Computational protocol: The Gut Entomotype of Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) and Their Effect on Host Nutrition Metabolism

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Protocol publication

[…] The variable region 4 (V4) of the bacterial 16S rRNA gene was amplified with the general 16S rRNA primers 515F (5′-GTGYCAGCMGCCGCGGTA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR reactions were carried out in a total volume of 30 μl which comprised 15 μl Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM forward and reverse primers and 10 ng template DNA. The thermal conditions set for PCR were as follows: initial denaturation for 1 min at 98°C followed by 30 cycles of denaturation for 10 s at 98°C, annealing for 30 s at 50°C and elongation for 60 s at 72°C, then a final extension for 5 min at 72°C. The amplified PCR products were mixed with the same volume of 1× buffer containing SYBR green and run on a 2% agarose gel. Next, DNA from an aliquot of each PCR reaction was purified using a Qiagen Gel Extraction Kit and equal volumes of the three reaction products per sample were mixed together and the sequencing libraries were generated with TrueSeq® DNA PCR-Free Sample Preparation Kit (Illumina). The libraries were sequenced using MiSeq platform after quality assessment on the [email protected] 2.0 fluorometer (Thermo Scientific) and Agilent Bio-analyzer 2100 system.The paired-end sequencing reads were merged through FLASH (V1.2.7) (). The raw tags were strictly filtered (<30 Phred score) to obtain high-quality clean tags using Quantitative Insight into Microbial Ecology (QIIME) software (V1.7.0) (; ) and were aligned with Gold database by running the UCHIME algorithm to remove the chimera for effective tags ().Operational taxonomic units (OTUs) were clustered using Uprase (V7.0.1001) to group the effective tags into OTUs with ≥97% sequence identity (ID) (). The most abundant unique sequence of each OTU cluster was selected as representative, and taxonomy of the non-chimaeric sequences was assigned by RDP Classifier (version 2.2) () using GreenGene database with default settings (). To investigate the species richness and community diversity of samples or groups, six diversity indices, Chao 1, ACE, Shannon, and Simpson’s were calculated with the software package QIIME, version 1.7.0. Beta diversity analysis of RPW gut bacterial communities was carried out through PCoA (Principal Coordinate Analysis) and PCA (Principal Component Analysis) using QIIME (Version 1.7.0). ANOSIM (Analysis of similarity) was used to estimate the differences between gut bacterial communities associated with RPW at different life stages and larvae from different host plants. [...] The larvae of R. ferrugineus were collected from the infested coconut palms C. nucifer in Hainan, China in mid-May 2014. The specimens were transported in plastic boxes along with host plant tissue to our laboratory. Upon arrival, larvae guts were dissected as above and homogenized by hand. The gut homogenates were poured on nutrient agar (NA) media in triplicate after serial dilution (10-1 to 10-6), and were incubated aerobically at 30°C for 24 h. Based on their color, size, and morphology, 200 single colonies were picked and repeatedly streaked on NA media to obtain pure cultures (). The purified single colonies were inoculated in nutrient broth (NB) and incubated at 37°C for 24 h. Thereafter, 500 μl bacterial solutions of each colony were processed for DNA extraction with the TIANamp Stool DNA Kit DP 328 (TIANGEN) according to the manufacturer’s instructions. The bacterial species were identified as described by with the universal bacterial primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492 R (5′-TACGGYTACCTTGTTACGACTT-3′). The taxonomy of each 16S rRNA gene sequence was assigned with NCBI BLAST and Sequence Match in the Ribosomal DNA Project (RDP). Our representative bacterial sequences and those retrieved from the GeneBank database were aligned with ClustalW using MEGA 5.05 software.To determine the ability of bacterial isolates to degrade cellulose, each of them was streaked on CMC (carboxymethyl-cellulose, Sigma) agar media and the plates were aerobically incubated at 37°C for 14 days. After growth, the incubated plates were covered with Congo red dye solution (). After dye removal, cellulose degradation was indicated by the clear zone around the colonies. The cellulolytic index was calculated based on the ratio of the diameter of the clear zone to the diameter of the bacterial colony. [...] Differences in the abundance of gut bacteria across developmental stages and host species at family and genus level, the cellulolytic ability of isolated bacterial species and that of nutrition indices were assessed by analysis of variance (ANOVA). The differences in abundance of gut bacteria and body weight between CK and AT groups were statistically detected by independent t-test. All analyses were performed using IBM SPSS Statistics (22.0). The significance level to threshold was set at 0.05 (P < 0.05). […]

Pipeline specifications

Software tools QIIME, UCHIME, RDP Classifier, BLASTN, Clustal W, SPSS
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Rhynchophorus ferrugineus
Chemicals Glucose