bioRxiv preprint doi: https://doi.org/10.1101/684803; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Synergic Interaction between Permethrin and Bt toxins discovered using RNA-seq Takuma Sakamoto1, Toshinori Kozaki2 and Norichika Ogata3* 1 Laboratory of Applied Entomology, Tokyo University of Agriculture and Technology, Tokyo, Japan. 2 Laboratory of Applied Entomology and Zoology, Faculty of Agriculture, Ibaraki University, Ibaraki, Japan. 3 Nihon BioData Corporation, 1-2-3 Sakado, Takatsu-ku, Kawasaki, Kanagawa, Japan. *Corresponding Author: [email protected] Abstract—Acting against the development of sometimes associated with increased susceptibility resistance to antibiotics and insecticides, involving to a second chemical1. This interaction phenomenon negatively correlated cross-resistance (NCR) is an is termed Negatively correlated cross-resistance alternative to use- and-discard approach. It is termed (NCR). New information on NCR would be useful in NCR that toxic chemicals interact with each other and integrated pest management. Up to now, several resistance of target organisms to one chemical is methods have been reported for screening and sometimes associated with increased susceptibility to a development of NCR toxins. However, only 17 toxin second chemical when; an allele confers resistance to one toxic chemical and hyper-susceptibility to another, pairs have been revealed to cause NCR in insects NCR occurs. However, only 11 toxin pairs have been (DDT and deltamethrina, DDT and phenylthioureab revealed to cause NCR in insects. Finding novel NCRs in Drosophila melanogaster, Bt toxins in Plodia is needed for integrated pest management. We interpunctella, Bt toxins Cry1Ac and Cry1F in analyzed permethrin, an insecticide, induced Helicoverpa zea, Bt toxins in Helicoverpa armigera, transcriptomes of cultured fat bodies of the silkworm Bt toxin and gossypol, Bt toxin and Steinernema Bombyx mori, a lepidopteran model insect. riobrave, Bt toxin and Heterorhabditis bacteriophora Differentially expressed gene analyses suggested in Pectinophora gossypiella, Bt and Bacillus thuringiensis (Bt) toxin was an NCR toxin of nucleopolyhedrovirus in Plutella xylostella, permethrin. NCR to permethrin and Bt toxins in Pyrethroids and N-alkylamidesc, AaIT and Thysanoplusia intermixta, the agricultural pest moth, pyrethroidsd in Musca domestica, AaIT and was examined; the children of permethrin survivor T. intermixta had increased susceptibility to Bt toxin. A pyrethroidse in Heliothis virescens, Pyrethroids and novel NCR toxin pair, permethrin and Bt toxin, was diazinonf in Haematobia irritans, N- discovered. The screening and developmental method propylcarbamate and N-methylcarbamateg, for negatively correlated cross-resistance toxins Nephotettix cincticeps, Organo-phosphates and established in this study was effective, in vitro synthetic pyrethroids in Tetranychus urticae)2. Given screening using model organisms and in vivo that next generation sequencing technology has verification using agricultural pests. recently been developed, a plan to identify resistant genes by omics analyses and seek drugs which target Keywords—Negatively correlated Cross-Resistance; Differential Expression; Digital Gene Expression; Permethrin; Bt toxins; resistant genes via a knowledge base has become Bombyx mori feasible3. Thus, it would become possible to predict NCR toxins from omics analyses and then apply I. INTRODUCTION agents to induce resistance, and then use the next Insecticides are essential in food production and medicine for the children of survivors. In this study, health protection. For instance, some 300 pesticides permethrin was selected for omics analyses. are in use to control about 900,000 different kinds of The moth, Thysanoplusia intermixta is a pest which insects living within a global population of feeds on Asteraceae4 (e.g., burdock) and 7,400,000,000. Insecticides interact with each other Umbelliferae (e.g., carrot). The genome of T. and resistance of insects to one chemical is intermixta is unknown. Here, we selected a bioRxiv preprint doi: https://doi.org/10.1101/684803; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. lepidopteran model insect, the silkworm Bombyx microscopic inspection. Infection-free tissues were mori, for omics analyses. The silkworm genome was used in the following induction assays. revealed in 20085 and is well annotated. To avoid confusion with omics analyses after surgical removal Chemicals of the guts and gut lumen, the primary cultured fat All the chemicals used in this study were of bodies of the silkworm in MGM-450 insect medium6 analytical grade. cis-permethrin (Wako Pure were used. Permethrin concentrations in the media Chemical, Osaka, Japan) and trans-permethrin followed a previous study where an appropriate (Wako Pure Chemical, Osaka, Japan) were dissolved permethrin concentration (0.25 mM) was obtained, in acetone. This solution was diluted with three times resulting in biologically significant genes by its volume of ethanol just prior to mixing with the referencing transcriptome diversity as the index of medium. The final concentrations of cis-permethrin the extent of transcriptome changes7. To investigate and trans-permethrin were 0.25 mM. No antibiotics the effects of permethrin on transcriptomes, we were used in the assays to maintain the primary sequenced 6 transcriptomes from larval fat-body culture. tissues exposed to permethrin. Freshly isolated tissues were cultured for 80 hours in MGM-450 Induction Assay insect medium, and then cultured for 10 hours in The original medium was replaced with the cis- medium supplemented with 0.25 mM cis-permethrin permethrin or trans-permethrin-containing medium or 0.25 mM trans-permethrin. The resistant gene was in the induction assays. The primary culture tissues identified by differentially expressed gene analyses. were incubated with 0.25mM cis-permethrin or Fortunately, the identified resistant gene was the trans-permethrin for 10 hours. The final target of the Bacillus thuringiensis (Bt) toxin8. The concentrations of the inducers and the duration of Bt toxin was predicted as a NCR toxin following the incubation were determined by the previous report7. permethrin. For validation of the predicted NCR Inductions were terminated by soaking the tissues in toxin, the probabilities of survival were compared for 0.75 ml of TRIzol LS (Invitrogen, CA, USA), and the natural population of Thysanoplusia. intermixta the tissues were kept at -80˚C until analyzed. and the children of permethrin survivor T. intermixta. NCR to permethrin and Bt toxins, very common RNA Isolation insecticides9, in T. intermixta is reported in this study. Total RNA was extracted using TRIzol LS after fertilization. (Invitrogen) and the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) following the II. MATERIALS AND METHODS manufacturers’ instructions as previously described7. Comparative Transcriptomics Silkworm fat bodies (30 mg) soaked in 300 µL Establishment of Primary Culture TRIzol LS were homogenized. We isolated total The p50 strain of the silkworm, Bombyx mori, was RNA from the homoginates as previously described7. grown on fresh leaves of the mulberry, Morus The integrity of rRNA in each sample was checked bombycis. The larvae of the 5th instar were using an Agilent 2100 Bioanalyzer (Agilent aseptically dissected 3 days after the 4th ecdysis and Technologies, Santa Clara, CA, USA). the fat body was isolated. More than 100 chunks of the tissue (approx. 2 mm3) were excised from the fat Library preparation and sequencing: RNA-seq bodies of 24 larvae. Those tissue particles were Sequencing was performed according to the TruSeq incubated in cell culture dishes (ø = 35 mm; BD single-end RNA-sequencing protocols from Illumina Biosciences, NJ, USA) with MGM-450 for Solexa sequencing on a Genome Analyzer IIx supplemented by 10% FBS (Biowest, Nuaillé, with paired-end module (Illumina, San Diego, CA, France) with no gas change. The tissue was cultured USA). A total of 1 µg total RNA was used as the without antibiotics for 80 hours at 25˚C. The starting material for library construction, using the infection of the microbes was checked by TruSeq RNA Sample Preparation Kit v2. This involved poly-A mRNA isolation, fragmentation, bioRxiv preprint doi: https://doi.org/10.1101/684803; this version posted June 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. and cDNA synthesis before adapters were ligated to pentamolting larvae (10 larvae were native strain and the products and amplified to produce a final cDNA 10 larvae were the permethrin-survivor strain) were library. Approximately 400 million clusters were exposed to Bt toxins (Toaro suiwa-zai CT; OAT generated by the TruSeq SR Cluster Kit v2 on the Agrio Co., Ltd., Tokyo, Japan) from Bacillus Illumina cBot Cluster Generation System, and 36–65 thuringiensis serotype kurstaki, containing Cry IAa,