Band 40/41 Surface Acoustic Wave Filters on 42°YX-Lithium Tantalate Substrate with Suppression of Transverse Leakage.

The transverse leakage of leaky surface acoustic waves (LSAWs) occurs on 42°YX-lithium tantalate substrates (42LT), which increases the insertion loss, narrows the bandwidth and flattens the roll-off of band 40/41 SAW filters and duplexers. In this work, LSAW characteristics with different metal materials and thicknesses are calculated by the finite element method (FEM), which determines the IDT material and thickness used for band 40/41 SAW filter design. To deeply understand transverse leakage and suppress it, the effects of different gap and dummy lengths on transverse leakage are simulated and discussed. Then, a new technique of using a wider dummy without any additional lithographic or depositing processes is proposed to suppress the leakage. Its effectiveness is validated by both simulations and experiments. Then, the technique is extended to applications of band 40 and 41 SAW filters. The experimental results show that with the wider dummy structure, the band 40 and 41 SAW filters achieve a more than 0.2 dB improvement in the insertion loss, a wider bandwidth and a steeper roll-off characteristic. This technique may also be extended to the other band SAW filter applications.

CircRNA-regulated immune responses of asian honey bee workers to microsporidian infection.

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana workers' midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 days (AcT1) and 10 days (AcT2) post inoculation days post-inoculation with N. ceranae. PCR amplification result verified the back-splicing sites within three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691, and 3723 respectively. Further, 83 52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group. Source genes of DEcircRNAs in workers' midgut at seven dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA (ceRNA) network analysis showed that 23 13) DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group could target 18 14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 164) internal ribosome entry site and 54 26) ORFs were identified from DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at seven dpi with N. ceranae were associated with several cellular immune pathways including endocytosis and ubiquitin-mediated proteolysis. Ultimately, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers' midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.

Deciphering the CircRNA-Regulated Response of Western Honey Bee (Apis mellifera) Workers to Microsporidian Invasion.

Vairimorpha ceranae is a widespread fungal parasite of adult honey bees that leads to a serious disease called nosemosis. Circular RNAs (circRNAs) are newly discovered non-coding RNAs (ncRNAs) that regulate biological processes such as immune defense and development. Here, 8199 and 8711 circRNAs were predicted from the midguts of Apis mellifera ligustica workers at 7 d (Am7T) and 10 d (Am10T) after inoculation (dpi) with V. ceranae spores. In combination with transcriptome data from corresponding uninoculated midguts (Am7CK and Am10CK), 4464 circRNAs were found to be shared by these four groups. Additionally, 16 circRNAs were highly conserved among A. m. ligustica, Apis cerana cerana, and Homo sapiens. In the Am7CK vs. Am7T (Am10CK vs. Am10T) comparison group, 168 (306) differentially expressed circRNAs (DEcircRNAs) were identified. RT-qPCR results showed that the expression trend of eight DEcircRNAs was consistent with that in the transcriptome datasets. The source genes of DEcircRNAs in Am7CK vs. Am7T (Am10CK vs. Am10T) were engaged in 27 (35) GO functional terms, including 1 (1) immunity-associated terms. Moreover, the aforementioned source genes were involved in three cellular immune-related pathways. Moreover, 86 (178) DEcircRNAs in workers' midguts at 7 (10) dpi could interact with 75 (103) miRNAs, further targeting 215 (305) mRNAs. These targets were associated with cellular renewal, cellular structure, carbohydrate and energy metabolism, and cellular and humoral immunity. Findings in the present study unraveled the mechanism underlying circRNA-mediated immune responses of western honey bee workers to V. ceranae invasion, but also provided new insights into host-microsporidian interaction during nosemosis.

Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome utilizing PacBio long reads combined with Illumina short reads.

Ascosphaera apis is a widespread fungal pathogen of honeybee larvae that results in chalkbrood disease, leading to heavy losses for the beekeeping industry in China and many other countries. This work was aimed at generating a full-length transcriptome of A. apis using PacBio single-molecule real-time (SMRT) sequencing. Here, more than 23.97 Gb of clean reads was generated from long-read sequencing of A. apis mycelia, including 464,043 circular consensus sequences (CCS) and 394,142 full-length non-chimeric (FLNC) reads. In total, we identified 174,095 high-confidence transcripts covering 5141 known genes with an average length of 2728 bp. We also discovered 2405 genic loci and 11,623 isoforms that have not been annotated yet within the current reference genome. Additionally, 16,049, 10,682, 4520 and 7253 of the discovered transcripts have annotations in the Non-redundant protein (Nr), Clusters of Eukaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, 1205 long non-coding RNAs (lncRNAs) were identified, which have less exons, shorter exon and intron lengths, shorter transcript lengths, lower GC percent, lower expression levels, and fewer alternative splicing (AS) evens, compared with protein-coding transcripts. A total of 253 members from 17 transcription factor (TF) families were identified from our transcript datasets. Finally, the expression of A. apis isoforms was validated using a molecular approach. Overall, this is the first report of a full-length transcriptome of entomogenous fungi including A. apis. Our data offer a comprehensive set of reference transcripts and hence contributes to improving the genome annotation and transcriptomic study of A. apis.

Transcriptome data of control and Ascosphaera apis infected Apis mellifera ligustica larval guts.

Ascosphaera apis is an obligate fungal pathogen of honeybee larvae that leads to chalkbrood, which causes heavy losses for the apiculture in China and many other countries. In this article, guts of 4-, 5-, 6-day-old Apis mellifera ligustica larvae challenged by A. apis (AmT1, AmT2, AmT3) and normal 4-day-old larval guts (AmCK) were sequenced using next-generation sequencing technology. On average, 29,196,197, 28,690,943, 29,779,715 and 30,496,725 raw reads were yielded from these four groups; an average of 29,540,895 clean reads were obtained after quality control. In addition, the mapping ratio of clean reads in treatment and control groups to the Apis mellifera genome were over 97.16%. For more insight please see "Uncovering the immune responses of Apis mellifera ligustica larval gut to Ascosphaera apis infection utilizing transcriptome sequencing" [1]. The raw data were submitted to the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession numbers: SRR4084091, SRR4084092, SRR4084095, SRR4084096, SRR4084097, SRR4084098, SRR4084099, SRR4084100, SRR4084101, SRR4084102, SRR4084093, SRR4084094.

Systematic identification of circular RNAs and corresponding regulatory networks unveil their potential roles in the midguts of eastern honeybee workers.

Currently, knowledge of circular RNAs (circRNAs) in insects including honeybee is extremely limited. Here, differential expression profiles and regulatory networks of circRNAs in the midguts of Apis cerana cerana workers were comprehensively investigated using transcriptome sequencing and bioinformatics. In total, 9589 circRNAs (201-800 nt in length) were identified from 8-day-old and 11-day-old workers' midguts (Ac1 and Ac2); among them, 5916 (61.70%) A. cerana cerana circRNAs showed conservation with our previously indentified circRNAs in Apis mellifera ligucstica workers' midguts (Xiong et al., Acta Entomologica Sinica 61:1363-1375, 2018). Five circRNAs were confirmed by RT-PCR and Sanger sequencing. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451, and novel_circ_001980 were highly expressed in both Ac1 and Ac2. In addition, the source genes of circRNAs were involved in 34 GO terms including organelle and cellular process and 141 pathways such as endocytosis and Wnt signaling pathway. Moreover, 55 DEcircRNAs including 34 upregulated and 21 downregulated circRNAs were identified in Ac2 compared with Ac1. circRNA-miRNA regulatory networks indicated that 1060 circRNAs can target 74 miRNAs; additionally, the DEcircRNA-miRNA-mRNA networks suggested that 13 downregulated circRNAs can bind to eight miRNAs and 29 miRNA-targeted mRNAs, while 16 upregulated circRNAs can link to 9 miRNAs and 29 miRNA-targeted mRNAs. These results indicated that DEcircRNAs as ceRNAs may play a comprehensive role in the growth, development, and metabolism of the worker's midgut via regulating source genes and interacting with miRNAs. Notably, eight DEcircRNAs targeting miR-6001-y were likely to be key participants in the midgut development. Our findings not only offer a valuable resource for further studies on A. cerana cerana circRNA and novel insights into understanding the molecular mechanisms underlying the midgut development of eastern honeybee but also provide putative circRNA candidates for functional research in the near future and novel biomarkers for identification of eastern honeybee species including A. cerana cerana and honeybee diseases such as chalkbrood and microsporidiosis.

MicroRNA dataset of normal and Nosema ceranae-infected midguts of Apis cerana cerana workers.

Nosema ceranae is a widespread fungal pathogen of honeybees, which is infective to all castes in the colony, including queens, drones and workers. Nosemosis caused by N. ceranae poses a big challenge for apiculture all over the world. Here, midguts of normal and N. ceranae-infected Apis cerana cerana workers at 7 and 10 days post infection were sequenced utilizing small RNA sequencing (sRNA-seq) technology. Totally, more than 150.54 Mb raw reads were produced in this article, and over 144.26 Mb high-quality clean reads with a mean ratio of 95.83% were obtained after strict filtering and quality control. For more insight please see "Comparative identification of microRNAs in Apis cerana cerana workers' midguts responding to Nosema ceranae invasion" (Chen et al., 2019). Raw data are available in NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA487111. Our data can be used for investigating differentially expressed microRNAs (miRNAs) and piRNAs and their regulatory roles engaged in A. c. cerana response to N. ceranae infection, and for offering potential candidates for uncovering the molecular mechanisms regulating eastern honeybee-microsporidian interactions.

A comprehensive transcriptome data of normal and Nosema ceranae-stressed midguts of Apis mellifera ligustica workers.

Honeybees are pivotal pollinators of crops and wild flora, and of great importance in supporting critical ecosystem balance. Nosema ceranae, a unicellular fungal parasite that infects midgut epithelial cells of honeybees, can dramatically reduce honeybee population and productivity. Here, midguts of Apis mellifera ligustica workers at 7 d and 10 d post inoculation (dpi) with sucrose solution (Ac7CK and Ac10CK) and midguts at 7 dpi and 10 dpi with sucrose solution containing N. ceranae spores (Ac7T and Ac10T) were sequenced using strand-specific cDNA library construction and next-generation sequencing. A total of 1956129858 raw reads were gained in this article, and after quality control, 1946489304 high-quality clean reads with a mean Q30 of 93.82% were obtained. The rRNA-removed clean reads were then aligned to the reference genome of Apis mellifera with TopHat2. For more insight please see "Genome-wide identification of long non-coding RNAs and their regulatory networks involved in Apis mellifera ligustica response to Nosema ceranae infection" [1]. Raw data were deposited in NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA406998. These data can be used for comparative analysis to identify differentially expressed coding RNAs and non-coding RNAs involved in A. m. ligustica responses to N. ceranae stress, and for investigation of molecular mechanisms regulating host N. ceranae -response.

Genome-Wide Identification of Long Non-Coding RNAs and Their Regulatory Networks Involved in Apis mellifera ligustica Response to Nosema ceranae Infection.

Long non-coding RNAs (lncRNAs) are a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins, and lncRNAs have been proven to play pivotal roles in a wide range of biological processes in animals and plants. However, knowledge of expression patterns and potential roles of honeybee lncRNA response to Nosema ceranae infection is completely unknown. Here, we performed whole transcriptome strand-specific RNA sequencing of normal midguts of Apis mellifera ligustica workers (Am7CK, Am10CK) and N. ceranae-inoculated midguts (Am7T, Am10T), followed by comprehensive analyses using bioinformatic and molecular approaches. A total of 6353 A. m. ligustica lncRNAs were identified, including 4749 conserved lncRNAs and 1604 novel lncRNAs. These lncRNAs had minimal sequence similarities with other known lncRNAs in other species; however, their structural features were similar to counterparts in mammals and plants, including shorter exon and intron length, lower exon number, and lower expression level, compared with protein-coding transcripts. Further, 111 and 146 N. ceranae-responsive lncRNAs were identified from midguts at 7-days post-inoculation (dpi) and 10 dpi compared with control midguts. Twelve differentially expressed lncRNAs (DElncRNAs) were shared by Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups, while the numbers of unique DElncRNAs were 99 and 134, respectively. Functional annotation and pathway analysis showed that the DElncRNAs may regulate the expression of neighboring genes by acting in cis and trans fashion. Moreover, we discovered 27 lncRNAs harboring eight known miRNA precursors and 513 lncRNAs harboring 2257 novel miRNA precursors. Additionally, hundreds of DElncRNAs and their target miRNAs were found to form complex competitive endogenous RNA (ceRNA) networks, suggesting that these DElncRNAs may act as miRNA sponges. Furthermore, DElncRNA-miRNA-mRNA networks were constructed and investigated, the results demonstrated that a portion of the DElncRNAs were likely to participate in regulating the host material and energy metabolism as well as cellular and humoral immune host responses to N. ceranae invasion. Our findings revealed here offer not only a rich genetic resource for further investigation of the functional roles of lncRNAs involved in the A. m. ligustica response to N. ceranae infection, but also a novel insight into understanding the host-pathogen interaction during honeybee microsporidiosis.

Comparative Identification of MicroRNAs in Apis cerana cerana Workers' Midguts in Responseto Nosema ceranae Invasion.

Here, the expression profiles and differentially expressed miRNAs (DEmiRNAs) in the midguts of Apis cerana cerana workers at 7 d and 10 d post-inoculation (dpi) with N. ceranae were investigated via small RNA sequencing and bioinformatics. Five hundred and twenty nine (529) known miRNAs and 25 novel miRNAs were identified in this study, and the expression of 16 predicted miRNAs was confirmed by Stem-loop RT-PCR. A total of 14 DEmiRNAs were detected in the midgut at 7 dpi, including eight up-regulated and six down-regulated miRNAs, while 12 DEmiRNAs were observed in the midgut at 10 dpi, including nine up-regulated and three down-regulated ones. Additionally, five DEmiRNAs were shared, while nine and seven DEmiRNAs were specifically expressed in midguts at 7 dpi and 10 dpi. Gene ontology analysis suggested some DEmiRNAs and corresponding target mRNAs were involved in various functions including immune system processes and response to stimulus. KEGG pathway analysis shed light on the potential functions of some DEmiRNAs in regulating target mRNAs engaged in material and energy metabolisms, cellular immunity and the humoral immune system. Further investigation demonstrated a complex regulation network between DEmiRNAs and their target mRNAs, with miR-598-y, miR-252-y, miR-92-x and miR-3654-y at the center. Our results can facilitate future exploration of the regulatory roles of miRNAs in host responses to N. ceranae, and provide potential candidates for further investigation of the molecular mechanisms underlying eastern honeybee-microsporidian interactions.

Genome-Wide Identification of Circular RNAs in Fungal Parasite Nosema ceranae.

Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs (ncRNAs) that play key roles in microRNA function and transcriptional regulation. Though a large number of circRNAs had been identified in animals and plants, however, little is known regarding circRNAs in Nosema ceranae, a widespread fungal parasite of honeybee. In this study, using deep sequencing technology and bioinformatic analysis, we predicted 204 circRNAs from N. ceranae spore samples, including 174 exonic circRNAs and 30 intergenic circRNAs. In addition, the expression of seven N. ceranae circRNAs was confirmed by RT-PCR assay. Furthermore, regulation networks of circRNAs were constructed, and 15 circRNAs were found to act as sponges of the corresponding three miRNAs. GO categorization and pathway enrichment analysis suggested that the circRNAs are likely to play significant roles in N. ceranae spore. This is the first report of circRNAs generated by a microsporidia species. Our results provide novel insights into understanding the basic biology of N. ceranae.

Systematic investigation of circular RNAs in Ascosphaera apis, a fungal pathogen of honeybee larvae.

Ascosphaera apis is a widespread fungal pathogen of honeybee larvae, which causes heavy losses in apiculture. To date, knowledge about non-coding RNA (ncRNA) including circular RNA (circRNA) in A. apis is lacking. In this study, A. apis mycelia and spores were sequenced using RNA-seq technology. A total of 551 circRNAs were predicted on the basis of bioinformatic analyses, and most of the circRNAs were 200-600 bp in length, which were different from animal and plant circRNAs. In addition, the expression of six circRNAs in A. apis were confirmed using divergent and convergent PCR. Moreover, circRNA-microRNA regulation networks in A. apis were constructed, and further investigation showed that A. apis circRNAs could regulate gene expression by competitively binding miRNAs. GO and KEGG pathway enrichment analyses of the miRNAs target genes of circRNAs demonstrated that these A. apis circRNAs are likely to play key roles in metabolism, environmental response and gene expression.