the activity of the queen bee's ovaries decreased, while the metabolism of glycerophospholipids remained or stored more lipid molecules, awaiting environmental changes for the queen bee to resume egg laying promptly. Furthermore, we observed that Bombella in the queen bee's gut may regulate the queen's ovarian metabolism through tryptophan metabolism. These findings provide novel insights into the interplay among queen egg laying, gut microbiota, and ovarian metabolism. IMPORTANCE With Varroa mite infestation, beekeepers often confine the queen bee in cages for control or breeding. However, the impact on the queen bee is largely unknown. We evaluated queen bee quality by restricting egg laying and analyzing ovarian metabolites and gut microbiota. In this study, we provided a comprehensive explanation of the expression of ovarian genes, the diversity of gut microbiota, and changes in ovarian metabolism in the queen bee. Through integrated analysis of the queen bee's gut microbiota and ovarian metabolism, we discovered that the gut microbiota can regulate the queen bee's ovarian metabolism. These findings provide valuable insights into the interplay among egg laying, gut microbiota, and the reproductive health of the queen bee. Understanding these relationships can contribute to the development of better strategies for Varroa mite control and queen bee breeding.

Honeybee (Apis mellifera) resistance to deltamethrin exposure by Modulating the gut microbiota and improving immunity.

Honeybees (Apis mellifera) are important economic insects and play important roles in pollination and maintenance of ecological balance. However, the use of pesticides has posed a substantial threat to bees in recent years, with the more widely used deltamethrin being the most harmful. In this study, we found that deltamethrin exposure significantly reduced bee survival in a dose-dependent manner (p = 0.025). In addition, metagenomic sequencing further revealed that DM exposure significantly reduced the diversity of the bee gut microbiota (Chao1, p < 0.0001; Shannon, p < 0.0001; Simpson, p < 0.0001) and decreased the relative abundance of core species of the gut microbiota. Importantly, in studies of GF-bees, we found that the colonization of important gut bacteria such as Gilliamella apicola and Lactobacillus kunkeei significantly increased bee resistance to DM (survival rate increased from 16.7 to 66.7%). Interestingly, we found that the immunity-genes Defensin-2 and Toll were significantly upregulated in bees after the colonization of gut bacteria. These results suggest that gut bacteria may protect against DM stress by improving host immunity. Our findings provide an important rationale for protecting honeybees from pollutants from the perspective of gut microbes.

The composition of bacteria in gut and beebread of stingless bees (Apidae: Meliponini) from tropics Yunnan, China.

Stingless bees are the main pollinators in tropical and subtropical regions. However, there are only a few studies on the structure and composition of bacteria in the gut and beebread of stingless bees, especially in China. To address this shortage of information, we characterized the microbiota of three common species of stingless bees (Lepidotrigona terminata, Lepidotrigona ventralis and Tetragonula pagdeni) and beebread samples of T. pagdeni. The results showed that the gut of stingless bees contained a set of dominant bacteria, including Acetobacter-like, Snodgrassella, Lactobacillus, Psychrobacter, Pseudomonas, Bifidobacterium and other species. The gut microbiota structures of the three stingless bees were different, and the abundances of bacterial species in the gut varied between communities of the same bee species. The reasons for this are manifold and may include food preference, age and genetic differences. In addition, the abundances of Lactobacillus, Carnimonas, Escherichia-Shigella, Acinetobacter and other species were high in the beebread of stingless bees. In conclusion, our findings reveal the bacteria composition and structure of the gut and beebread of stingless bees in China and deepen our understanding of the dominant bacteria of the gut and beebread of stingless bees.

The Succession of the Gut Microbiota in Insects: A Dynamic Alteration of the Gut Microbiota During the Whole Life Cycle of Honey Bees (Apis cerana).

The Asian honey bee Apis cerana is a valuable biological resource insect that plays an important role in the ecological environment and agricultural economy. The composition of the gut microbiota has a great influence on the health and development of the host. However, studies on the insect gut microbiota are rarely reported, especially studies on the dynamic succession of the insect gut microbiota. Therefore, this study used high-throughput sequencing technology to sequence the gut microbiota of A. cerana at different developmental stages (0 days post emergence (0 dpe), 1 dpe, 3 dpe, 7 dpe, 12 dpe, 19 dpe, 25 dpe, 30 dpe, and 35 dpe). The results of this study indicated that the diversity of the gut microbiota varied significantly at different developmental stages (ACE, P = 0.045; Chao1, P = 0.031; Shannon, P = 0.0019; Simpson, P = 0.041). In addition, at the phylum and genus taxonomic levels, the dominant constituents in the gut microbiota changed significantly at different developmental stages. Our results also suggest that environmental exposure in the early stages of development has the greatest impact on the gut microbiota. The results of this study reveal the general rule of gut microbiota succession in the A. cerana life cycle. This study not only deepens our understanding of the colonization pattern of the gut microbiota in workers but also provides more comprehensive information for exploring the colonization of the gut microbiota in insects and other animals.

Exposure to benzo[a]pyrene triggers distinct patterns of microRNA transcriptional profiles in aquatic firefly Aquatica wuhana (Coleoptera: Lampyridae).

Larval aquatic fireflies in fresh water are adversely affected by water pollutants such as benzo(a)pyrene (BaP). However, their response to BaP stress at the microRNA (miRNA)-regulatory level remains unknown. Here, transcriptomes containing 31,872 genes and six miRNA transcriptional profiles were obtained for Aquatica wuhana larvae, and comparative analysis was performed between larvae exposed to BaP (0.01 mg/L) and unexposed controls. Fifteen of 114 miRNAs identified via bioinformatics were detected as differentially expressed (DEMs) upon BaP exposure. Analysis results of predicted target genes of DEM suggests that BaP exposure primarily triggered transcriptional changes of miRNA associated with five major regulatory categories: 1) osmotic balance, 2) energy metabolic efficiency, 3) development, 4) xenobiotic metabolism (oxidative stress), and 5) innate immune response. Based on six innate immune- and xenobiotic metabolism-related pathways enriched by the predicted DEM targets, 11 key BaP-responsive DEMs were further screened to investigate dynamic changes of expression in response to BaP stress at five time points, and also to validate the miRNA sequencing data using quantitative real-time PCR. This study provides valuable information for the protection of firefly resources and supplements the understanding of miRNA regulatory mechanisms in response to water deterioration.

Comparative analysis of the gut microbiota of Apis cerana in Yunnan using high-throughput sequencing.

Gut microbes play an important role in host disease and health. The Asian honey bee Apis cerana is an important pollinator of agricultural crops in China. However, there are still few studies on the structure and composition of the microbiota in the intestine of A. cerana, especially A. cerana in Yunnan. To understand the species and composition of the microbiota in the intestine of A. cerana in Yunnan, we used high-throughput sequencing technology to carry out 16S rRNA sequencing on 50 samples from Kunming, Xishuangbanna and Mengzi. The results show that both from the phylum level and the genus level, the structure and abundance of the microbiota in the gut of A. cerana from the three regions tended to be the same. At the phylum level, the abundance of Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Acidobacteria and other species was high in A. cerana from different areas. At the genus level, the abundance of Lactobacillus, Gilliamella, Snodgrassella, Apibacter, Candidatus Schmidhempelia and other species was high in A. cerana from different areas. Due to its unique geographical environment and climatic conditions, at the genus level, the diversity of bacterial communities in Xishuangbanna was significantly lower than that in the other two regions, which was about 100 genera less. In conclusion, our results reveal the composition and structure of the intestinal microbiota of bees in Yunnan and deepen our understanding of the intestinal microbiota of bees.

Comparative transcriptomic analysis of fireflies (Coleoptera: Lampyridae) to explore the molecular adaptations to fresh water.

Aquatic insects are well adapted to freshwater environments, but the molecular basis of these adaptations remains largely unknown. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of several species are aquatic. Here, larval and adult transcriptomes from Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) were generated to test whether the genes associated with metabolic efficiency and morphology have undergone adaptive evolution to fresh water. The aquatic fireflies had a significantly lower ratio of nonsynonymous to synonymous substitutions than the terrestrial insects, indicating a genomewide evolutionary constraint in the aquatic fireflies. We identified 341 fast-evolving genes and 116 positively selected genes in the aquatic fireflies. Of these, 76 genes exhibiting both fast evolution and positive selection were primarily involved in ATP production, energy metabolism and the hypoxia response. We identified 7,271 differentially expressed genes (DEGs) in A. leii (adults versus larvae) and 8,309 DEGs in L. praetexta (adults versus larvae). DEGs specific to the aquatic firefly (n = 1,445) were screened via interspecific comparisons (A. leii versus L. praetexta) and were significantly enriched for genes involved in metabolic efficiency (e.g., ATP production, hypoxia, and immune responses) and certain aspects of morphology (e.g., cuticle chitin, tracheal and compound eye morphology). These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies. This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.

The impact of mercury on the genome-wide transcription profile of zebrafish intestine.

Mercury is a widely used heavy metal that causes pollution to aquatic environments and severely affects the health of fish. Little is known about how heavy metal pollutants affect fish, particularly for gene expression within important organs such as the intestine. Herein, whole transcriptome sequencing was performed on zebrafish (Danio rerio) intestine tissue after HgCl2 (HGC, 30 µg/L) exposure. A total of 2,257 differentially expressed genes (DEGs) were identified, including 1,788 up- and 469 down-regulated genes. Functional enrichment analysis revealed that these DEGs were primarily related to xenobiotic biodegradation, biomacromolecule metabolism, development, oxidative defense, and immune response. Ten key HGC-responsive DEGs were screened to survey the dynamic changes of expression in response to HGC exposure at different time points, and were also used to validate RNA sequencing data using quantitative real-time PCR (qPCR). Results indicate that the expression of genes encoding UGT1AB, GSTT1B, GSTO1, GSTM2, UGT5G1, GSTT1A, GSTR, GSTM3, GSTA1, and GSTP2 were significantly upregulated in response to the HGC exposure, and potentially help to counteract the adverse effects of HGC. This study provides insight into fish molecular toxicological responses to heavy metals and method on environmental risk assessment.

Colonization of the gut microbiota of honey bee (Apis mellifera) workers at different developmental stages.

The role of the gut microbiome in animal health has become increasingly evident. Although the structure of the gut microbiome of A. mellifera is well known, little is known about the dynamic change across different developmental stages. In this study, we explored the dynamic changes of the gut microbiota of A. mellifera at different developmental stages covering the whole life cycle using high-throughput 16S rRNA gene sequencing. The results indicated that the core (shared) gut microbiota changes significantly among different developmental stages. The diversity of the bacterial community in workers among different ages was significantly different. In addition, by comparing the core gut microbiota among different-aged workers, we found that newly emerged workers had fewer core microbiota. Three genera, Gilliamella, Frischella, and Snodgrassella, were significantly colonized at 1 day poste mergence (dpe); Lactobacillus, Bifidobacterium, Commensalibacter were significantly colonized at 3 dpe and significantly reduced with Gilliamella. Lactobacillus kunkeei and Bartonella were significantly colonized at 12 dpe and were significantly decreased with Lactobacillus helsingborgensis. Commensalibacter and Bifidobacterium were significantly decreased at 25 dpe, and Bacteroides, Escherichia-Shigella, and Porphyromonadaceae were significantly decreased between 19 and 25 dpe. Our results reveal the succession of the gut microbiota of workers from birth to senescence, which provides a theoretical basis for further exploring the roles of gut microbiota during different developmental stages.

MicroRNA profile of immune response in gills of zebrafish (Danio rerio) upon Staphylococcus aureus infection.

The gills of fish are large mucosal surfaces that are very important portals for pathogen entry. Investigations have shown that microRNAs (miRNAs) are key regulators of immune response to bacterial infections in the gills of fish; however, how miRNA expression changes in response to infection by Gram-positive bacteria remains largely unknown. To further investigate the immunological role of miRNAs in fish gills under pathogen stress induced by Gram-positive bacterial infection, this study investigated Staphylococcus aureus (SA)-induced changes in the miRNAs levels in gills of adult zebrafish (Danio rerio). miRNA microarrays were used to analyze expression profiles of known miRNA in the gills of zebrafish in response to SA infection and compared these to uninfected control fish. A total of 30 differentially expressed miRNAs (DEMs) were identified. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses were performed. The results indicated that DEM targets were primarily involved in innate immune processes, apoptosis, defense responses, and antibacterial responses. Pathways involving bacterial infection, innate immunity, metabolic process, disease, and apoptosis were mediated by DEMs. Furthermore, real-time quantitative PCR experiments for nine key SA-responsive DEMs that regulated the "SA infection" pathway validated the accuracy of microarray results. Dynamic variations in gene expression were surveyed in detail for these key SA-responsive DEMs for PBS control and at 6, 12, 24, and 48 h after SA challenge in detail. This study provides novel insight into the mechanisms underlying the miRNA regulation during the SA-induced immune response in zebrafish gills, and provides basic knowledge on the innate immune response against Gram-positive bacterial infection in bony fish.

Genome-wide transcriptional response of microRNAs to the benzo(a)pyrene stress in amphioxus Branchiostoma belcheri.

Amphioxus, a cephalochordate found in sand habitats in shallow in-shore seawaters, has been widely used as a model in comparative immunology of chordates. However, the role of microRNAs (miRNAs) in amphioxus under abiotic stress, particularly xenobiotics with strong toxicity, remains largely unknown. Here, a widespread marine contaminant, benzo(a)pyrene (BaP) is used to evaluate its toxic effects on miRNA expression of amphioxus. Six small RNA libraries were sequenced from Branchiostoma belcheri. A total of 144 known and 157 novel miRNAs were identified using deep sequencing and bioinformatics approaches. A total of 58 differentially expressed miRNAs (DEMs) were screened, including 25 up- and 33 down-regulated DEMs under BaP stress. Target genes possibly regulated by DEMs were predicted, and their functional enrichment analyses were performed. Targets of DEMs are primarily involved in xenobiotic and cellular homeostasis, catabolic and transport process. They could be largely linked to nine immune- and toxin detoxification-related pathways, including metabolism of xenobiotics by cytochrome P450, drug metabolism-other enzymes, and drug metabolism-cytochrome P450, etc. Furthermore, quantitative real-time PCR (qRT-PCR) analysis for 12 key BaP-responsive DEMs validates the accuracy of deep sequencing. Experiments were then conducted to investigate their expression responses to BaP stress at different time intervals in detail to further determine their expression dynamic in responses of B. belcheri towards BaP exposure. This study, to the best of our knowledge, investigates the regulatory roles of miRNAs in the toxicological response of amphioxus for the first time, providing valuable information for the protection of lone existing cephalochordate amphioxus.

A Phylogenomic Framework and Divergence History of Cephalochordata Amphioxus.

Amphioxus, or cephalochordates, are often used as the living invertebrate proxy of vertebrate ancestors and are widely used as evolutionary biology models of chordates. However, their phylogeny, divergence history, and speciation characteristics remain poorly understood, and phylogenomic studies to explore these problems lacking entirely from the literature. Here, we determined a new transcriptome of Branchiostoma japonicum. Combined with mass sequences of all other 18 species, a 19-way phylogeny was constructed via multiple methods (ML, BI, PhyloBayes, and ASTRAL), consistently supporting a phylogeny of [(B. belcheri + B. japonicum) + (B. lanceolatum + B. floridae) + Asymmetron lucayanum] in amphioxus. Congruent phylogenetic signals were found across mitochondrial genes, 12S RNA, and complete mitochondrial genomes according to previous reports, indicating that 12S RNA may have potential as a molecular marker for phylogenetic analysis in amphioxus. Molecular dating analysis indicated a radiation of the cephalochordates during the Cretaceous (∼104-61 million years ago), supporting an association between the diversification and speciation of cephalochordates with continental drift and associated changes in their respective habitats during this time. The identified functional enrichment analysis for species-specific domains indicated that their function mainly involves immune response, apoptosis, and lipid metabolism and utilization, signaling that pathogens and changes of energy requirements are an important driving force for amphioxus speciation. This study represents the first large-scale phylogenomic analysis of most major amphioxus genera based on phylogenomic data, providing a new perspective on both phylogeny and divergence speciation of cephalochordates.