Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection.

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs' expression between uninoculated and A. apis-inoculated larval guts were confirmed based on molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. c. cerana larvae against A. apis invasion. KEY POINTS: • The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection. • Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis-challenged host guts.

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.

Regulatory Roles of Long Non-Coding RNAs Relevant to Antioxidant Enzymes and Immune Responses of Apis cerana Larvae Following Ascosphaera apis Invasion.

Long non-coding RNAs (lncRNAs) play an essential part in controlling gene expression and a variety of biological processes such as immune defense and stress-response. However, whether and how lncRNAs regulate responses of Apis cerana larvae to Ascosphaera apis invasion has remained unclear until now. Here, the identification and structural analysis of lncRNAs in the guts of A. cerana worker larvae were conducted, and the expression profile of larval lncRNAs during the A. apis infection process was then analyzed, followed by an investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in the host response. In total, 76 sense lncRNAs, 836 antisense lncRNAs, 184 intron lncRNAs, 362 bidirectional lncRNAs, and 2181 intron lncRNAs were discovered in the larval guts. Additionally, 30 known and 9 novel lncRNAs were potential precursors for 36 and 11 miRNAs, respectively. In the three comparison groups, 386, 351, and 272 DElncRNAs were respectively identified, indicating the change in the overall expression pattern of host lncRNAs following the A. apis invasion. Analysis of cis-acting effect showed that DElncRNAs in the 4-, 5-, and 6-day-old comparison groups putatively regulated 55, 30, and 20 up- and down-stream genes, respectively, which were involved in a series of crucial functional terms and pathways, such as MAPK signaling pathway, and cell process. Analysis showed that 31, 8, and 11 DElncRNAs as potential antisense lncRNAs may interact with 26, 8, and 9 sense-strand mRNAs. Moreover, investigation of the competing endogenous RNA (ceRNA) network indicated that 148, 283, and 257 DElncRNAs were putatively regulated. The expression of target genes by targeting corresponding DEmiRNAs included those associated with antioxidant enzymes and immune responses. These results suggested that DElncRNAs played a potential part in the larval guts responding to the A. apis infection through a cis-acting manner and ceRNA mechanisms. Our findings deepen our understanding of interactions between A. cerana larvae and A. apis and offer a basis for clarifying the DElncRNA-mediated mechanisms underlying the host response to fungal invasion.

Diverse Regulatory Manners and Potential Roles of lncRNAs in the Developmental Process of Asian Honey Bee (Apis cerana) Larval Guts.

Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. Here, on the basis of our previously obtained deep-sequencing data from the 4-, 5-, and 6-day-old larval guts of A. cerana workers (Ac4, Ac5, and Ac6 groups), an in-depth transcriptome-wide investigation was conducted to decipher the expression pattern, regulatory manners, and potential roles of lncRNAs during the developmental process of A. cerana worker larval guts, followed by the verification of the relative expression of differentially expressed lncRNAs (DElncRNAs) and the targeting relationships within a competing endogenous RNA (ceRNA) axis. In the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, 527 and 498 DElncRNAs were identified, respectively, which is suggestive of the dynamic expression of lncRNAs during the developmental process of larval guts. A cis-acting analysis showed that 330 and 393 neighboring genes of the aforementioned DElncRNAs were respectively involved in 29 and 32 functional terms, such as cellular processes and metabolic processes; these neighboring genes were also respectively engaged in 246 and 246 pathways such as the Hedgehog signaling pathway and the Wnt signaling pathway. Additionally, it was found that 79 and 76 DElncRNAs as potential antisense lncRNAs may, respectively, interact with 72 and 60 sense-strand mRNAs. An investigation of competing endogenous RNA (ceRNA) networks suggested that 75 (155) DElncRNAs in the Ac4 vs. Ac5 (Ac5 vs. Ac6) comparison group could target 7 (5) DEmiRNAs and further bind to 334 (248) DEmRNAs, which can be annotated to 33 (29) functional terms and 186 (210) pathways, including 12 (16) cellular- and humoral-immune pathways (lysosome pathway, necroptosis, MAPK signaling pathway, etc.) and 11 (10) development-associated signaling pathways (Wnt, Hippo, AMPK, etc.). The RT-qPCR detection of five randomly selected DElncRNAs confirmed the reliability of the used sequencing data. Moreover, the results of a dual-luciferase reporter assay were indicative of the binding relationship between MSTRG.11294.1 and miR-6001-y and between miR-6001-y and ncbi_107992440. These results demonstrate that DElncRNAs are likely to modulate the developmental process of larval guts via the regulation of the source genes' transcription, interaction with mRNAs, and ceRNA networks. Our findings not only yield new insights into the developmental mechanism underlying A. cerana larval guts, but also provide a candidate ceRNA axis for further functional dissection.

First Characterization and Regulatory Function of piRNAs in the Apis mellifera Larval Response to Ascosphaera apis Invasion.

piRNAs are a class of small non-coding RNAs that play essential roles in modulating gene expression and abundant biological processes. To decode the piRNA-regulated larval response of western honeybees (Apis mellifera) to Ascosphaera apis infection, the expression pattern of piRNAs in Apis mellifera ligustica larval guts after A. apis inoculation was analyzed based on previously obtained high-quality small RNA-seq datasets, followed by structural characterization, target prediction, regulatory network investigation, and functional dissection. Here, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts after inoculation with A. apis, with 411 ones shared. These piRNAs shared a similar length distribution and first base bias with mammal piRNAs. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups. Targets of the DEpiRNAs were engaged in diverse pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. These targets were involved in three energy metabolism-related pathways, eight development-associated signaling pathways, and seven immune-relevant pathways such as the Jak-STAT signaling pathway. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. The effective overexpression and knockdown of piR-ame-945760 in A. apis-infected larval guts were achieved by feeding a specific mimic and inhibitor. Furthermore, piR-ame-945760 negatively regulated the expression of two target immune mRNAs, SOCS5 and ARF1, in the larval gut during the A. apis infection. These findings indicated that the overall expression level of piRNAs was increased and the expression pattern of piRNAs in larval guts was altered due to the A. apis infection, DEpiRNAs were putative regulators in the A. apis-response of A. m. ligustica worker larvae. Our data provide not only a platform for the functional investigation of piRNAs in honeybees, especially in bee larvae, but also a foundation for illuminating the piRNA-involved mechanisms underlying the host response to the A. apis infection.

ame-miR-34 Modulates the Larval Body Weight and Immune Response of Apis mellifera Workers to Ascosphara apis Invasion.

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. mellifera. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (p < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (p < 0.001), while that in the inhibitor-miR-34 group was significantly downregulated (p < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

Systematic Characterization and Regulatory Role of lncRNAs in Asian Honey Bees Responding to Microsporidian Infestation.

Long noncoding RNAs (lncRNAs) are pivotal regulators in gene expression and diverse biological processes, such as immune defense and host-pathogen interactions. However, little is known about the roles of lncRNAs in the response of the Asian honey bee (Apis cerana) to microsporidian infestation. Based on our previously obtained high-quality transcriptome datasets from the midgut tissues of Apis cerana cerana workers at 7 days post inoculation (dpi) and 10 dpi with Nosema ceranae (AcT7 and AcT10 groups) and the corresponding un-inoculated midgut tissues (AcCK7 and AcCK10 groups), the transcriptome-wide identification and structural characterization of lncRNAs were conducted, and the differential expression pattern of lncRNAs was then analyzed, followed by investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in host response. Here, 2365, 2322, 2487, and 1986 lncRNAs were, respectively, identified in the AcCK7, AcT7, AcCK7, and AcT10 groups. After removing redundant ones, a total of 3496 A. c. cerana lncRNAs were identified, which shared similar structural characteristics with those discovered in other animals and plants, such as shorter exons and introns than mRNAs. Additionally, 79 and 73 DElncRNAs were screened from the workers' midguts at 7 dpi and 10 dpi, respectively, indicating the alteration of the overall expression pattern of lncRNAs in host midguts after N. ceranae infestation. These DElncRNAs could, respectively, regulate 87 and 73 upstream and downstream genes, involving a suite of functional terms and pathways, such as metabolic process and Hippo signaling pathway. Additionally, 235 and 209 genes co-expressed with DElncRNAs were found to enrich in 29 and 27 terms, as well as 112 and 123 pathways, such as ABC transporters and the cAMP signaling pathway. Further, it was detected that 79 (73) DElncRNAs in the host midguts at 7 (10) dpi could target 321 (313) DEmiRNAs and further target 3631 (3130) DEmRNAs. TCONS_00024312 and XR_001765805.1 were potential precursors for ame-miR-315 and ame-miR-927, while TCONS_00006120 was the putative precursor for both ame-miR-87-1 and ame-miR-87-2. These results together suggested that DElncRNAs are likely to play regulatory roles in the host response to N. ceranae infestation through the regulation of neighboring genes via a cis-acting effect, modulation of co-expressed mRNAs via trans-acting effect, and control of downstream target genes' expression via competing endogenous RNA networks. Our findings provide a basis for disclosing the mechanism underlying DElncRNA-mediated host N. ceranae response and a new perspective into the interaction between A. c. cerana and N. ceranae.

Unveiling the circRNA-Mediated Immune Responses of Western Honey Bee Larvae to Ascosphaera apis Invasion.

Western honey bee (Apis mellifera), a eusocial insect with a superior economic and ecological value, is widely used in the beekeeping industry throughout the world. As a new class of non-coding RNAs (ncRNAs), circular RNAs (circRNAs) participate in the modulation of considerable biological processes, such as the immune response via diverse manners. Here, the identification, characteristic investigation, and molecular verification of circRNAs in the Apis mellifera ligustica larval guts were conducted, and the expression pattern of larval circRNAs during the Ascosphaera apis infection was analyzed, followed by the exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 2083 circRNAs in the larval guts of A. m. ligustcia were identified, with a length distribution ranging from 106 nt to 92,798 nt. Among these, exonic circRNAs were the most abundant type and LG1 was the most distributed chromosome. Additionally, 25, 14, and 30 up-regulated circRNAs as well as 26, 25, and 62 down-regulated ones were identified in the A. apis-inoculated 4-, 5-, and 6-day-old larval guts in comparison with the corresponding un-inoculated larval guts. These DEcircRNAs were predicted to target 35, 70, and 129 source genes, which were relative to 12, 23, and 20 GO terms as well as 11, 10, and 27 KEGG pathways, including 5 cellular and humoral immune pathways containing apoptosis, autophagy, endocytosis, MAPK, Toll, and Imd signaling pathways. Furthermore, complex competing endogenous RNA (ceRNA) regulatory networks were detected to be formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The Target DEmRNAs were engaged in 24, 20, and 25 functional terms as well as 62, 80, and 159 pathways, including several vital immune defense-associated pathways, namely the lysosome, endocytosis, phagosome, autophagy, apoptosis, MAPK, Jak-STAT, Toll, and Imd signaling pathways. Finally, back-splicing sites within 15 circRNAs and the difference in the 9 DEcircRNAs' expression between un-inoculated and A. apis-inoculated larval guts were confirmed utilizing molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions, but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. m. ligustica larvae against A. apis invasion.

ALD-induced TiO2/Ag nanofilm for rapid surface photodynamic ion sterilization.

The daily life of people in the intelligent age is inseparable from electronic device, and a number of bacteria on touch screens are increasingly threatening the health of users. Herein, a photocatalytic TiO2/Ag thin film was synthesized on a glass by atomic layer deposition and subsequent in situ reduction. Ultraviolet-visible (UV-Vis) spectra showed that this film can harvest the simulated solar light more efficiently than that of pristine TiO2. The antibacterial tests in vitro showed that the antibacterial efficiency of the TiO2/Ag film against S. aureus and E. coli was 98.2% and 98.6%, under visible light irradiation for 5 min. The underlying mechanism was that the in-situ reduction of Ag on the surface of TiO2 reduced the bandgap of TiO2 from 3.44 to 2.61 eV due to the formation of Schottky heterojunction at the interface between TiO2 and Ag. Thus, TiO2/Ag can generate more reactive oxygen species for bacterial inactivation on the surface of electronic screens. More importantly, the TiO2/Ag film had great biocompatibility with/without light irradiation. The platform not only provides a more convenient choice for the traditional antibacterial mode but also has limitless possibilities for application in the field of billions of touch screens.

Noggin, an inhibitor of bone morphogenetic protein signaling, antagonizes TGF-ß1 in a mouse model of osteoarthritis.

Osteoarthritis (OA) is the most common joint disease worldwide; however, disease-modifying treatments are lacking because of the complicated pathological mechanisms. As a breakthrough, aberrant activation of transforming growth factor-ß 1 (TGF-ß1)in subchondral bone has been confirmed as an essential pathomechanism for OA progression, and has become a potential therapeutic target. In addition to R&D on neutralizing antibodies, small-molecule antagonists and chemical medicines, native antagonists of TGF-ß1 could be exploited as another promising approach. Noggin (NOG) is an antagonist of bone morphogenetic proteins (BMPs) and was reported to effectively attenuate OA by protecting cartilage and preventing pathological subchondral bone remodeling. However, the underlying mechanisms have not been fully clarified. We first detected the distribution of NOG in knee joints of an OA mouse model, which showed upregulation at early stage of OA but downregulation later in the subchondral bone and no significant change in the articular cartilage. Furthermore, the interaction between NOG and TGF-ß1 was verified, which in turn suppressed the downstream SMAD2/3 activity of TGF-ß1. Moreover, the proliferation and chondrogenesis of mesenchymal stem cells (MSCs) were not significantly influenced by NOG. Taken together, the results showed that NOG antagonized TGF-ß1 but did not repress MSC proliferation and chondrogenesis; thus, it seems promising for OA treatment.