Study On The Localization Of SAS-6 And Detection Method Of Nosema Bombycis

Microsporidia are a group of obligate intracellular eukaryotes that infect almost all of vertebrates and invertebrates.Nosema bombycis is the first identified microsporidia and is the main pathogen causing pebrine disease.Because it can be transmitted through the food and eggs,which often brings huge economic losses to the sericulture industry,affecting the stability of the silk industry.Therefore pebrine is listed as the only stipulated quarantine objective in silkworm eggs production.To date,microscopic check to female silkworm moths against N.bombycis has been the main inspection method in practice.However,its accuracy is affected by many factors.It is essential to establish a simple,rapid and accurate N.bombycis detection technique for the prevention and treatment of pebrine disease.In this article,we first optimized the traditional fluorescent staining method to detect N.bombycis by using Fluorescent Brightener 28(FB28)and Propidium Iodide(PI).The results show that the mature spores were stained in blue by FB28 and their nucleus were stained in red by PI,but the immature spores could only be stained by PI.N.bombycis spores could be stained and discriminated in infected midguts of larvae,eggs,female moths and newly-hatched silkworm by using this method,moreover N.bombycis spores could also be discriminated in infected female moths diluted 1000 times.The nucleus of spores stained were smaller than the nucleus of midgut cells stained and the former is brighter than the latter.The chitin debris shapes of eggs,moths and newly-hatched silkworm were different from spores and the chitin debris could not be stained by PI.N.bombycis spores were difficult to be observed from 1 d to 2 d after midguts of larvae were infected.At the point of3 d,a few immature spores in local position were discriminated.At the point of 4 d,mature spores in local position were stained and discriminated.From 5 d to 6 d,many mature and immature spores in large areas were detected.These results suggest that this method is suitable for the early diagnosis of silkworms infected with N.bombycis.Using this method can filter out distractions of Bombyx mori cells and chitin debris to improve the detection accuracy and can also improve the sensitivity of detection in the case of low concentration of N.bombycis.Although the fluorescence staining method is more accurate and sensitive than the ordinary microscopy method,the observation requires a fluorescence microscope and also disadvantage including long staining time.In order to further simplify the detection process and improve detection sensitivity and accuracy,this paper combined immunological techniques,molecular biology techniques and AuNPs to establish a simple and efficient method for N.bombycis detection.We first combined the immunological technique and colorimetric nanogold to establish a new detection assay for N.bombycis.Total proteins of N.bombycis spores were extracted by glass bead crushing,and the prepared antibody can specifically detect a variety of N.bombycis proteins.The optimum pH with prepared gold nanoparticle probes was 8.5.The optimum amount of protein with prepared AuNPs(20 nm)probes was 5μL 0.5 mg/mL antibody per 500μL solution.The colorimetric assay could detect the lowest amount of N.bombycis protein 10 ng.Although the AuNPs colorimetric method is accurate and sensitive,and the detection results can be visually identified,there are also disadvantages such as time-consuming sample processing and loss of sample protein.To further optimize detection methods,we combined LAMP and colorimetric nanogold to establish the LAMP-AuNP method,which is a simple and sensitive detection assay for N.bombycis.The DNA templates of microsporidia were extracted by FTA card.Nucleic acid sequences that are complementary to the nucleotide sequence of the target are modified to the surface of AuNPs by thiolation.The amplification product can be visually detected via hybridization at 63°C for 5 min with a ssDNA-labelled nanogold probe,followed by salt-induced AuNP aggregation.This method needs approximately 65 min.LAMP-AuNP could detect 10 spores per ml while PCR assay only could detect 10~2 spores per ml.When LAMP-AuNP was used to detect N.bombycis in silkworm eggs,the limit for infected silkworm eggs was 700,in which only one egg was infected with N.bombycis while that of PCR assay is 200 silkworm eggs.The LAMP-AuNP assay was only specific for N.bombycis and can eliminate bacteria,fungi,virus interference.Centrioles in eukaryotes function as the cell’s microtubule-organizing center(MTOC)to nucleate spindle assembly.The evolutionarily conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis.Microsporidia possess the spindle plaque instead of centriole as their MTOC to nucleate spindle assembly.However,little is known about the components of spindle plaques in microsporidia.In our present study,we identified the SAS-6 in the microsporidium Nosema bombycis for the first time and named it as NSAS-6.The NSAS-6 gene contains a complete ORF of 1104 bp in length that encodes a 367-amino acid polypeptide.NSAS-6 consists of a conserved N-terminal domain and a coiled-coil domain.The high identity of SAS-6homologous sequences from microsporidia indicates that SAS-6 is a conserved protein in microsporidia.Immunolocalization in sporoplasms,intracellular stages and mature spores showed that NSAS-6 probably localizes to the nucleus of N.bombycis and exists throughout the life cycle of N.bombycis.Moreover,we found that the NSAS-6 replicates during nuclear division in the early proliferative stage of N.bombycis.These results suggest that NSAS-6 is required in cell morphogenesis and cell division in N.bombycis.The function and structure of NSAS-6 should be the focus for further studies,which is essential to elucidate the role of SAS-6 in spindle plaque assembly.