Tribolium castaneum strains and maintenance
Wild-type Tribolium castaneum strain San Bernardino and GFP recombinant strain Pig-19 (Lorenzen et al. 2003) were reared in darkness on a whole-grain flour diet supplemented with 5 % yeast powder (basal medium) on glass Petri dishes (20 cm diameter) at 32 °C and 70 % relative humidity, as described elsewhere (Altincicek et al. 2008).
Fresh broccoli florets (Brassica oleracea) were cut into small pieces and lyophilized overnight and then ground to fine powder using a mortar and pestle. We mixed 1–100 mg of powder with enough basal medium to make the total weight up to 1 g, resulting in final concentrations of 0.1–10 % (w/w). The medium was mixed thoroughly to ensure an even distribution of the vegetable powder.
The same procedure was used to spike the feed with isolated isothiocyanates (Sigma, Deisenhofen, Germany) except that the fine chemicals were first dissolved in 70 % ethanol to make 0.2–20 μg/ml stocks, 5 ml of which was pipetted onto 6-cm Petri dishes loaded with 1 g basal medium to achieve final concentrations of 0.0001–0.01 % (w/w). The liquid was allowed to evaporate overnight under a laminar flow hood and then ground to fine powder using a mortar and pestle. Control plates were treated with 70 % ethanol only.
Measurements of food intake
Beetles were starved for 6 h before 30 beetles per group were randomly assigned to feed on one of the broccoli concentrations during 2, 4 or 6 h either at 32 °C or at 42 °C, after which individuals were frozen at −18 °C. Food intake was determined by measuring the wet weight of 5 individuals on a microbalance with 6 replicates per concentration per species.
Life span determination
Synchronized adult beetles were obtained by distributing 30 pupae per plate (two plates per condition) on Petri dishes (92 mm diameter) containing the basal medium (control) or basal media supplemented either with broccoli or with isothiocyanates. Pupae were hatched completely after 4 days, and beetles remained on the plates thereafter at 32 °C and 70 % humidity. Beetles were transferred to freshly prepared media every 14 days and scored for dead beetles directly before the media changes.
For the thermotolerance assay, beetles were hatched and treated as described above. Media changes were done here every 7 days. After 14 days of feeding at 32 °C, beetles were transferred to an incubator at 42 °C at 70 % humidity, and henceforward, surviving animals were scored every 12 h.
Bacterial strains and GFP cloning
Feeding with dsRNA-expressing bacteria is a standard protocol to achieve RNAi in C. elegans. Accordingly, we evaluated the efficiency of this method in T. castaneum by constructing an E. coli clone expressing GFP dsRNA to achieve GFP knockdown in the GFP-expressing T. castaneum strain Pig-19. The GFP-coding sequence from pPD95 75 (Addgene, Cambridge, UK) was inserted into the pGEM-T-easy vector (Promega, Mannheim, Germany) using the BamHI and ApaI sites and introduced into E. coli strain TOP 10F’ (Invitrogen, Karlsruhe, Germany). A 225-bp insert was then transferred to vector L4440 (MRC GeneService Ltd., Cambridge, UK) and introduced into E. coli strain HT115 (DE3). The integrity of the construct was verified by custom sequencing with M13 and T7 sequencing primers (JLU, Microbiology Institute, Gießen, Germany). The expression of gene-specific dsRNAs was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (Sigma, Deisenhofen, Germany) for 1 h at 37 °C to trigger interference. The bacterial cells were then washed and 5 × 108 bacterial cells were mixed with 1 g of the basal flour medium after 1 h desiccation at 37 °C or after lyophilization.
Generation of gene-specific RNAi constructs
Total RNA was prepared by grinding 200 beetles under liquid nitrogen in a mortar and using 200 mg of powder for RNA extraction with the Roti-Quick kit (Roth, Karlsruhe, Germany) according to the manufacturer’s instructions. We reverse transcribed 2 μg of total RNA (1 h at 37 °C) using the M-MLV Reverse Transcription kit (Promega, Mannheim, Germany) and gene-specific reverse primers for nrf-2 (Tc-nrf-2-B3356, accession number XM_963549; 5′-GCT ACA CAT TCA CAG GGA TAC-3′), jnk-1 (Tc-jnk-1-B1172, accession number XM_969870; 5′-TAC CGC ATT GAA TCC GTC C-3′), foxo-1 (Tc-foxo-1-B1353, accession number XM_970107; 5′-ACT TCC CTC CAT ACT CAA CTC-3′); sirt-1 (Tc-sirt-1-B726, accession number XM_961975; 5′-CGC CAG TCG AAC GTA AAT CCC-3′) and sirt-3 (Tc-sirt-3-B600, accession number XM_963962; 5′-CCT ACA CGC TAA ACA ATG TCC CG-3′). The number in each primer name corresponds to its annealing position within the corresponding cDNA.
A 2-μl aliquot from each reverse transcription reaction was then used for PCR amplification with REDTaq DNA Polymerase (Sigma, Deisenhofen, Germany) and gene-specific primers containing a flanking T7 promoter sequence (5′-TAA TAC GAC TCA CTA TAG GG-3′) represented in the following sequences as ‘T7′. The primers for nrf-2 were Tc-nrf-2-F2587-T7 (5′-T7-TCG CCG TAC CAA TACAGT C-3′) and Tc-nrf-2-B3091-T7 (5′-T7-GTC AGT CAC TCG CAT TCA TC-3′). Those for jnk-1 were Tc-jnk-1-F179-T7 (5′-T7-ATG TGA CGC ACG CTA AAA G-3′) and Tc-jnk-1-B835-T7 (5′-T7-AGG GAA ACA GCA CAT CGG G-3′). Those for foxo-1 were Tc-foxo-1-F756-T7 (5′-T7-CAC CAC TCC TAG TCC TAG TTC-3′) and Tc-foxo-1-B1095 (5′-T7-ATG CGG ATA CGA AGG CGA C-3′). Those for sirt-1 were Tc-sirt-1-F221-T7 (5′-T7-AGA CCT CAC CTC ACA CTG ACC-3′) and Tc-sirt-1-B676-T7 (5′-T7-ACG AAA CAG AGA CTC CAG CGC C-3′). And those for sirt-3 were Tc-sirt-3-F52-T7 (5′-T7-CCA GAA GCA CAG TCG GAG GAA G-3′) and Tc-sirt-3-B587-T7 (5′-T7-CAA TGT CCC GTG TAA AAA GTG CC-3′).
Amplifications were carried out using a Thermocycler Primus 25 (peqlab, Erlangen, Germany) and each reaction comprised an initial denaturation at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C (cycles 1–5), 60 °C (cycles 6–10) or 65 °C (cycles 11–35) for 30 s and extension at 72 °C for 90 s followed by a final elongation at 72 °C for 10 min. PCR products were verified by agarose gel electrophoresis and inserted into vector pCR-2.1 using the TOPO-TA-cloning kit (Invitrogen, Karlsruhe, Germany). Constructs were introduced into E. coli strain TOP10F’ (included in the kit), and their integrity was verified by reamplification and sequencing.
dsRNA synthesis and injection
Gene-specific DNA templates in the pCR-2.1 vector were amplified by PCR using 10 ng of the gene-specific RNAi plasmid and corresponding primer pairs as listed above. A fragment of the Galleria mellonella insect metalloproteinase inhibitor (IMPI) gene was amplified as a control using primers 5′-T7-AGA CGG TGG AGC CTG CGA TAA TG-3′ and 5′-T7-AGA CGA CGG TGG AGG GGA GTC AA-3′. PCR products were purified with the MinElute PCR Purification Kit (Qiagen, Hilden, Germany), and 1 μg of purified template DNA was used to produce dsRNA by in vitro transcription using the MEGAscript®RNAi kit (Ambion, Austin, USA) according to the manufacturer’s instructions. RNA concentration was determined by spectrophotometry using the Nanodrop ND-1000 (peqlab, Erlangen, Germany), and RNA integrity was monitored by 1.4 % denaturing agarose gel electrophoresis.
For RNAi experiments, approximately 0.1 μg of the indicated dsRNA (1 μg/μl) was injected laterally between abdominal segments 3 and 4 into pupae by using a nanoliter 2000 microinjector (WPI, Sarasota, USA). After injection, pupae were transferred to 6-cm Petri dishes containing basal medium with or without supplements.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from 80 beetles per group using Trizol reagent (Invitrogen). One-step real-time RT-PCR was carried out in triplicate using 1 μl of RNA template, the Brilliant II SYBR Green 1-step Q-RT-PCR-Kit and appropriate primers in a CFX 96 Real-Time PCR Detection System (BioRad, München, Germany). Changes in the target gene expression were calculated according to Pfaffl (2001) using equation 2−ΔΔCT. For each sample, the fold change in the target gene was normalized to rpS18 RNA and was expressed in relation to control gene expression. To determine RNAi efficiency, unique primer pairs recognizing only cDNA derived from endogenous mRNA were designed to avoid cross-reaction between genomic DNA and the recombinant dsRNA. The primers for rpS18 were Tc-rpS18-F100 (5′-GCA AAG TCA TGT TTG CCC TCA C-3′) and Tc-rpS18-B226 (5′-TTC TCG ACC TCT TCG TCG GAA C-3′), those for nrf-2 were Tc-nrf-2-F1420 (5′-CAG CAC AAC CAT ACG TAC CAC C-3′) and Tc-nrf-2-B1524 (5′-CAA ATG CTC CTC GTC ACC CTT C-3′), those for jnk-1 were Tc-jnk-1-F25 (5′-ACC GTT GAG GTG GGC GAC AC-3′) and Tc-jnk-1-B201 (5′-CGC CCT TTT AGC GTG CGT CA-3′), those for foxo-1 were Tc-foxo-1-F109 (5′-CCC AAC GAA GAG GGC AAC AAG TGC-3′) and Tc-foxo-1-B217 (5′-GGT TGC CCC AGG CGT TCC GT-3′), those for sirt-1 were Tc-sirt-1-F903 (5′-TCG GGA GCC TTT ACC GCA TTG-3′) and Tc-sirt-1-B1008 (5′-GCC TTC CTT CCA AAC ACC GCC A-3′) and those for sirt-3 were Tc-sirt-3-F190 (5′-TCC TGG GCT CGT CTT TAG TTG TCC-3′) and Tc-sirt-3-B5378 (5′-CCC CAA TCC ACG CAA CAT CCC G-3′).
Relative mRNA levels compared to the control (imagos injected with IMPI dsRNA) were estimated in dsRNA-injected imagos after feeding with the control medium over 14 days at 32 °C and were 0.21 ± 0.03 (p < 0.05) for nrf-2, 0.33 ± 0.06 (p < 0.05) for jnk-1, 0.10 ± 0.02 (p < 0.01) for foxo-1, 0.09 ± 0.04 (p < 0.01) for sirt-1 and 0.16 ± 0.04 (p < 0.01) for sirt-3.
Beetles were analyzed using a MZ16F fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with DFC300FX Leica fluorescence camera. Beetles were fixed dorsally onto adhesive foil after folding the wings to allow the detection of muscle tissue fluorescence. Digital images were recorded using the Leica Application Suite software v2.8.1.
Statistical analysis was performed with GraphPad Prism software (v5.01, GraphPad, La Jolla, CA, USA) to present the longevity data as Kaplan–Meier survival curves. Survival curves were compared using the log-rank test with a significance threshold of p < 0.05. Maximum life spans were determined as the average age of the oldest decile of surviving subjects. For statistical analysis of differences between two groups, a Student’s unpaired t test was used. For each variable, at least 3 independent experiments were carried out.