Computational protocol: Relative Abundance of Carsonella ruddii (Gamma Proteobacterium) in Females and Males of Cacopsylla pyricola (Hemiptera: Psyllidae) and Bactericera cockerelli (Hemiptera: Triozidae)

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Protocol publication

[…] DNA was extracted from all samples using a modified cetyltrimethlyammonium bromide (CTAB) method (, ). The insects were ground in 500 µl of CTAB buffer using a micropestle in a 1.5 ml microfuge tube. The samples were incubated in 65°C for 30 min and then maintained at room temperature for 3 min before adding 500 µl of ice-cold chloroform. After centrifugation at 16,000 × g for 10 min, the aqueous layer was transferred to 0.6 volume of isopropanol containing 1 µl of glycogen. Samples were held on ice for 20 min to precipitate DNA. After centrifugation at 16,000 × g for 10 min, the resulting pellet was washed twice with ice-cold 70% ethanol, air-dried, and dissolved in 50 µl of nuclease-free water. Samples used for qPCR were precipitated in isopropanol for a second time and dissolved in 50 µl of nuclease-free water.To design qPCR primers, a region of 16S of Carsonella was first amplified using DNA from a group of five field-collected C. pyricola or five B. cockerelli obtained from the colony. Approximately 930-bp region of 16S was amplified using the universal primers for eubacteria, 27F-AGA GTT TGA TCM TGG CTC AG and 1494R-TAC GGC TAC CTT GTT ACG AC according to . The excised PCR products were purified using GenElute minus EtBr spin columns (Sigma, St. Louis, MO) and cloned using a TOPO TA cloning kit with TOP10 Escherichia coli chemically competent cells (Invitrogen, Carlsbad, CA). Plasmid DNA was extracted from selected colonies using the QIAprep spin mini prep kit (Qiagen, Valencia, CA), and DNA clones were sequenced by MC Laboratories (MC Lab, San Francisco, CA). Sequences were analyzed using BLASTn () function on the NCBI website (http://www.ncbi.nlm.nih.gov/, last accessed 2 March 2015). Sequences identified as Carsonella (GenBank accessions KR045611 and KR045612) were used to design qPCR primers for Carsonella of C. pyricola and B. cockerelli using Primer3Plus software (). The Carsonella-specific PCR primers were CarF-AAG AAG AGA TTA GAA TTT C and CarR-AAA TAG TTG ACA TCG TTT AC. To confirm the target specificity of this primer set, conventional PCR was performed in 10 µl reactions using Invitrogen Amplitaq Gold 360 PCR Master Mix, 5 µM forward and reverse primers, and DNA isolated from five C. pyricola or five B. cockerelli adults. The thermal-cycling conditions were 95°C for 10 min, 40 cycles for 95°C of 30 s, 60°C for 30 s, and 72°C for 35 s, followed by 72°C for 7 min. The resulting 170-bp PCR products from each psyllid species were cloned, sequenced, and analyzed as described above (three clones per adult).The quantity and quality of DNA from experimental insects (see “Insects” subsection) was analyzed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA); none of the samples exhibited a 260/280 ratio above 1.7. Real-time qPCR was performed on an Applied Biosystems 79HT real-time PCR system (Applied Biosystems, Waltham, MA). Each 20 µl reaction included 10 µl of SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA), 1 µl of each primer (final concentration of 250 nM), 10 ng/µl template DNA, and nuclease-free water. The PCR conditions were 40 cycles of 95°C for 15 s and 60°C for 1 min. Three technical replications were included for each sample, and each technical replication was performed on a separate plate. A 10-fold serial dilution from 20 to 0.04 ng/µl of vector+16 S insert was used to determine the relationship between copies of 16S of Caronella and CT. The concentration of copies of 16S of Carsonella was estimated according to , and linear regression (PROC REG; SAS Institute 2012, Cary, NC) was used to model the relationship between relative abundance of Carsonella and CT. The resulting linear regression model was used to estimate the number of copies of 16S of Carsonella present in each experimental insect from the mean CT values. The efficiency of the qPCR reactions was determined using the equation, efficiency = 10(−1/slope) − 1. To estimate repeatability of the assay, the coefficient of variation of the average CT values of technical replications calculated as the ratio of the standard deviation to mean.Carsonella titers within whole insects were compared among insect sexes and ages using the GLIMMIX procedure of SAS 9.3. For each analysis, the log of the estimated number of copies of 16S of Carsonella was included as the dependent variable. Sex, age, and the sex by age interactions were included as the fixed effects in analysis of Carsonella in C. pyricola. For analysis of Carsonella titers in B. cockerelli, sex was included as the fixed effect, and repetition was included as a random variable. For each analysis, data were examined for heterogeneity of variance and nonnormality of errors by inspecting residual and normal quantile-quantile plots, respectively. Based on these plots, data were modeled assuming a Gaussian distribution using the DIST=G option of the MODEL statement. […]

Pipeline specifications

Software tools BLASTN, Primer3
Application qPCR
Organisms Candidatus Carsonella ruddii, Bactericera cockerelli