Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.

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.

Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts.

Circular RNAs (circRNAs) are a class of novel non-coding RNAs (ncRNAs) that play essential roles in the development and growth of vertebrates through multiple manners. However, the mechanism by which circRNAs modulate the honey bee gut development is currently poorly understood. Utilizing the transcriptome data we obtained earlier, the highly expressed circRNAs in the Apis mellifera worker 4-, 5-, and 6-day-old larval guts were analyzed, which was followed by an in-depth investigation of the expression pattern of circRNAs during the process of larval guts development and the potential regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 1728 expressed circRNAs were detected in the A. mellifera larval guts. Among the most highly expressed 10 circRNAs, seven (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the 4-, 5-, and 6-day-old larval guts. In addition, 21 (46) up-regulated and 22 (27) down-regulated circRNAs were, respectively, screened in the Am4 vs. Am5 (Am5 vs. Am6) comparison groups. Additionally, nine DEcircRNAs, such as novel_circ_000340, novel_circ_000758 and novel_circ_001116, were shared by these two comparison groups. These DEcircRNAs were predicted to be transcribed from 14 and 29 parental genes; these were respectively annotated to 15 and 22 GO terms such as biological regulation and catalytic activity as well as 16 and 21 KEGG pathways such as dorsoventral axis formation and apoptosis. Moreover, a complicated competing endogenous RNA (ceRNA) network was observed; novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs engaged in several developmental signaling pathways (e.g., TGF-beta, hedgehog, and wnt signaling pathway) and immune pathways (e.g., phagosome, lysosome, and MAPK signaling pathway). The results demonstrated that the novel_circ_000838-ame-miR-6000a-3p axis may plays a critical regulatory part in the larval gut development and immunity. Furthermore, back-splicing sites of six randomly selected DEcircRNAs were amplified and verified by PCR; an RT-qPCR assay of these six DEcircRNAs confirmed the reliability of the used high-throughput sequencing data. Our findings provide a novel insight into the honey bee gut development and pave a way for illustration of the circRNA-modulated developmental mechanisms underlying the A. mellifera worker larval guts.

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.

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.

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.

Complete mitochondrial genome and phylogenetic analysis of Huangshan Black chicken (Gallus gallus).

In this study, we sequenced the complete mitochondrial genome (mitogenome) of the Huangshan Black chicken (HBC). Results showed that the complete HBC mitogenome was 16,785 bp in size, comprising 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and 1 non-coding control region (D-loop). The overall nucleotide composition was 32.5% for C, 30.3% for A, 23.7% for T, and 13.5% for G. Phylogenetic analysis showed that the HBC mitogenome was clustered with Xianju chicken, which belonged to the haplogroup D2. Our results therefore demonstrate that the origin of HBC corresponds to haplogroup D2 distribution and might have at least one maternal lineage originated from Southeast Asia.

Expression of genes related to lipid transport in meat-type ducks divergent for low or high residual feed intake.

OBJECTIVE: This study examined the effects of divergence in residual feed intake (RFI) on expression profiles of key genes related to lipid transport in the liver and duodenal epithelium and their associations with feed efficiency traits in meat-type ducks. METHODS: A total of 1,000 male ducks with similar body weight (1,042.1±87.2 g) were used in this study, and their individual RFI was calculated from 21 to 42 d of age. Finally, the 10 highest RFI (HRFI) and 10 lowest RFI (LRFI) ducks were chosen for examining the expression of key genes related to lipid transport in the liver and duodenal epithelium using quantitative polymerase chain reaction. RESULTS: In the liver, expression levels of albumin (ALB), CD36 molecule (CD36), fatty acid hydroxylase domain containing 2 (FAXDC2), and choline kinase alpha (CHKA) were significantly higher in LRFI ducks than in HRFI ducks (p<0.01); negative correlations (p<0.05) between expression levels of ALB, CD36, FAXDC2, and CHKA and RFI were detected in the liver. Additionally, ALB expression was strongly positively correlated (p<0.05) with CD36, FAXDC2, CHKA, and apolipoprotein H (APOH) expression in the liver. In duodenal epithelium, we found that mRNA levels of ALB, CD36, FAXDC2, and APOH were significantly higher in LRFI ducks than in HRFI ducks (p<0.01); RFI was strongly negatively correlated (p<0.05) with ALB, FAXDC2, and APOH expression, while ALB expression was strongly positively correlated with APOH expression (p<0.01) in duodenal epithelium. Furthermore, expression levels of both ALB and FAXDC2 genes were significantly associated with feed conversion ratio and RFI in both liver and duodenal epithelium (p<0.05). CONCLUSION: Our findings therefore suggest that ALB and FAXDC2 genes might be used as potential gene markers designed to improve feed efficiency in future meat-type duck breeding programs.

Influence of free-range days on growth performance, carcass traits, meat quality, lymphoid organ indices, and blood biochemistry of Wannan Yellow chickens.

This study investigated the effects of free-range days on growth performance, carcass traits, meat quality, lymphoid organ indices, and blood biochemical parameters of Wannan Yellow chickens. A total of 1,000 one-day-old male Wannan Yellow chickens were reared to 56 D. At 56 D of age, 960 birds with similar body weight (BW) were randomly allocated to free-range treatment at 56, 70, 84, and 98 D of age (assigned to free-range treatment for 42, 28, 14, and 0 D, respectively); 6 replicates with 40 chickens per treatment. In the free-range system, chickens were reared in indoor floor pens with an outdoor free-range paddock measuring 4 × 7 m (28 m2, 1.5 birds/m2). Results showed that BW of birds decreased significantly in the first 2 wk after birds were assigned to free-range treatment compared with those in the conventional treatment (P < 0.05). Average daily gain (ADG) and average daily feed intake (ADFI) decreased significantly (P < 0.05) for chickens assigned to free-range treatment from 56 to 70 D of age, while feed conversion ratio increased significantly (P < 0.05). Breast yield increased linearly with increasing free-range days (P < 0.05), whereas leg and foot yields decreased linearly (P < 0.05). Drip loss and L* value of thigh muscle decreased linearly with increasing free-range days (P < 0.05), while shear force improved linearly (P < 0.05). Additionally, the absolute thymus weight and thymus to BW ratio showed significant increasing and then decreasing quadratic responses to increasing free-range days (P < 0.05). Furthermore, serum glucose, total protein, cholesterol, and triglyceride content declined linearly, while high-density lipoprotein cholesterol (HDL-C) content increased linearly with increasing free-range days (P < 0.05). In conclusion, increasing free-range days had positive effects on breast yield, shear force, thymus weight, and HDL-C content, but negatively affected leg yield, foot yield, drip loss, L* value of thigh muscle, glucose, total protein, cholesterol, and triglyceride levels of Wannan Yellow chickens.

Association analysis between feed efficiency and expression of key genes of the avTOR signaling pathway in meat-type ducks.

Genes involved in the target of rapamycin (TOR) signaling pathway are implicated in nutrient translation, cell proliferation and differentiation, and anabolism, which can affect both growth and feed intake. However, the role of TOR signaling in the regulation of feed intake and feed efficiency in poultry is not clear. In the present study, a total of 1000 ducks, of similar initial weight, were chosen and transferred to individual cages to determine their residual feed intake (RFI) from the age of 21 to 42 days. Subsequently, 60 ducks, which were divided into high (HRFI) and low (LRFI) groups according to their RFI, were chosen to analyze the TOR signaling activities in the liver. The differential expression level of genes involved in the TOR signaling pathway was assayed by the real-time polymerase chain reaction. In the liver, the expression of AKT, avTOR, avLST8, and S6K1 was significantly higher in LRFI ducks than in HRFI ducks; avTOR and AKT were negatively associated with the feed conversion ratio and RFI. Furthermore, PI3K was moderately positively associated with AKT; AKT was strongly positively associated with PI3K, avTOR, avLST8, and S6K1; and avTOR was strongly positively associated with S6K1. In conclusion, the activation of avTOR signaling in the liver of LRFI ducks might be ascribed to higher energy state or more active nutrient transport (amino acids), or both, than those in the liver of HRFI ducks. The results of the present study indicate that AKT and avTOR of TOR signaling might be used as candidate genes to assess molecular regulation of feed efficiency.

A new polymorph of magnesium oxalate dihydrate.

In the asymmetric unit of the title compound, catena-poly[[diaqua-magnesium(II)]-µ-oxalato], [Mg(C(2)O(4))(H(2)O)(2)](n), there is one Mg atom in an octa-hedral coordination with site symmetry 222, a unique C atom of the oxalate anion lying on a twofold axis, an O atom of the anion in a general position and a water O atom at a site with imposed twofold rotation symmetry. The Mg(2+) ions are ligated by water mol-ecules and bridged by the anions to form chains that are held together by O-H⋯O hydrogen bonds. The structure of the title compound has already been reported in a different space group [Lagier, Pezerat & Dubernat (1969 ▶). Rev. Chim. Miner.6, 1081-1093; Levy, Perrotey & Visser (1971 ▶). Bull. Soc. Chim. Fr. pp. 757-761].

Poly[disodium [diaqua-tri-µ(2)-oxalato-dimagnesium(II)]].

The title compound, {Na(2)[Mg(2)(C(2)O(4))(3)(H(2)O)(2)]}(n), is isotypic with its Co analogue. There are two crystallographically independent oxalate groups in the asymmetric unit, one lying on an inversion center and the other on a general position. Mg(2+) ions are ligated by H(2)O mol-ecules and bridged by tri- and tetra-dentate oxalate ligands, forming ladder-like double chains that are held together via O-H⋯O hydrogen bonds, with Na(+) cations located between the chains to balance the charge.

Lithium dioxobis[trioxoiodato(V)]vanadate, Li[VO2(IO3)2].

The title compound represents a new structure type, in which distorted VO6 octahedra are bridged by iodate groups to form infinite two-dimensional [VO2(IO3)2]- layers that are separated by octahedrally coordinated Li+ cations.

Gallium tris(iodate), Ga(IO3)3.

Ga(IO(3))(3) crystallizes in the space group P6(3), with the Ga atom at a site with imposed threefold symmetry. The crystal structure consists of slightly distorted GaO(6) octahedra that are bridged by I atoms of IO(3)(-) groups, giving rise to a three-dimensional polar network. The framework contains unoccupied hexagonal channels running parallel to the hexagonal [001] direction. The iodate groups have their stereochemically active non-bonded electron pairs pointing in the same direction along [001], which creates the polarity in the structure. The I-O bond distances and O-I-O angles are normal, being in the ranges 1.783 (3)-1.847 (2) A and 94.68 (11)-99.61 (12) degrees , respectively.

Cerium triiodate, Ce(IO(3))(3).

The crystal structure of Ce(IO3)3 consists of one-dimensional chains of edge-sharing CeO9 polyhedra which are crosslinked into two-dimensional layers through bridging IO3- groups. The layers are held together via long I...O contacts, resulting in an extended three-dimensional network. The I-O bond distances and O-I-O angles are normal, lying in the ranges 1.806 (4)-1.846 (4) A and 89.9 (2)-100.9 (2) degrees, respectively. The three crystallographically independent iodate groups all show different coordination modes.