Identification, Diversity and Evolution of MITEs in the Genomes of Microsporidian Nosema Parasites.

Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous DNA transposons, which are widespread in most eukaryotic genomes. However, genome-wide identification, origin and evolution of MITEs remain largely obscure in microsporidia. In this study, we investigated structural features for de novo identification of MITEs in genomes of silkworm microsporidia Nosema bombycis and Nosema antheraeae, as well as a honeybee microsporidia Nosema ceranae. A total of 1490, 149 and 83 MITE-related sequences from 89, 17 and five families, respectively, were found in the genomes of the above-mentioned species. Species-specific MITEs are predominant in each genome of microsporidian Nosema, with the exception of three MITE families that were shared by N. bombycis and N. antheraeae. One or multiple rounds of amplification occurred for MITEs in N. bombycis after divergence between N. bombycis and the other two species, suggesting that the more abundant families in N. bombycis could be attributed to the recent amplification of new MITEs. Significantly, some MITEs that inserted into the homologous protein-coding region of N. bombycis were recruited as introns, indicating that gene expansion occurred during the evolution of microsporidia. NbS31 and NbS24 had polymorphisms in different geographical strains of N. bombycis, indicating that they could still be active. In addition, several small RNAs in the MITEs in N. bombycis are mainly produced from both ends of the MITEs sequence

The Microsporidian Polar Tube and Spore Wall

All of the members of the microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. This chapter reviews the data on the organization, structure, and function of this invasion organelle. The application of immunological and molecular techniques and recent genome sequencing data has resulted in the identification of multiple polar tube and spore wall proteins (SWPs). The interactions of these identified proteins in the formation and function of the polar tube and spore wall remain to be determined. Inside the spore, the polar tube is filled with material and is often termed the polar filament; however, this chapter uses the term polar tube to refer to this structure when it is within the spore as well as when it forms a hollow tube after germination and is found outside the spore. The chapter presents details on the spore activation and discharge

Several rounds of amplifications of MITE families <i>in N</i>. <i>bombycis</i>.

<p>(A) The distribution profile of genetic distance for copies of MITE families. X-axis represents the interval of genetic distance; Y-axis represents the numbers of any pairwise copies. (B) The tree diagram for the copies of <i>Nbh2</i> families with neighbor-joining (NJ) method.</p

MITE-derived small RNAs in <i>Nosema bombycis</i>.

<p>(A) Length distribution of small RNAs generated by MITE sequences. (B) Density (sense, black; antisense, red) of small RNA tags assigned to MITE sequences. Frequency is shown along the Y-axis. Relative nucleotide position within the consensus sequence is indicated along the X-axis.</p

Presence/absence of MITEs polymorphisms in four geographical strains of <i>N</i>. <i>bombycis</i>.

<p>(A) The product of <i>NbS3</i>-inserted PCR amplification in four strains, SD1 and SD2 represent one pair of segmental duplication in <i>N</i>. <i>bombycis</i>. (B) The product of <i>NbS24</i>-inserted PCR amplification in three strains and the illustration. M, DNA marker, CQ1: Chongqing isolate, YN: Yunnan isolate, GD: Guangdong isolate, GX: Guangxi isolate. Black arrowhead: MITE-inserted PCR-amplified products. Gray arrowheads: PCR-amplified product without MITEs insertion. Rectangular arrowheads: genes in the syntenic region among several <i>N</i>. <i>bombycis</i> isolates and <i>N</i>. <i>antheraeae</i>. Same color rectangles correspond to homologous genes.</p

Lipidomic Profiling Reveals Distinct Differences in Sphingolipids Metabolic Pathway between Healthy Apis cerana cerana larvae and Chinese Sacbrood Disease

Chinese sacbrood disease (CSD), which is caused by Chinese sacbrood virus (CSBV), is a major viral disease in Apis cerana cerana larvae. Analysis of lipid composition is critical to the study of CSBV replication. The host lipidome profiling during CSBV infection has not been conducted. This paper identified the lipidome of the CSBV–larvae interaction through high-resolution mass spectrometry. A total of 2164 lipids were detected and divided into 20 categories. Comparison of lipidome between healthy and CSBV infected-larvae showed that 266 lipid species were altered by CSBV infection. Furthermore, qRT-PCR showed that various sphingolipid enzymes and the contents of sphingolipids in the larvae were increased, indicating that sphingolipids may be important for CSBV infection. Importantly, Cer (d14:1 + hO/21:0 + O), DG (41:0e), PE (18:0e/18:3), SM (d20:0/19:1), SM (d37:1), TG (16:0/18:1/18:3), TG (18:1/20:4/21:0) and TG (43:7) were significantly altered in both CSBV_24 h vs. CK_24 h and CSBV_48 h vs. CK_48 h. Moreover, TG (39:6), which was increased by more than 10-fold, could be used as a biomarker for the early detection of CSD. This study provides evidence that global lipidome homeostasis in A. c. cerana larvae is remodeled after CSBV infection. Detailed studies in the future may improve the understanding of the relationship between the sphingolipid pathway and CSBV replication

Diversity of Bacterial Communities Associated with Solitary Bee Osmia excavata Alfken (Hymenoptera: Megachilidae)

Insect-associated microorganisms play important roles in the health and development of insects. This study aimed to investigate the similarities and differences in bacterial community structure and composition between the larval gut of Osmia excavata, nest soil, and brood provision from the nest tube. We sequenced larvae gut and their environments&rsquo; microorganisms of O. excavata from four locations based on full-length 16S rRNA gene amplicons. The results showed 156, 280, and 366 bacterial OTUs from gut, brood provision, and nest soil, respectively, and three groups shared 131 bacterial OTUs. In the gut, the top two dominant bacteria were Sodalis praecaptivus (68.99%), Lactobacillus micheneri (17.95%). In the brood provision, the top two dominant bacteria were S. praecaptivus (26.66%), Acinetobacter nectaris (13.05%), and in the nest soil, the two most abundant bacteria were Gaiella occulta (4.33%), Vicinamibacter silvestris (3.88%). There were significant differences in diversity between the brood provision groups and the nest soil groups, respectively. Three of the four locations did not differ for gut microbial diversity. Bacteria similar to other solitary bees also existed in the gut of the larvae. Results indicated when the habitat environments were similar, the bacterial community diversity of the gut of O. excavata was similar, despite significant differences among brood provisions and soils, respectively

Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while β-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research

Expression of <i>β</i>-Tubulin.

<p>(A) Validation of pCold I-<i>β-tubulin</i> vector by PCR and Bam HI/Sal I enzyme digestion. ~1300 bp products were amplified by PCR or cleaved from recombinant vector. (B) SDS-PAGE of proteins expressed in <i>Escherichia coli</i> Rosetta. Recombinant <i>β-</i>Tubulin protein was induced to express at 37°C and 16°C. pCold I vector transformed <i>E</i>. <i>coli</i> Rosetta were induced for expression at 37°C as a control. (C) Immunoblot for <i>β-</i>Tubulin in total protein of <i>Nosema bombycis</i> mature spore. The antibody recognized a 50 kDa band which was consistent with prediction.</p

Location of <i>β</i>-Tubulin in intracellular microsporidian <i>N</i>. <i>bombycis</i>.

<p>Images were taken by laser scanning confocal microscopy using filter sets for Alexa fluo 488 labeling <i>β-</i>Tubulin proteins and DAPI staining nucleus. Immunofluorescence assay with <i>β-</i>Tubulin antiserum demonstrated that the membrane and cell plasma location contained in <i>N</i>. <i>bombycis</i> cells in the proliferative phase. (Bars = 5 μm)</p