Comparative identification of microRNAs inApis cerana ceranaworkers’ midguts responding toNosema ceranaeinvasion

Abstract

30 MicroRNAs (miRNAs) are endogenous small noncoding RNAs that post 31 transcriptionally regulate gene expression and are involved in many biological 32 processes including host-pathogen interactions. However, the potential role of 33 miRNAs in the responses of eastern honeybees to Nosema ceranae invasion is 34 completely unknown. Here, the expression profiles and differentially expressed 35 miRNAs (DEmiRNAs) in the midguts of Apis cerana cerana workers 7 and 10 days 36 post infection (dpi) with N. ceranae were investigated via small RNA sequencing and 37 bioinformatics. In total, 529 miRNAs highly conserved between various species and 38 25 novel miRNAs with varied expressions were identified for the first time. In 39 addition, stem-loop RT-PCR confirmed the expression of 16 predicted miRNAs, 40 validating their existence. Eight up-regulated miRNAs and six down-regulated 41 miRNAs were detected in midguts at 7 dpi, while nine and three miRNAs were 42 significantly up-regulated and down-regulated, respectively, in midguts at 10 dpi. In 43 addition, Venn analysis showed that five DEmiRNAs were shared, while nine and 44 seven DEmiRNAs were specifically expressed in midguts at 7 and 10 dpi, 45 respectively. Gene ontology analysis suggested that a portion of the DEmiRNAs and 46 corresponding target genes were involved in various biological processes, cellular 47 components, and molecular functions including immune system processes and 48 response to stimulus and signaling. Moreover, KEGG pathway analysis shed light on 49 the potential functions of some DEmiRNAs in the regulation of target genes engaged 50 in material and energy metabolism, cellular immunity such as endocytosis and 51 phagosome, and the humoral immune system, including the Jak-STAT and MAPK 52 signaling pathways. Further investigation demonstrated a complex regulation network 53 between DEmiRNAs and their target mRNAs, with miR-598-y, miR-252-y, miR-92-x 54 and miR-3654-y at the center of the network, implying their key parts in host 55 responses. This comprehensive miRNA transcriptome analysis demonstrated that N. 56 ceranae invasion influenced the expression of miRNAs in the midguts of A. c. 57 ceranae workers; the results can not only facilitate future exploration of the regulatory 58 roles and mechanisms of miRNAs in hosts' responses, especially their immune 59 3 responses to N. ceranae, but also provide potential candidates for further investigation 60 of the molecular mechanisms underlying eastern honeybee-microsporidian 61 interactions. 62

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

“…1C). The shared miRNA profile is from normal and N. ceranae -infected midguts of A. c. cerana workers [1]. On average, more than 12.54 Mb raw reads in each group were yielded from sRNA-seq, and over 12.02 Mb (95.83%) clean reads were gained after strict filtering and quality control (Table 1).…”

“…Small RNA libraries were constructed according to the general protocol [1]. Briefly, total RNA of each midgut sample in N. ceranae -treated and control groups were extracted using TRIzol Reagent followed by removal of DNA contaminants; only values of 28S/18S ≥ 0.7 and RIN ≥7.0 were considered qualified for the subsequent small RNA library construction.…”

“…Additionally, Pearson correlation coefficients between different biological replicas within each control and N. ceranae -infected group were above 0.9768 and 0.9912, respectively (Fig. 2) [1]. In total, 14 differentially expressed miRNAs (DEmiRNAs) were observed in midgut at 7 days post inoculation (dpi) with N. ceranae (AcT1) compared with corresponding normal midgut (AcCK1), including eight up-regulated and six down-regulated miRNAs (Table 2); while 12 miRNA with differential expressions were detected in midgut at 10 dpi with N. ceranae (AcT2) compared with corresponding normal midgut (AcCK2), including nine up-regulated and three down-regulated ones (Table 3).…”

Nanopore-based genome-wide DNA methylation sequencing data of Nosema ceranae-inoculated and un-inoculated eastern honeybee workers’ midguts

“…The inoculation and rearing of A. c. cerana workers as well as midgut sample preparation were performed according to the established procedure in our lab [4, 5]. Briefly, (1) To offer newly emerged Nosema-free workers, combs of sealed brood from a healthy colony were kept in an incubator at 34 ± 0.5 °C, 50% RH; the emergent workers were seriously transferred into cages in groups (n=35) and kept in the incubator at 32 ± 0.5 °C, 50% RH.…”

Identification and Comparative Profiling of microRNAs at Different Diapause Stages of Galeruca Daurica Adults

“…MicroRNAs (miRNAs) are a class of small noncoding RNAs of approximately 22 nt in length, which regulate gene expression at the post-transcriptional level by targeting sites in mRNA, and have been shown to regulate a variety of physiological processes throughout insect development [4]. Up to date, thousands of miRNAs have been identi ed from insect species, including Drosophila melanogaster [5], Bombyx mori [6,7], Locusta migratoria [8], Manduca sexta [9][10][11], Plutella xylostella [12,13], Helicoverpa armigera [14,15], Mayetiola destructor [16], Nilaparvata lugens [17], Blattella germanica [18], Aphis gossypii [19], Lissorhoptrus oryzophilus [20], Leptinotarsa decemlineata [21], Grapholita molesta [22], Atrijuglans hetaohei [23], Apis cerana cerana [24], Laodelphax striatellus [25], and Plodia interpunctella [26]. More recent evidence has shown that miRNAs may play an important role in regulation of insect diapause.…”