متاژنومیکس میکروبی: مطالعه میکروبیوم یک نماتد بومی مرتبط با حشرات از طریق تکنولوژی توالی‌یابیNGS

The metagenomic technique, part of next-generation sequencing (NGS) technology, offers an ideal and culture-independent approach. This method presents new possibilities to uncover the microbial composition of various organisms, including entomopathogenic and entomophilic nematodes, and untangle the complex interactions between these nematodes and their microbiomes. The authors' previous investigations have already revealed the capabilities of the indigenous nematode isolate, Acrobeloides sp., in terms of penetration, reproductive capacity, and notable virulence against some economically important pests such as Zeuzera pyrina. To acquire a comprehensive understanding of the bacterial communities associated with the infective juveniles (IJs) of the native Acrobeloides nematode, the microbiota of the IJs was profiled through NGS metabarcoding amplicon sequencing. According to the manufacturer's protocol, genomic DNA was extracted from the tissues using the DNeasy Blood and Tissue kit. Subsequently, the concentration and purity of the extracted DNA were evaluated using a NanoDrop 2000 spectrophotometer, and the integrity of the DNA was confirmed through agarose gel electrophoresis (1% gel). The Illumina MiSeq platform was utilized for the metagenomic DNA library preparation and downstream NGS process, targeting amplicons from the V3 and V4 hypervariable regions of the 16S rDNA to amplify a 466-bp portion. Following quality control on the raw read fastq files, sequences with low quality, as well as adapter and primer sequences, were removed. The cleaned paired-end reads were then analyzed using QIIME 2. The Amplicon Sequence Variants (ASV) were taxonomically assigned with the SILVA 138 SSURef NR99 taxonomy database, as a reference database, to analyze the microbial communities present in this nematode across various taxonomic levels, encompassing phylum to species classifications. To evaluate the richness and evenness of the microbiota within the Acrobeloides IJs sample, alpha diversity was calculated using three different indices: observed Operational Taxonomic Units (OTUs), Shannon diversity index, and Simpson diversity index. Analysis of the phylogenetic distribution of microbial taxa in Acrobeloides IJs demonstrated that the most abundant bacteria belonged to the phyla Proteobacteria, Bacteriodota, and Firmicutes, accounting for 42.7%, 29.4%, and 18.3% of the community, respectively. Notably, the genus Alcaligenes sp. stood out as the most abundant (11.9%), followed by Myxococcus, Acinetobacter, and Pseudomonas, which were significantly represented in the microbiome of Acrobeloides IJs. The results of evaluating the biodiversity of the Acrobeloides microbiome using the Shannon, Simpson, and Chao1 indices, along with identifying bacteria at the genus level, highlighted a rich and diverse microbial community within Acrobeloides IJs. The present study's findings represent the first record of the bacterial flora within indigenous Acrobeloides nematode populations, affirming the presence of insect-pathogenic bacteria in its microbiome.