Downregulation of imidacloprid resistant genes alters the biological parameters in Colorado potato beetle, Leptinotarsa decemlineata Say (chrysomelidae: Coleoptera)

Journal Pre-proof Downregulation of imidacloprid resistant genes alters the biological parameters in Colorado potato beetle, Leptinotarsa decemlineata Say (chrysomelidae: Coleoptera) Muhammad Nadir Naqqash, Ayhan Gökçe, Emre Aksoy, Allah Bakhsh PII:

S0045-6535(19)32096-X

DOI:

https://doi.org/10.1016/j.chemosphere.2019.124857

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CHEM 124857

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ECSN

Received Date: 21 July 2019 Revised Date:

10 September 2019

Accepted Date: 13 September 2019

Please cite this article as: Naqqash, M.N., Gökçe, A., Aksoy, E., Bakhsh, A., Downregulation of imidacloprid resistant genes alters the biological parameters in Colorado potato beetle, Leptinotarsa decemlineata Say (chrysomelidae: Coleoptera), Chemosphere (2019), doi: https://doi.org/10.1016/ j.chemosphere.2019.124857. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Title: Downregulation of imidacloprid resistant genes alters the biological parameters in Colorado potato beetle, Leptinotarsa decemlineata Say (Chrysomelidae: Coleoptera) Authors: Muhammad Nadir Naqqash*1, Ayhan Gökçe1, Emre Aksoy2 and Allah Bakhsh*2 1

Department of Plant Production & Technologies, Ayhan Şahenk Faculty of Agricultural

Sciences and Technologies, Niğde Omer Halisdemir University, Niğde, Turkey 2

Department of Agricultural Genetic Engineering, Ayhan Şahenk Faculty of Agricultural

Sciences and Technologies, Niğde Omer Halisdemir University, Niğde, Turkey Corresponding Author: Dr. Allah Bakhsh Department of Agricultural Genetic Engineering Faculty of Agricultural Sciences and Technologies Niğde Omer Halisdemir University, Turkey Tel: +90 507 027 4481 Email: [email protected]; [email protected]

1

Title: Downregulation of imidacloprid resistant genes alters the biological

2

parameters in Colorado potato beetle, Leptinotarsa decemlineata Say

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(Chrysomelidae: Coleoptera)

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Abstract

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Colorado potato beetle, Leptinotarsa decemlineata Say (coleoptera: chrysomelidae), is the

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important pest of potato all over the world. This insect pest is resistant to more than 50 active

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compounds belonging to various chemical groups. Potential of RNA interference (RNAi) was

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explored to knock down transcript levels of imidacloprid resistant genes in Colorado potato

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beetle (CPB) under laboratory conditions. Three important genes belonging to cuticular protein

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(CP), cytochrome P450 monoxygenases (P450) and glutathione synthetase (GSS) families

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encoding imidacloprid resistance were targeted. Feeding bio-assays were conducted on various

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stages of imidacloprid resistant CPB lab population by applying HT115 expressing dsRNA on

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potato leaflets. Survival rate of insects exposed to CP-dsRNA decreased to 4.23%, 15.32% and

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47.35% in 2nd, 3rd and 4th instar larvae respectively. Larval weight and pre-adult duration were

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also affected due to dsRNAs feeding. Synergism of RNAi with imidacloprid conducted on the

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2nd instar larvae, exhibited 100% mortality of larvae when subjected to reduced doses of GSS

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and CP dsRNAs along with imidacloprid. Utilization of

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imidacloprid resistant CPB population reveal that dsRNAs targeting CP, P450 and GSS enzymes

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could be useful tool in management of imidacloprid resistant CPB populations.

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Keywords: Colorado potato beetle, detoxification enzymes, resistance management, synergism

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1.0.

Introduction

three different dsRNAs against

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Colorado potato beetle (CPB) is the most devastating insect-pest of potato in America, Asia and

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Europe. The larvae and adults of CPB are serious defoliators of many members of Solanaceae

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family including potato, tomato, eggplant and nightshade (Jacques, 1988). Annual yield losses

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range between 30–50% due to CBP which sometimes causes no economic yield in some fields

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(Zhou et al., 2012). Heavy reliance on insecticides and co-evolution of this insect with secondary

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metabolite rich plant family have led CPB to develop amazing resistance ability against

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insecticides being actively used to manage it (Bishop and Grafius, 1996). It develops resistance

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to new insecticide shortly after its introduction at commercial level (Forgash, 1985; Mota-

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Sanchez and Wise, 2017). Enhanced resistance (> 100-folds) to insecticides has been recorded in

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a very short time e.g. only 3 generations (Ioannidis et al., 1992). Decreased susceptibility to

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neonicotinoids was reported in only 2 years on Long Island, New York, USA after their

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introductions (Zhao et al., 2000). It has developed resistance to more than 56 active ingredients

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(Mota-Sanchez and Wise, 2017).

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Resistance mechanisms of insects to synthetic and natural insecticides are diverse and their

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management requires through understanding. However, some mechanisms are common to both

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synthetic and bio-insecticides. These include decreased penetration (Argentine et al., 1994),

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target site insensitivity (Malekmohammadi and Galehdari, 2016), metabolic detoxification (Li

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et al., 2007), and increased excretion (Dermauw and Van Leeuwen, 2014); however literature

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emphasizes more on metabolic resistance among these different mechanisms (Dermauw et al.,

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2012). Breakdown of insecticide molecules by detoxification enzymes followed by excretion

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is termed as metabolic resistance, and is characterized by enhanced activity of detoxification

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enzymes (Dermauw and Van Leeuwen, 2014).

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The first barrier to all the insecticides is its cuticle. So, an important member of cuticular protein

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family (CP) was selected which generally provides penetration resistance in insects

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(Muthukrishnan et al., 2018). The CP has various other functional roles viz. barrier to various

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biotic and abiotic stresses, growth and ecdysis as well (Arakane et al., 2016; Noh et al., 2016;

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Balabanidou et al., 2018). After entering the insect body, phase-I reactions carry out the basic

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detoxification with the help of cytochrome P450 enzymes (Feyereisen, 2006) so other target was

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selected from this family. The P450 enzymes of insects metabolize the exogenous chemicals of

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synthetic origins like insecticides and/or natural origin like plant secondary metabolites and also

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they mediate various growth hormones like ecdysteroids, shade etc. (Yang et al., 2008;

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Feyereisen 2012; Guo et al., 2012). While, phase-II reactions carry out the detoxification after

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the phase-I reactions and Glutathione synthetase (GSS) is the key player of detoxification

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mechanism during phase-II reactions (Stohs et al., 2000). It is a key member of complex

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glutathione system with an imperative role in the regulation of cell defense to various biotic and

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abiotic stressors (Panini et al., 2016; Zhang et al., 2016; Dang et al., 2017).

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RNA interference (RNAi), an effective gene-silencing tool, has been used in a various organisms

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as a powerful strategy of functional genomics, especially in living organisms which do not

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support stable transgenesis, like insects. It is well proven that RNAi works well in order

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Coleoptera (Tomoyasu et al., 2008; Terenius et al., 2011). Various experiments conducted on

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coleopteran insects like the red flour beetle, the western corn rootworm and CPB have shown the

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impact of RNAi in both functional genomics and insect-pest management (Palli, 2012; Hussain

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et al., 2019).

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Although a few studies are conducted on use of RNAi as synergists with imidacloprid. There was

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lack of information on lethal and sub-lethal effects of downregulation of genes conferring

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resistance to imidacloprid at various immature stages of CPB. Also, use of these targets as

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synergists with imidacloprid was needed to be explored. In the current study, three important

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insecticide resistance related genes viz. P450, GSS and CP were targeted to study their lethal and

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sub-lethal effects in four CPB larval instars.

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2.0. Materials & Methods

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2.1. Insect culture

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2.1.1. Susceptible population

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The CPB population was maintained according to the modified methodology of Gökçe et al.,

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(2006). For starting the colony, about 40 CPB adults were collected from the pesticide free

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potato fields and brought to laboratory. Eggs obtained were separated in 90 mm Petri plates

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(VWR, USA) on filter paper (Whatman® Sigma-Aldrich, US). After hatching, larvae were

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transferred into rearing boxes prepared from plastic boxes (90mm×180mm×120mm) until they

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entered pupation. Before pupation, 4th larval instars were shifted to 147.85 ml volume plastic

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cups (Yöm Plastic Company, Istanbul, TR) filled with soil to provide them a medium for

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pupation. After adult emergence, they were allowed to lay eggs on new plants. In this way, CPB

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was reared for more than 3 successive years without any selection pressure. This strain was used

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as a reference strain.

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2.1.2. Imidacloprid resistant population

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The imidacloprid resistant CPB population was reared according to the modified methodology of

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Gökçe et al. (2006). CPB population used for the experiments was 25.6 folds resistant to

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imidacloprid as compared to the susceptible CPB population. LD50 value of the lab susceptible

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was 102.52 µg mL−1 (83.06-128.86 µg mL−1), while that of the lab resistant CPB population was

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2628.51 µg mL−1 (1903.50-4340.26 µg mL−1). Slope was more steep with 2.94±0.39 value in the

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lab susceptible CPB population than the lab resistant CPB population (Slope= 1.85±0.33).

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Method of rearing of CPB populations was same, however susceptible population was reared

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without selection pressure while resistant population was subjected to imidacloprid selection

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pressure for 6 generations. For this purpose, 2nd instar larvae of each generation were topically

94

treated with imidacloprid during each generation, dead larvae were discarded and surviving

95

larvae were used for next generation.

96

2.2. Targeted genes selection

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Considering the importance of three important genes viz. cuticular protein, glutathione

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synthetase and cytochrome P450 were as target. The targeted gene sequences were imported

99

from GenBank (cytochrome p450 (accession number: GEEF01131148), a cuticular protein

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(accession number: GEEF01064138), and a glutathione synthetase (accession number:

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GEEF01119768) ; the information about gene annotation and accession numbers has been

102

provided in Supplementry table-1. Primers for these genes were modified by adding sites of

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restriction enzymes i.e. KpnI and BglII (Supplementary table 2). The TRIzol method was used

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for extraction of total RNA from CPB with some modifications (Simms et al., 1993). RNA was

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converted to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Cat

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No 1622). Afterwards, cDNA was used as template to amplify targeted fragments using gene

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specific primers.

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2.3. Recombinant plasmids construction

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The methods for the construction of T-tailed L4440 vector were described by Kamath and

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Ahringer (2003). The purified gel-eluted genes fragments and L4440 vector were digested with

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KpnI and BglII restriction enzymes (Thermo Scientific) to create sticky ends of gene fragments

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and vector. The samples were incubated for 30 minutes at 37oC followed by deactivation of

113

enzyme activity at 65oC for 5 minutes. The digested genes fragments and vector were run on 1%

114

agarose gel at 80V for 45 min and observed under UV-light. Digested genes fragments and

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plasmid were ligated using T4 DNA Ligase (Promega) following the instructions of

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manufacturer’s protocol. For ligation, equal concentration of vector and insert (1:1) were added.

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Ligation buffer (1.5 µL), T4 ligase (1 µL) and water were added to adjust the volume to 15 µL.

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Ligation temperature was 22oC for 2 hours, 15oC for 3 hours and 4oC overnight.

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2.4. dsRNA synthesis in bacteria

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Transformation of cloned L4440 vectors was performed to competent cells of HT115 (DE3)

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strain according to the Zhu et al. (2011). Single positive colony of HT115 containing vector

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L4440 with CP, GSS and P450 was inoculated in LB broth (10 ml) along with 40 µL of

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ampicillin (25 mg/mL) and cultured overnight. The bacterial culture was diluted to 100X with

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LB medium and was grown to OD600 = 0.4. For dsRNA induction, IPTG (50 µM) was added to

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concentration of 1mM and culture was incubated at 37 °C for about 5 hours on shaking. At the

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end solution was given heat shock at 80 °C for about 20 min and saved at -20 °C.

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2.5. Identification of dsRNA produced in bacteria

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Total RNA from bacteria was extracted to analyze the proper synthesis of dsRNA in bacteria,

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TRI reagent was used for RNA extraction. Extracted total RNA was treated with DNase-I to

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remove DNA. The pellet was dissolved in double distilled water (50 µL) and concentration was

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measured by loading 4 µg of extracted RNA on the 1% agarose TBE gel, ethidium bromide was

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used for staining of the gel. Volume of samples was the normalized accordingly as described by

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Zhu et al. (2011).

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2.6. Direct lethal effects of dsRNA on CPB larvae

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Effects of dsRNAs on survival, development duration and weight gain were studied under

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laboratory conditions with the lab resistant CPB population. All the dsRNA feeding bioassays

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were performed using the modified methodology of Baum et al. (2007) and Zhu et al. (2011).

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Effect of dsRNAs on mortality of CPB larval instars i.e. 1st, 2nd, 3rd and 4th instar was studied in

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this experiment. This experiment was conducted as a preliminary trial before studying the effect

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of dsRNAs on biological parameters of CPB larvae. Same sized larvae were collected from the

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lab resistant CPB population and pre-starved for 6 hours before initiation of feeding bioassay.

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Three similar size fresh potato leaflets were selected and kept on top of filter paper in a 90 mm

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plastic Petri plate (VWR, USA). Each potato leaflet was treated with 500 µL (1 µg) of bacterial

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suspension expressing dsRNA against targeted gene fragment. The dsRNA was spread on

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surface of leaflets by the help of glass spreader to equally distribute the dsRNA and the leaflets

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were left to dry under the laminar flow. Each time the glass spreader was cleaned with ethanol

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and rinsed with distilled water to avoid any contamination. All the procedure of dsRNA

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application and drying was carried out in laminar flow (CHC Biolus, Korea). After drying in

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laminar flow, the larvae were shifted to each petri plate and were transferred in insect growth

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chamber at 28±1°C under a 16: 8 h light–dark photoperiod and 50-60% relative humidity. Fresh

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potato leaves treated with dsRNA were supplied to the larvae daily. The 1st and 2nd instar larvae

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were fed on dsRNA treated leaves for 6 days. While, 3rd and 4th instar larvae were fed for 3 days.

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In the control group, each potato leaflet was treated with 500 µL of empty vector and supplied to

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the control group larvae. The mortality was recorded after 3 and 6 days. Randomized block

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experimental design was used and 3 replications were performed on different days for each

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dsRNA. Total 30 larvae were used for each dsRNA and the control group.

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2.7. Effect of dsRNA on survival of different CPB larval instars and development period

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The survival rate of different CPB larval instars (2nd, 3rd and 4th); and pupal duration were

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calculated until the emergence of adults from pupae. Due to higher mortality in the 1st instar CPB

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larvae in direct effects of dsRNA, this stage was not included in this experiment. Methodology

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followed for this experiment was according to the group rearing method of life table (Chi and

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Liu, 1985; Chi, 1988). In initial 3 days, the larvae were reared on dsRNA treated potato leaflets.

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The treatment of potato leaflets and incubation of the larvae were performed as described above.

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After three days, the larvae were transferred into new Petri plates and fed with non-treated fresh

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potato leaflets in the insect growth chamber at 28±1°C under a 16: 8 h light–dark photoperiod

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and 50-60% relative humidity. In the control group, the larvae were fed with empty vector

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treated potato leaflets during whole experiment. Survival of 2nd, 3rd and 4th instar was recorded

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until the emergence of adults from pupae. Duration of larval and pupal periods was also

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calculated. The larval period was calculated until all the larvae pupated in a treatment. Lesser

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number of adults emerged from the treated 2nd instar CPB larvae so pupal duration was not

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recorded for this stage. The experiment was set up in randomized block design and repeated on

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three different times. Total 30 larvae were used for each stage and each treatment.

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2.8. Feeding effects of dsRNA on CPB larval weight gain

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Effect of the dsRNA treatments on weight gain of CPB larvae (3rd and 4th instar) was tested

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under laboratory conditions. Due to higher mortality and lesser feeding at 1st and 2nd instar CPB

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larvae, these stages were not included in this experiment. Prior to the feeding of larvae with the

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dsRNA treated leaflets, the initial weight of each CPB larvae was measured with a sensitive

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balance (Model: ATX224; SHIMADZU). The larvae were transferred to the petri plates

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containing one of the dsRNA treated leaflets. The treatment of the potato leaflets and incubation

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of the insects were carried out according to the methodology described above. Weight of 3rd and

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4th instar larvae was measured after 3 days of feeding (Sintim et al., 2009). Increase in weight

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was calculated by using formula:

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WG = FW-IW

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Where, WG is the weight gained; FW is the final weight; and IW is the initial weight

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2.9. Synergistic effect of dsRNA with imidacloprid

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The 2nd instar larvae were used in this experiment because this stage was used for providing

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imidacloprid selection pressure during each generation. Also, this stage is mostly preferred for

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experiments by researchers due to various reasons (Zhu et al. 2011). The equal size of larvae

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were collected from the lab resistant population and pre-starved for 6 hours before initiation of

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feeding bioassay. Reduced dose of 300 µL (dsRNA=0.66 µg) of bacterial suspension was

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applied to each potato leaflets as described above. Rest of the procedure and incubation of larvae

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were similar to the methodology above. After 3 days of dsRNA feeding, the dead larvae were

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recorded and discarded from the plates. The remaining larvae were topically treated with 1 µL

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imidacloprid at 187.5 µg mL-1 dose (Confidor® 350 SC; Bayer Crop Science, Germany) with the

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help of micro-syringe attached to a hand micro-applicator (Hamilton Company, Reno, NV). The

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larvae were then transferred to the new petri plates containing the untreated fresh potato leaflets

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as food source. The larvae were incubated at 28±1°C temperature under a 16: 8 h light–dark

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photoperiod and 50-60% relative humidity. There were two control groups (positive and negative

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control) in the experiment. In the positive control group, the insect were fed on empty vector

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treated potato leaflets for the initial period and then they were treated with imidacloprid at 187.5

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µg mL-1 dose. In the negative control, the larvae were fed on non-treated potato leaflets and also

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there was no imidacloprid application for this group. Mortality of larvae was recorded 24 h

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intervals for three days. Randomized complete block experimental design was used and 3

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replications were performed on three different days for each ds RNA and positive and negative

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control. Total 30 larvae were used for each group.

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2.10. Real-Time Quantitative PCR (qRT-PCR)

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It was hypothesized that mortality in targeted CPB larvae will occur due to downregulation of

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the targeted genes following feeding on dsRNA-treated leaves. To check the hypothesis, mRNA

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levels of targeted genes in insect’s larvae were measured by quantitative real-time PCR (qRT-

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PCR).

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For this purpose, extraction of total RNA was performed from larvae feeding on the dsRNAs for

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designated time period for qRT-PCR analysis. Total RNA was extracted by TRIzol method. First

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stranded cDNA was made from the RNA (1µg) primed by oligo dT using MMLV reverse

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transcriptase. The primers for all genes were designed by using NCBI primer blast tool and given

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in supplementary table 2. Furthermore, qRT-PCR was performed in 20 µl volume by using gene

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specific primers (0.5 µM), 10 µl of SYBR Green Master Mix (Biorad, USA) and water. The

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ribosomal protein gene (Accession no. EB76117) of CPB were used as reference gene for data

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normalization... Analysis of data was done using Real-Time PCR Detection System (Qiagen,

219

Netherlands). The gene expression was calculated following the method as described by Livak

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and Schmittgen (2001).

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2.11. Statistical Analysis

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The mortality data recorded for different stages of CPB fed on dsRNA were corrected with

223

Abbott’s formula (Abbott, 1925). The data were then subjected to arcsine transformation for

224

normalization (Zar, 1999). The transformed data was analyzed with one-way analysis of variance

225

(ANOVA) at the 5% significance level (P ≤ 0.05) and then the Tukey multiple comparison test

226

was used to find the difference between treatments (P ≤ 0.05). Similarly, the data regarding

227

survival rate were first transformed to arcsine and then subjected to ANOVA at the 5%

228

significance level. The difference between treatments were analyzed with the Tukey multiple

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comparison test (P≤0.05). The effect of dsRNA on larval and pupal duration time and weight

230

gain was analyzed with ANOVA and means were compared by the Tukey multiple comparison

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test at P ≤ 0.05. Paired t-test was used for the analysis of qRT-PCR results. All the data were

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analyzed using Statistix 8.1 (Analytical Software, 2005).

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3.0. Results

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3.1. Plasmid construction and Quantification of dsRNA

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Using standard molecular cloning methods all three plasmids viz. CP-L4440, P450-L4440 and

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GSS-L4440 were constructed and confirmed by colony PCR and restriction analysis (Data not

237

shown). The total dsRNAs when extracted from bacteria showed expected size of CP, GSS and

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P450 gene fragments as 421,435 and 335 bp respectively (Figure 1 A and B).

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3.2. Mortality effect of dsRNAs on various larval stages of CPB

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Three dsRNAs targeting CP, P450 and GSS caused various levels of mortality at different larval

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stages of CPB feeding on potato leaflets (Supplementary table 3 and 4). The highest mortality

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rates among the tested stages were observed on 1st instar larvae. After 6 days the mortality

243

increased in parallel to the incubation time. Following 6 days of feeding, the mortality of 1st

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instar larvae fed on different dsRNAs were 100.0%, 95.9% and 90.9% for CP, P450 and GSS

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respectively and they were significantly higher than the control after 6 days (P ≤ 0.05). Similarly,

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the mortality of CPB 2nd instar larvae was 67.4%, in CP treatment as it was 53.8% in P450 and

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37.6% in GSS treatment. There was no mortality in the control group at this stage, after 6 days of

248

incubation (Supplementary table 3).

249

There was significant difference between the treatments in feeding bioassay conducted on 3rd

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instar. In the CP treatment, the mortality rate was 50.6% and it was followed by P450 and GSS

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with 31.3% and 15.3% mortality rate, respectively. There was no mortality in the control group.

252

Unlike previous stages, the results with CBP 4th instar larvae showed that there was no

253

significant difference in larval mortality among dsRNA treatments (P >0.05). The highest

254

mortality was observed on 4th instar larvae fed with CP targeted dsRNA treated leaflets and it

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was 12.2%. The other two dsRNA caused only 9.4% (GSSs) and 1.1% (P450) mortality after 3

256

days of feeding (Supplementary table 4).

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3.3. Effects of dsRNAs on survival of various larval stages of CPB

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Survival rate of 2nd instar larvae fed on dsRNA treated potato leaflets varied significantly

259

between treatments (P ≤ 0.05). After 3 days, significantly lower survival rate (37.6%) was

260

observed in case of larvae fed on CP targeted dsRNA while higher survival rate was observed in

261

GSS treatment with 76.9%. The survival rate, during the start of adult eclosion from pupae,

262

decreased to 4.2%, 15.3% and 18.1% in CP, P450 and GSS treatment and they were significantly

263

lower than the control group (Figure 2A).

264

Survival rate of 3rd instars CPB larvae exposed to dsRNAs was calculated up to adult emergence.

265

The survival rate varied significantly after the initial feeding on 3rd instar (P ≤ 0.05). Lower

266

survival rate (43.0%) of 3rd instar larvae fed on CP-dsRNA was observed, while higher survival

267

rate (70.4%) was calculated in GSS-dsRNA treatment. After pupation, the survival rate of the

268

insects showed a similar trend and the survival rate due to GSS-dsRNA was 31.3% and was

269

24.9% for P450. Significantly lesser number of adults i.e. 15.3% emerged in case of CP targeting

270

dsRNA treatment (Figure 2B).

271

There was no significant difference between the survival rates in the dsRNAs in 4th instar larvae

272

during pre-adult stage (P >0.05). Significantly lower number of adults (47.4%) emerged from the

273

pupae which were exposed to dsRNA targeting CP when they were 4th stage (P ≤ 0.05). The

274

adult emergence rate from pupae was 50.9% in P450 treatment (Figure 2C).

275

3.4. Effects of dsRNA on weight gain of CPB different stages

276

Weight gain in the control was significantly higher with 36.7 mg followed by weight increase in

277

P450 treatment (28.7 mg). Significantly lower weight gain was calculated in CP-dsRNA (18.3

278

mg) (Figure 3A).

279

Varying weight gain in the 4th instar larvae of CPB was observed after 3 days of exposure. A

280

higher increase in weight was recorded in control (90.4 mg) and P450 treatment (85.1 mg). It

281

was followed by weight gain in the larvae fed on GSS treatment with 52.1 mg. While,

282

significantly lower increase in weight was observed in larvae with decreased expression of CP

283

(31.4 mg) (Figure 3B).

284

3.5. Effect of dsRNAs on CPB pre-adult durations

285

Developmental time for CPB is an important parameter especially regarding assessment of

286

population increase in a growing period of potato plant. There was significant difference between

287

developmental time of the 2nd instar larvae to reach pupae as compared to the control (P ≤ 0.05).

288

Lesser time was taken by 2nd instar to reach pupal stage after feeding on CP, GSS and P450

289

based dsRNAs i.e. 7.8, 8.1 and 8.2 days, respectively. All of them were statistically lesser than

290

the control larvae where larval period was 9.1 days (Figure 4A). Due to higher mortality in pupal

291

stage, it was not possible to calculate pupal duration for 2nd instar.

292

Developmental time of 3rd instar larvae varied significantly between the dsRNA feeding 3rd

293

instar larvae and control (P ≤ 0.05). Numbers of days required to reach pupal stage for 3rd instar

294

treated with CP-dsRNA, GSS-dsRNA and P450 dsRNA were 5.5, 5.8 and 6.0 days, respectively.

295

Control larvae took significantly more time (7.0 days) to reach pupal stage (Figure 4B). There

296

was significant difference regarding larval period among the treatments (P ≤ 0.05). Lesser

297

number of days (3.6 days) was required by 4th instar CPB larvae to reach pupal stage, fed on CP-

298

dsRNA. To reach pupal stage, numbers of days required by 4th instar larvae fed on dsRNA

299

targeting P450 and GSS were 3.9 and 4.1 days, respectively (Figure 4C).

300

Pupal duration varied significantly among the treatments (P ≤ 0.05). There was prolonged pupal

301

duration in the larvae which were fed on dsRNA synthesized to target CP i.e. 12.3 days. Lesser

302

pupal duration was observed in P450 with 10.7 days and control with 10.2 days (Figure 5A).

303

Pupal duration recorded in the 4th instar larvae after their exposure to dsRNA for 3 days varied

304

between the treatments (P ≤ 0.05). Pupal duration recorded in CP treatment was more i.e. 11.2

305

days. While, the pupal duration in the control was 10.1 days (Figure 5B).

306

3.6. Real-Time Quantitative PCR (qRT-PCR)

307

The qRT-PCR was performed to find relative change in targeted gene expression in different

308

CPB larval instars fed on dsRNA and respective control.

309

Experiment conducted on the 1st instar larvae revealed that the larvae fed on leaves treated with

310

dsRNA targeting all the genes were significantly down-regulated as compared to the control

311

(untreated) as shown in Figure 6A. The control group was measured separately for each gene

312

with its respective primers. The expression of control group was taken as 1.0000 in all the cases.

313

Relative gene expression in case of different dsRNA treatments varied significantly between the

314

treatments. It was found to be 0.0300, 0.0000 and 0.0700 in CP, GSS and P450, respectively (P ≤

315

0.05).

316

Experiment on 2nd instar larvae showed that the relative gene expression significantly varied

317

between the treatments as compared to the control. A significant under-expression (0.0100)

318

occurred in larvae exposed to treated leaves with P450-dsRNA Similarly, significantly lower

319

expression was also calculated in the dsRNA targeting CP (0.0500) and GSS (0.0900) as

320

compared to the control (P ≤ 0.05) (Figure 6B).

321

Experiment conducted on 3rd instar showed dramatically lower gene expression in case of all the

322

genes as compared to the control. All the treatments viz. CP, GSS and P450 shown 0.0000,

323

0.0100 and 0.0300, respectively (P ≤ 0.05) (Figure 6C).

324

The relative expression of dsRNAs varied significantly between the treatments and the control.

325

Significantly less expression of CP (0.0010), P450 (0.0000) and GSS (0.0004) were observed as

326

compared to the control (P ≤ 0.05) (Figure 6D).

327

3.7. Synergism of dsRNA with imidacloprid

328

Synergism experiment was conducted on 2nd instar of the lab resistant population, which was

329

25.6X resistant to imidacloprid. Mortality rate in larvae fed on dsRNAs and the control varied

330

significantly (P ≤ 0.05) after 3 days initial feeding. CP feeding caused 34.5% mortality as

331

mortality rate recorded in P450 and GSS treatments was 24.9% and 15.3%, respectively. The

332

mortality rates of CPB 2nd instar larvae increased following reduced dose of imidacloprid. The

333

mortality rates were 50.6, 44.1 and 47.4 in CP, P450 and GSS targeting dsRNA treatments after

334

24 hours on imidacloprid application. It was clear especially in P450 and GSS treatments

335

because there were 2 and 3 fold increased in 24 hours. The mortality rates in treatments showed

336

similar trend and continued to increase both 48 and 72 hours after application. The CP and GSS

337

targeting dsRNA synergism with imidacloprid caused 100.0% mortality after 72 hours of

338

imidacloprid application as it was 97.6% in P450 treatment. Reduced dose imidacloprid caused

339

4.2% mortality in the positive control (Table 1).

340

4.0. Discussion

341

The CPB has become resistant to more than 50 synthetic insecticides (Mota-Sanchez and Wise,

342

2017).

343

common in CPB. So, keeping in view the importance of imidacloprid resistance encoding genes

344

lethal and sub-lethal effects of silencing three important CPB genes viz. CP, P450 and GSS were

345

studied. Our findings showed that mortality rate caused by dsRNAs varied between CPB larval

346

stages, while, a higher mortality (90.9-100.0% and 37.6-67.4%) was observed in the 1st and 2nd

347

instar whereas 15.3-50.6% in the 3rd and 1.1-12.9% in the 4th instar larvae. These results are

348

comparable to the findings of Amiri and Bakhsh (2019) and Hussain et al. (2019) who reported

349

that earlier instars of CPB are more susceptible than the later instars. This phenomenon could be

350

related with co-regulation of genes at later stages. Co-regulation of some closely related genes is

351

also reported by Cornman et al. (2008) and Togawa et al. (2008) who reported that some genes

352

are stage specific. Closely related genes probably replaced the function of our target gene at the

353

3rd and 4th instar larvae, which may lead to the lower mortalities seen in these stages as depicted

354

from the percent identity matrix of P450 and GSS (Data not shown).

355

Our study showed that among 3 different dsRNAs, larvae fed on CP-dsRNA underwent higher

356

mortality i.e. 50.6%-100.0% in 3 larval instars. These findings are in accordance with the

Among these insecticides, neonicotinoids especially imidacloprid resistance is very

357

findings of Jasrapuria et al. (2012) and Mun et al. (2015). The CP plays a vital role in insect

358

growth, development of penetration resistance to various insecticides and tolerance of

359

environmental factors so that its downregulation could result in higher mortality in insect species

360

(Jasrapuria et al., 2012). Similarly, decrease of CPB larvae survival and fitness fed on dsRNA

361

was also reported by Jin et al. (2015). Down-regulation of CP resulted in significant decrease in

362

survival rate in 2nd, 3rd and 4th instar CPB larvae. Decreased survival especially during adult

363

eclosion from pupae is comparable to the findings of Jasrapuria et al. (2012) and Mun et al.

364

(2015) who reported a decrease in adult eclosion due to downregulation of cuticular protein in

365

beetles.

366

Less weight gain was recorded in larvae fed on CP-dsRNA viz. 18.3 and 31.4 mg in 3rd and 4th

367

instar larvae of CPB. Decrease in weight to dsRNA feeding was previously reported by Jin et al.

368

(2015) in H. armigera and Zhu et al. (2011) in CPB, who found that the weight gain was less in

369

larvae fed with dsRNA. Less weight gain can be attributed to the fact that growth and

370

development of insects are highly reliant on the ability of insect to remodel their exocuticle

371

(Ahmad et al., 2006; Qiao et al., 2014). Effect on larval and pupal duration due to CP-dsRNA, is

372

comparable to the findings of Qiao et al (2014), who reported that larval growth in silkworm can

373

be affected by the change in cuticle protein. Similarly, pupal duration viz. 12.3 and 11.2 days in

374

3rd and 4th instar treated CPB larvae was significantly affected due to CP-dsRNA feeding. It may

375

be attributed to the fact that CP related transcripts are more active right after pupation in normal

376

insects. These set of genes are associated with first ecdysone pulse, with variation in their time of

377

expression (Arakane et al., 2008).

378

Synergism experiment showed a remarkable potential of the targeted genes as synergists with

379

imidacloprid. Only one larva survived in P450 dsRNA treatment while all the tested insects died

380

in CP and GSS treatments. These results are in accord with the findings of Clements et al.

381

(2017). The CP has important role in growth and also penetration resistance to various

382

insecticides so downregulation of CP resulted in complete decline of the exposed population

383

(Hadley, 1982; Clements et al., 2017). While, role of GSS in phase-II reactions is well

384

established and the phase II reactions are quite important in detoxification of neonicotinoids. Yu

385

and Killiny (2018) reported the increase in susceptibility of Asian citrus psyllid to thiamethoxam

386

due to decreased expression of GSTs. Resistant insects can undergo mortality if phase II

387

reactions are hindered by any source like gene silencing. This finding confirms our results

388

(synergist effect of GSS with imidacloprid). Use of P450 as synergist is also reported by

389

Bautista et al. (2009) who found decrease in resistance of P. xylostella to permethrin due to

390

downregulation of a P450 gene. Although there is diversity of CP, P450 and GSS in a highly

391

resistant insect like CPB, but 2nd instar is considered as the susceptible stage of its life cycle as

392

reported by Zhu et al. (2011). So, downregulation of any of the resistant gene at this stage can be

393

lethal and significantly enhance the susceptibility of CPB to imidacloprid. Also, at this stage

394

some closely related genes lying close to the targeted gene in phylogenetic tree and percent

395

identity matrix can be down-regulated by targeted one gene.

396

Down-regulation of these genes in feeding bioassays ranging from 0.0000-0.0900 was verified

397

with qRT-PCR. The 3rd and 4th larval instars showed more downregulation of the genes and that

398

could be due to more food consumption and thus more intake of dsRNA. Our results are in

399

accordance with the studies by Zhu et al. (2011) and Hussain et al. (2019).

400

Conclusion

401

RNAi technology was utilized in the control of imidacloprid resistant CPB population by

402

knocking-down 3 important imidacloprid resistance associated genes; cuticular protein,

403

glutathione synthetase and cytochrome P450 monoxygenase. This is the first study reporting the

404

effects of dsRNA on mortality, growth and survival of different larval instars of CPB. Bacterially

405

expressed dsRNA was used to conduct oral feeding bioassays on different larval instars of CPB.

406

The mortality rates were greater at the earlier stages. Decreased survival rate of exposed CPB

407

larvae was observed in all the dsRNAs. Similarly, body weight and pre-adult duration were also

408

affected due to dsRNAs. Synergistic effect of all the dsRNAs with imidacloprid on 2nd instar

409

CPB larvae produced high mortality with reduced dose of the both treatments. Further research

410

on the use of dsRNA in field and its implementation can significantly decrease the cost of

411

development of new insecticides. It can be a milestone in resistance management of CPB and

412

various other notorious insect pests. Suppressing of resistant genes to produce a susceptible

413

population of CPB by gene silencing can be useful in devising novel control strategies.

414

Acknowledgements

415

We acknowledge Doğuş Group and Tübitak to support this research work. We also acknowledge

416

Prof. Dr. Hsin Chi and Dr. Halil Toktay for their support and guidance during research work.

417

Author Contribution

418

AB and AG conceived the idea and designed the study. MNN constructed recombinant vector

419

and performed all bioassays as a part of his doctoral studies. EA made significant contribution to

420

molecular and application assays of dsRNAs.

421

Conflict of Interest

422

There is no conflict of interest regarding this manuscript among the authors

423

424

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in

the

Colorado

potato

beetle

(Coleoptera:

Chrysomelidae). J.

Econ.

Legends of the tables Table 1. Synergistic effect of dsRNAs with imidacloprid on CPB 2nd instars larvae

Table 1. Synergistic effect of dsRNAs with imidacloprid on CPB 2nd instars larvae Treatment

Mortality (%) (Mean±SEM) before imidacloprid 34.50±0.11a** 24.92±0.13ab 15.33±0.21b 0.00±0.00c

Mortality data as % (Mean±SEM*) after imidacloprid application

24 HAT*** 48 HAT 72 HAT CP 50.59±0.11a 73.71±0.14a 100.00±0.00a p-450 44.12±0.07a 63.67±0.11a 97.56±1.07a GST 47.35±0.21a 70.36±0.13a 100.00±0.00a Positive 0.00±0.00b 1.07±1.07b 4.23±1.07b control Negative 0.00±0.00c 0.00±0.00b 0.00±0.00b 0.00±0.00b control * SEM = Standard Error Mean **Mean values followed by the different letter in the same column are statistically different (P ≤ 0.05) *** HAT= Hours after treatment

Legends of the figures Figure 1. (A) shows the dsRNA of P450 gene fragment in lane 1 and 2, while 100 bp plus DNA ladder (Thermo Scientific) while (B) is showing dsRNA of GSS in lane 1, CP in lane 2 and 3 while 500 bp plus DNA ladder (Thermo Scientific) in lane 3 Figure 2. Survival of CPB (A) 2nd instar, (B) 3rd instar and (C) 4th instar larvae after 3 days of feeding on 3 different dsRNAs Figure 3. Weight gain (mg) in 3rd (A) and 4th larval instar (B) of CPB after 3 days of 3 different dsRNAs feeding Figure 4. Larval duration of CPB (A) 2nd instar, (B) 3rd instar and (C) 4th instar larvae after 3 days of feeding on 3 different dsRNAs Figure 5. Pupal duration of (A) 3rd instar and (B) 4th instar larvae of CPB after 3 days of feeding on 3 different dsRNAs Figure 6. Effect of feeding dsRNA on target-gene expression (Mean ± SE) in CPB A) 1st instar, B) 2nd instar, C) 3rd instar and D) 4th larvae after feeding assay

Figure 1. (A) shows the dsRNA of P450 gene fragment in lane 1 and 2, while 100 bp plus DNA ladder (Thermo Scientific) while (B) is showing dsRNA of GSS in lane 1, CP in lane 2 and 3 while 500 bp plus DNA ladder (Thermo Scientific) in lane 3

Survival rate (%)

100 90 80 70 60 50 40 30 20 10 0

A

CP GSS p-450 Control

0

5

10

15

20

25

Number of days 100

B

90 80 70 Survival rate (%)

60

CP

50

GSS

40 30

p-450

20

Control

10 0 0

5

10

15

20

25

Number of days 100

C

Survival rate (%)

90 80 70 60

CP

50

GSS

40 30

p-450

20

Control

10 0 0

5

10

15

20

25

Number of days

Figure 2. Effect of 3 different dsRNAs on survival rate (%) of (A) 2nd instar, (B) 3rd instar and (C) 4th instar larvae of CPB

180

A

180

160

160 120 100

a

b

c

d

60

Weight (mg)

Weight (mg)

a

B

140

140

80

a

b

120

c

100 80

Initial weight

60

Final Weight

40

40

20

20 0

0 Control

P450

GSS

Treatment

CP

Control

P450

GSS

CP

Treatment

Figure 3. Weight gain (mg) in 3rd (A) and 4th larval instar (B) of CPB after 3 days of 3 different dsRNAs feeding *Different letters on error bars represent statistical difference (P ≤ 0.05)

Larval duration (days)

2nd instar 10

b

b

CP

GSS

b

A

a

8 6 4 2 0 P450

Control

dsRNA

Larval duration (days)

3rd instar 8 7 6 5 4 3 2 1 0

B a

b

CP

b

b

GSS

P450

Control

dsRNA

C

Larval duration (days)

4th instar 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

b

CP

ab

GSS

ab

a

P450

Control

dsRNA

Figure 4. Larval duration of CPB (A) 2nd instar, (B) 3rd instar and (C) 4th instar larvae after 3 days of feeding on 3 different dsRNAs * Different letters on error bars represent statistical difference (P ≤ 0.05)

3rd instar 14

a

ab

b

12 Pupa duration (days)

A

b

10 8 6 4 2 0 CP

GSS

P450

Control

dsRNA

4th instar

B

Pupal duration (days)

14 12

a

ab

b

b

GSS

P450

Control

10 8 6 4 2 0 CP

dsRNA

Figure 5. Pupal duration of (A) 3rd instar and (B) 4th instar larvae of CPB after 3 days of feeding on 3 different dsRNAs *Different letters on error bars represent statistical difference (P ≤ 0.05)

B

Relative gene expression

1.2 1

1.2

A

*

1

0.8

0.8

0.6

1st instar

0.6

2nd instar

0.4

0.4

0.2

0.2 0

0 Control

Treatment

GSS Treatment

C

D

CP

GSS

Control

p-450

1.2

CP

p-450

1.2

* Relative gene expression

*

1

1

0.8

0.8

0.6

3rd instar

0.6

0.4

0.4

0.2

0.2

0

*

4th instar

0 Control

CP

GSS Treatment

p-450

Control

CP

GSS Treatment

Figure 6. Effect of feeding dsRNA on target-gene expression (Mean ± SE) in CPB A) 1st instar, B) 2nd instar, C) 3rd instar and D) 4th larvae after feeding assay

p-450

Highlights •

Lethal and sub-lethal effects of down-regulating imidacloprid resistance genes were studied

•

Survival, weight and pre-adult duration were affected due to dsRNA feeding

•

Targeted genes were significantly down-regulated

•

Synergism of dsRNAs and imidacloprid resulted in complete decline of the resistant CPB population