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. Author manuscript; available in PMC: 2023 Mar 6.
Published in final edited form as: Arch Insect Biochem Physiol. 2020 Apr 29;104(4):e21683. doi: 10.1002/arch.21683

Double‐stranded RNAs targeting inhibitor of apoptosis gene show no significant cross‐species activity

Shankar C R R Chereddy 1, Dhandapani Gurusamy 1, Jeffrey L Howell 1, Subba R Palli 1
PMCID: PMC9987616  NIHMSID: NIHMS1870354  PMID: 32350930

Abstract

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double‐stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species‐specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.

Keywords: Inhibitor of apoptosis, Leptinotarsa decemlineata, Nezara viridula, nontarget effects, Tribolium castaneum

1 |. INTRODUCTION

The use of double‐stranded RNA (dsRNA) to induce knockdown of a target gene through the RNA interference (RNAi) pathway has become a valuable tool in understanding the functions of a plethora of genes (Fjose, Ellingsen, Wargelius, & Seo, 2001). Since its discovery in the nematode, Caenorhabditis elegans, significant progress has been made in integrating the inner workings of the RNAi pathway into mainstream research and has contributed to advances in science (Fire et al., 1998). RNAi has also become the subject of widespread interest in the agrochemical industry due to its potential as a pest control method. The growing need for an alternative to chemical pesticides due to resistance development in pests and their nontarget effects on other organisms in the ecosystem has increased interest toward RNAi‐based pest control methods (Baum et al., 2007; Burand & Hunter, 2013; Huvenne & Smagghe, 2010; Zhu & Palli, 2020). Furthermore, studies have shown that the dsRNA molecules have a short half‐life, which further reduces their environmental impact (Dubelman et al., 2014).

The major targets for designing dsRNAs for pest management are lethal genes that are highly conserved in pathways across taxa (Yu et al., 2013). The apoptosis pathway, which is highly conserved across species, is a good target for RNAi‐based control of pests (Crawford et al., 2012). The process of apoptosis occurs naturally during development to maintain homeostasis and also drives immune responses in the case of cell damage (Norbury & Hickson, 2001). Apoptosis in cells is caused by caspases, which were discovered in 1993 in C. elegans (Yuan, Shaham, Ledoux, Ellis, & Horvitz, 1993). Caspases function by cleaving nuclear, mitochondrial and plasma membrane proteins to activate them, ultimately leading to cell death (Martinez, Reif, & Pappas, 2010). Inhibitors of apoptosis proteins (IAP) bind and inactivate caspases, thereby preventing cell death (Deveraux & Reed, 1999; Kobayashi, Hatano, Otaki, Ogasawara, & Tokuhisa, 1999). Multiple IAP proteins containing baculoviral IAP repeat (BIR) domains have been identified in insects (Birnbaum, Clem, & Miller, 1994; Crook, Clem, & Miller, 1993; Hay, Wassarman, & Rubin, 1995) and shown to inhibit caspase activity (Shi, 2002). As many as five different iap genes have been identified in insect genomes (Srinivasula & Ashwell, 2008; Verhagen, Coulson, & Vaux, 2001; Yoon, Koo, George, & Palli, 2020).

The effectiveness of dsIAP in inducing knockdown of the iap gene, apoptosis, and mortality in several insect species has been reported recently (Cao, Gatehouse, & Fitches, 2018; Dhandapani, Gurusamy, Howell, & Palli, 2019; Igaki, Yamamoto‐Goto, Tokushige, Kanda, & Miura, 2002; Mogilicherla, Howell, & Palli, 2018; Powell, Bradish, Gatehouse, & Fitches, 2017; Rodrigues, Dhandapani, Duan, & Palli, 2017; Yoon, Shukla, Gong, Mogilicherla, & Palli, 2016; Yoon et al., 2018). However, the cross‐species activity of dsIAP is not studied extensively. Injection of dsRNA targeting the Drosophila melanogaster iap1 gene into two nontarget dipteran pests, Musca domestica and Delia radicum induced knockdown of iap1 orthologs and mortality in these insects despite the absence of 21 bp conserved regions (Powell et al., 2017). In this study, we looked at the degree of nontarget effects caused by dsRNAs targeting iap genes from nine insect species: Spodoptera frugiperda (Sf), Leptinotarsa decemlineata (Ld), Tribolium castaneum (Tc), Anoplophora glabripennis (Ag), Lasioderma serricorne (Ls), Aedes aegypti (Aa), Halyomorpha halys (Hh), Murgantia histrionica (Mh) and Nezara viridula (Nv) from four insect orders. We also compared dsRNA targeting regions of iap genes across the nine species tested to determine the degree of sequence similarity. We used in vitro, in vivo, and computational methods to study potential nontarget effects of nine iap genes in four cell lines and three insect species. Interestingly, no significant nontarget effects were detected even among closely related species, such as the three stink bug species under these testing conditions.

2 |. MATERIALS AND METHODS

2.1 |. Transcriptome analysis

The transcriptome for A. aegypti was obtained from Vectorbase; the version of the transcriptome used for the analysis was L5.2 (Giraldo‐Calderón et al., 2014). Transcriptomes for H. halys, T. castaneum, L. decemlineata, and A. glabripennis were obtained from the NCBI database. As the curated databases for S. frugiperda, L. serricorne, M. histrionica, and N. viridula are not available, we assembled transcriptomes for these insects using the TRINITY transcriptome assembler.

2.2 |. Small interfering RNA (siRNA) fragment analysis

To analyze the similarity of siRNA produced by small interference iap gene fragments (siIAP) across species, we created local blast directories of transcriptomes for all nine species using CLC Bio. All possible siIAPs produced by the dsIAPs were obtained using a python script. These siIAPs were blasted against the transcriptomes of all species (Camacho et al., 2009).

2.3 |. IAP domain analysis

To perform the domain analysis, we used the nucleotide sequences of nine iap gene sequences. We obtained the open reading frames for these nucleotide sequences using the ExPASY (Gasteiger et al., 2003). After obtaining the open reading frames, an NCBI Conserved domain search was performed to identify conserved domains and their locations within each iap gene. The MUSCLE alignment program was used to align iap gene sequences (Edgar, 2004).

2.4 |. Target gene amplification, polymerase chain reaction (PCR) purification, and dsRNA synthesis

The dsRNA targeting the iap gene in each of the nine insects tested was designed at different times for experiments in our laboratory. The dsRNA targeting sequences (see Supplementary Information) and primer sequences (Supplementary Information Table S1) for all species tested are included. Species‐specific complementary DNA (cDNA) was used as a template to amplify fragments of iap genes from nine insects using gene‐specific primers (Supplementary Information Table S1) as described previously (Yoon et al., 2016). dsRNAs targeting the gene coding for green fluorescence protein (GFP, control) and iap genes were synthesized using a MEGAscript® T7 RNAi kit (Ambion). The integrity of the dsRNA was analyzed on 1% agarose gels, and the concentration was determined by using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific).

2.5 |. Insect cell culture, apoptosis phenotypes, and gene knockdown study

The L. decemlineata cell line (Lepd‐SL1 cells) and T. castaneum cells, BCIRL‐TcA‐CLG1 were received from Dr. Cynthia Goodman from Biological Control of Insecťs Research Laboratory (USDA‐ARS, Columbia, MO) and cultured, as described previously (George, Gaddelapati, & Palli, 2019; Roy & Palli, 2018; Shukla et al., 2016; Yoon et al., 2016). The A. aegypti, Aag‐2 cells (Cui, Sui, Xu, Zhu, & Palli, 2014) were cultured in Schneider's insect medium with L‐glutamine (Sigma‐Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Seradigm FBS, VWR® Life Science). Sf9 cells were derived from IPLB‐Sf21‐AE, an established cell line originally isolated from S. frugiperda ovaries. The plasmids containing the genes, Luciferase and SID‐1 (dsRNA transporter channel) from C. elegans (Xu et al., 2013) were stably expressed in Sf9 cells and named Sf9_LUC_CeSID‐1 and maintained in Sf‐900™ II SFM (Gibco™, Thermo Fisher Scientific). All the cell lines were maintained in a 27–28°C incubator. For apoptosis phenotypes and gene knockdown studies, cells were seeded in 96‐ and 24‐well plates in 100 and 250 μl culture medium, respectively and exposed to dsGFP or dsmalE as control and dsIAP targeting the iap genes in nine insect species.

2.6 |. In vivo assays

To study the effect of nontarget dsRNAs in insects, we selected three insect species: the Colorado potato beetle (CPB), the Red flour beetle (RFB; Coleopterans) and the Southern Green Stink Big (SGSB; Hemipteran). Four micrograms of dsGFP or dsIAP genes were spread on washed and TritonX‐100 treated eggplant leaf discs (7 mm diameter). The treated leaf discs were fed to third instar CPB larvae that were starved overnight. Four micrograms dsRNA/larva/day was fed over a 3‐day period to a total of 12 μg dsRNA/insect. The mortality was recorded on the fifth‐day post‐feeding. For SGSB feeding, the green been mediated dsRNA delivery method (Ghosh, Hunter, Park, & Gundersen‐Rindal, 2017; Ghosh, Hunter, Park, & Gundersen‐Rindal, 2018) was used. Certified organic green beans were washed and cut to a length of 5 cm, then immersed in capless 2 ml microcentrifuge tubes containing 300 μl nuclease‐free water and 20 μg dsGFP or dsIAP. The beans, dsRNA, and water were replaced daily for 3 days. The tubes were sealed with parafilm to prevent evaporation of the solution and to prevent insects from entering the solution. Beans were immersed in water containing dsRNA 3 hr before feeding. The tubes were then placed in a solid platform to keep them upright and enclosed within magenta jars (Sigma‐Aldrich). Second or third instar SGSB nymphs were starved overnight before feeding. Ten nymphs were released per magenta jar containing one green bean in a microcentrifuge tube containing dsRNA (20 μg daily for 3 days) solution. The mortality was recorded on the 14th day after the initiation of feeding. In the RFB assay, 1 μg dsRNA in 200 nl was injected into each larva. Afterward, the phenotypic changes and mortality were recorded daily until the 10th day after injection.

2.7 |. Extraction of total RNA, cDNA synthesis, and quantitative reverse transcription polymerase chain reaction (RT‐qPCR) analysis

For the gene knockdown study, total RNA was isolated from dsRNA‐treated cells, injected Tc larvae and fed Ld larvae and Nv nymphs using the TRI reagent (Molecular Research Center Inc., Cincinnati, OH). In the cell line, each well that was treated using dsRNA was considered as one replicate and RNA was extracted accordingly. We isolated RNA only from live insects and used one larva and nymph each in Ld and Nv and two larvae in Tc per replicate. The RNA was used for cDNA synthesis followed by a quantitative PCR assay. Two micrograms of total RNA were used to cDNA synthesize using the M‐MLV reverse transcriptase (Invitrogen, USA). RT‐qPCR was performed, as described previously (Mogilicherla et al., 2018). Relative mRNA levels of Ld_IAP1, Tc_IAP1, Aa_IAP1, Sf_IAP1, and Nv_IAP5 were determined by the 2−ΔΔCt method using Rp4 (Ld), Rp49 (Tc), SRP7 (Aa), 28S rRNA (Sf), and 60S RP L12 (Nv) as reference genes. The primers used for dsRNA synthesis and RT‐qPCR analysis are listed in Supporting Information Table S1.

2.8 |. Statistical analysis

The CLC Bio tool was used to create local blast directories of transcriptomes for all nine species and a python script was used to obtain all possible siIAP produced from the dsIAP. The siIAPs were then blasted against the transcriptomes of all species (Camacho et al., 2009). The open reading frames for nine IAP nucleotide sequences were obtained by using the ExPASY (Gasteiger et al., 2003). An NCBI Conserved domain search was performed to identify conserved domains and their locations within each iap gene and the MUSCLE alignment program was used to align iap gene sequences (Edgar, 2004). The delta delta Ct (ΔΔCt) method was used for RT‐qPCR data analysis and one‐tailed t test was used to compare the mean of a single variable. The significance of the target gene knockdown in cells/tissues was tested by one‐way analysis of variance (ANOVA) with Duncan multiple range test with the significance level set at p < .05 using IBM SPSS Statistics 20 software.

3 |. RESULTS

3.1 |. Transcriptomes analysis―Prediction of IAP domains and siRNA fragments

To identify conserved domains present in iap gene sequences from the nine insects selected, open reading frames of these genes were searched at the NCBI. The presence of conserved domains in nine iap genes tested are shown in Figure 1. The location of the dsRNA target region in each iap gene is also marked in Figure 1. The dsRNA targeting the iap gene in each of the nine insects tested was designed at various times for RNAi experiments in each insect (Supporting Information File S2). Therefore, the dsRNA targeting regions (Supporting Information File S2) are different in each iap gene tested. The nucleotide sequences of nine iap genes are aligned and shown in Supplementary information File S3. The dsRNA targeting regions are also marked in the aligned nucleotide sequences. To identify all possible siRNAs produced from each of the nine dsIAPs, a python code was used. The presence of complementary sequences for all identified siRNAs was checked by searching the transcriptomes of nine insects (Table 1). These searches revealed minimal similarity between siIAP and transcriptome sequences. For example, out of a total of 332 siIAPs produced from A. aegypti dsIAP, only four of them were perfectly matched in A. glabripennis transcriptome suggesting that region of the iap gene used has low nucleotide sequence conservation. In general, the siIAPs produced from each dsIAP tested showed less than 10 siRNAs that matched 100% with the transcriptome sequences searched (Table 1). The only exception to this general conclusion is the three stink bug species tested. Forty‐three and 95 siRNAs produced from H. halys dsIAP2 (Hh_dsIAP2) matched 100% with the sequences in the transcriptomes of M. histrionica and N. viridula, respectively. Similarly, 118 and 79 siRNAs produced from M. histrionica dsIAP2 (Mh_dsIAP2) matched 100% with the sequences in the transcriptomes of H. halys and N. viridula, respectively. Also, 51 and 57 siRNAs produced from N. viridula dsIAP5 (Nv_IAP5) matched 100% with the sequences in the transcriptomes of H. halys and M. histrionica, respectively.

FIGURE 1.

FIGURE 1

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Analysis of domains present in the coding sequences of iap genes from nine insect species. The coding sequences of iap genes from Spodoptera frugiperda (Sf), Leptinotarsa decemlineata (Ld), Tribolium castaneum (Tc), Anoplophora glabripennis (Ag), Lasioderma serricorne (Ls), Aedes aegypti (Aa), Halyomorpha halys (Hh), Murgantia histrionica (Mh), and Nezara viridula (Nv) were selected and the presence of conserved domains were analyzed using the NCBI Conserved domain search tool. The boxes indicate the location of conserved domains and the red arrows indicate the position of the dsRNA target sequences. BIR, baculoviral IAP repeat

TABLE 1.

siRNA fragment analysis in transcriptomes of nine insect species

 19‐Mer IAP Total siRNA fragments/complete siRNA match
Aa Ag Ls Ld Sf Tc Hh Mh Nv
 Aedes aegypti IAP1 332/332 332/0 332/1 332/0 332/2 332/3 332/0 332/9 332/0
 Anoplophora glabripennis IAP1 368/4 368/368 368/7 368/4 368/1 368/1 368/6 368/13 368/5
 Lasioderma serricorne IAP1 323/3 323/3 323/323 323/5 323/0 323/0 323/4 323/5 323/3
 Leptinotarsa decemlineata IAP1 385/4 385/5 385/11 385/385 385/1 385/4 385/0 385/7 385/8
 Spodoptera frugiperda IAP1 317/1 317/0 317/0 317/1 317/317 317/2 317/0 317/0 317/0
 Tribolium castaneum IAP1 415/5 415/2 415/2 415/1 415/4 415/415 415/0 415/1 415/1
 Halyomorpha halys IAP2 325/1 325/1 325/3 325/1 325/3 325/3 325/325 325/43 325/95
 Murgantia histrionica IAP2 363/3 363/3 363/3 363/13 363/8 363/9 363/118 363/363 363/79
 Nezara viridula IAP5 392/3 392/9 392/5 392/3 392/8 392/7 392/51 392 /57 392/392
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Abbreviations: IAP, inhibitor of apoptosis; siRNA, small interfering RNA.

3.2 |. dsIAPs induced knockdown of iap genes and apoptosis only in cell lines developed from target insects

Nine dsIAPs targeting iap genes from CPB, RFB, ALB, YFM, FAW, CB, BMSB, HB, and SGSB along with a control dsGFP/dsmalE were added to the culture medium of Lepd‐SL1, TcA, Aag‐2, and Sf9_LUC_CeSID‐1 cells. The CPB dsIAP knockeddown iap gene expression and induced apoptosis only in Lepd‐SL1 cells developed from this insect (Figure 2a and Supporting Information Figure S1a). The RFB dsIAP induced knockdown of the iap gene and apoptosis only in TcA cells developed from this insect (Figure 2b and Supporting Information Figure S1b). The YFM dsIAP induced knockdown of iap gene and apoptosis only in Aag‐2 cells developed from this insect (Figure 3a and Supporting Information Figure S2a). Also, the FAW dsIAP induced knockdown of the iap gene and apoptosis only in Sf9_LUC_CeSID‐1 cells developed from this insect (Figure 3b and Supporting Information Figure S2b). Other than the target‐specific dsIAPs, none of the other eight dsIAPs or control dsRNA induced significant knockdown of the iap gene or apoptosis in four cell lines tested.

FIGURE 2.

FIGURE 2

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Target gene knockdown analysis in the Colorado potato beetle (Leptinotarsa decemlineata) Lepd‐SL1 and red flour beetle (Tribolium castaneum) BCIRL‐TcA‐CLG1 (TcA) cell lines exposed to dsIAP. (a) 70,000 Lepd‐SL1 cells per well seeded in 24‐well plates in 250 μl EX‐CELL® 420 containing 10% FBS were exposed to 100 ng of dsRNA targeting iap genes from nine insects or dsGFP as a control. At 48 hr after treatment, the total RNA was extracted and used to determine relative IAP mRNA levels gene by RT‐qPCR normalized with RP4 as a reference gene. Mean + SE (n = 4) are shown. Data were analyzed by one‐way ANOVA with Duncan multiple range test with the significance level set at p < .05 using IBM SPSS Statistics 20 software. The numbers and arrow toward down on the top of the bars show the percent decrease in the IAP mRNA levels in dsIAP‐treated cells when compared to those in the control cells treated with dsGFP. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). (b) 70,000 TcA cells per well seeded in 24‐well plates with 250 μl EX‐CELL 420 containing 10% FBS and exposed to 10 μg of dsRNA targeting iap genes from nine insects or malE and the relative mRNA levels were determined by RT‐qPCR normalized with RP49 as a reference gene as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). Aa, Aedes aegypti; Ag, Anoplophora glabripennis; ANOVA, analysis of variance; dsRNA, double‐stranded RNA; FBS, fetal bovine serum; GFP, green fluorescence protein; Hh, Halyomorpha halys; IAP, inhibitor of apoptosis; Ld, Leptinotarsa decemlineata; Ls, Lasioderma serricorne; Mh, Murgantia histrionica; mRNA, messenger RNA; Nv, Nezara viridula; RT‐qPCR, quantitative reverse transcription polymerase chain reaction; Sf, Spodoptera frugiperda; SE, standard error; Tc, Tribolium castaneum

FIGURE 3.

FIGURE 3

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Target gene knockdown analysis in the yellow fever mosquito, Aedes aegypti Aag‐2 cells and fall armyworm, Spodoptera frugiperda Sf9 stable cells expressing Luciferase and CeSID‐1 genes exposed to dsIAP. (a) 100,000 Aag‐2 cells per well seeded in 24‐well plates in Schneider's insect medium containing 10% FBS were exposed to 30 μg of dsRNAs (10 μg/day) targeting GFP or IAP genes from nine different insects. On the sixth day after treatment, total RNA was extracted and IAP mRNA levels were determined by RT‐qPCR with SRP7 as a reference gene as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). (b) 50,000 Sf9_LUC + CeSID‐1 stable cells per well seeded in 24‐well plates in 250 μl Sf‐900™ II SFM and exposed to 2000 ng of dsRNAs targeting GFP or iap genes from nine insects. On the fifth day after treatment, total RNA was extracted and IAP mRNA levels were determined by RT‐qPCR with 28S rRNA as a reference gene as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). Aa, Aedes aegypti; Ag, Anoplophora glabripennis; dsRNA, double‐stranded RNA; GFP, green fluorescence protein; Hh, Halyomorpha halys; IAP, inhibitor of apoptosis; Ld, Leptinotarsa decemlineata; Ls, Lasioderma serricorne; Mh, Murgantia histrionica; mRNA, messenger RNA; Nv, Nezara viridula; RT‐qPCR, quantitative reverse transcription polymerase chain reaction; Sf, Spodoptera frugiperda; Tc, Tribolium castaneum

3.3 |. dsIAPs induce knockdown of iap genes and mortality only in target insects

The CPB larvae fed on nine dsIAPs and dsGFP showed knockdown of the iap gene and 100% mortality only in larvae fed on dsIAP targeting the CPB iap gene (Figure 4). Similarly, RFB larvae injected with nine dsIAPs and dsmalE showed knockdown of the iap gene and 100% mortality only in larvae injected with dsIAP targeting the RFB iap gene (Figure 5). Also, feeding nine dsIAPs and dsGFP to SGSB nymphs induced knockdown of the iap gene and 70% mortality only in nymphs fed on dsIAP targeting the SGSB iap gene (Figure 6). Except for the dsIAP produced from a fragment of the iap gene from the target species, none of the other eight dsIAPs or dsGFP induced significant knockdown of the iap gene or mortality.

FIGURE 4.

FIGURE 4

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Knockdown of iap gene and mortality induced by dsIAP in the Colorado potato beetle, Leptinotarsa decemlineata, larvae that fed on dsRNA targeting the iap gene in nine insects or GFP. (a) Four micrograms of dsRNA targeting GFP or iap gene from nine insects was spread on washed and TritonX‐100‐treated eggplant leaf disc and fed to one each of overnight starved third instar larvae. On the third day after feeding dsRNA, total RNA was extracted and used to determined the IAP mRNA levels by RT‐qPCR with RP4 as a reference gene, as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). (b) Fresh leaf disc with dsRNA was replaced daily for 3 days and mortality recorded on the fifth day. Experiments were repeated twice with each experiment consisting of 10 larvae for each dsRNA tested. The numbers on the top of the bars show percent mortality induced by dsIAPs. Aa, Aedes aegypti; Ag, Anoplophora glabripennis; dsRNA, double‐stranded RNA; GFP, green fluorescence protein; Hh, Halyomorpha halys; IAP, inhibitor of apoptosis; Ld, Leptinotarsa decemlineata; Ls, Lasioderma serricorne; Mh, Murgantia histrionica; mRNA, messenger RNA; Nv, Nezara viridula; RT‐qPCR, quantitative reverse transcription polymerase chain reaction; Sf, Spodoptera frugiperda; Tc, Tribolium castaneum

FIGURE 5.

FIGURE 5

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Knockdown of iap gene and mortality induced by dsIAP in the red flour beetle, Tribolium castaneum larvae injected with different insect IAP dsRNAs. (a) The dsRNA (1 μg/larva) targeting malE or iap gene from nine insects was injected into the last instar larvae. On the third day after treatment, the total RNA was extracted and IAP mRNA levels were determined by RT‐qPCR with RP49 as a reference gene as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). (b) The dsRNA (1 μg/larva) targeting malE or iap gene from nine insects were injected into the last instar larvae and the rate of mortality recorded from the third to 10th day after treatment. Experiments were repeated twice and each experiment consisted of 12 larvae/each dsRNA used. The numbers on the top of the bars show percent mortality induced by dsIAPs. Aa, Aedes aegypti; Ag, Anoplophora glabripennis; dsRNA, double‐stranded RNA; Hh, Halyomorpha halys; IAP, inhibitor of apoptosis; Ld, Leptinotarsa decemlineata; Ls, Lasioderma serricorne; Mh, Murgantia histrionica; mRNA, messenger RNA; Nv, Nezara viridula; RT‐qPCR, quantitative reverse transcription polymerase chain reaction; Sf, Spodoptera frugiperda; Tc, Tribolium castaneum

FIGURE 6.

FIGURE 6

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Knockdown of iap gene and mortality induced by dsIAP in the Southern Green Stink Bug, Nezara viridula, SGSB. (a) Certified organic beans were inserted in 2 ml tubes containing 300 μl of nuclease‐free water and 20 μg of dsRNA targeting GFP or iap gene from nine insects were added to water and one 10 SGSB nymph were fed on each bean for 3 days. The dsRNA solution was replaced daily. Total RNA was isolated on the fifth day after initiation of feeding dsRNA and used to determine IAP mRNA levels by RT‐qPCR using 60S RP L12 as a reference gene as described in Figure 2a legend. Means followed by the same letter are not significantly different (Duncan multiple range test (p < .05). (b) Certified organic beans were inserted in 2 ml tubes containing 300 μl of nuclease‐free water and 20 μg of dsRNA targeting iap gene in nine insects (20 μg of dsRNA in water was added to the tube on the first, second, and third day). Ten second/third instar nymphs were fed on each bean and mortality was recorded on the 14th day after treatment. Mean + SE (n = 20) are shown. The numbers on the top of the bars show percent mortality induced by dsIAP. Aa, Aedes aegypti; Ag, Anoplophora glabripennis; dsRNA, double‐stranded RNA; GFP, green fluorescence protein; Hh, Halyomorpha halys; IAP, inhibitor of apoptosis; Ld, Leptinotarsa decemlineata; Ls, Lasioderma serricorne; Mh, Murgantia histrionica; mRNA, messenger RNA; Nv, Nezara viridula; RT‐qPCR, quantitative reverse transcription polymerase chain reaction; SE, standard error; Sf, Spodoptera frugiperda; Tc, Tribolium castaneum

4 |. DISCUSSION

RNAi‐based pest management methods are currently under development (Zhu & Palli, 2020). A product to control Western corn rootworm (WCRW) has recently been approved and several other products are under development. Potential nontarget effects of dsRNA used in pest control have been discussed since RNAi‐based pest management was first proposed. Initial testing of dsRNA nontarget effects followed established protocols used for testing of nontarget pest control agents, especially biological insecticides such as Bacillus thuringiensis toxins (Bachman et al., 2013). The concept of designing dsRNAs that target a conserved or variable region of a target gene provides flexibility in targeting one or multiple related species of insect pests depending on the needs. However, the possibility of using a highly conserved region for designing the dsRNAs could lead to undesirable nontarget effects that can influence the populations of other beneficial insects (Lundgren & Duan, 2013; Roberts, Devos, Lemgo, & Zhou, 2015). To understand and evaluate the possibility of nontarget effects, there is a need to study the effects of multiple dsRNAs targeting genes in the conserved pathways across species. Multiple iap genes have been identified in several insects and the iap gene has been identified as an efficient target for RNAi in many insects (Cao et al., 2018; Dhandapani et al., 2019; Igaki et al., 2002; Mogilicherla et al., 2018; Powell et al., 2017; Rodrigues et al., 2017; Yoon et al., 2016, 2018). Here, we assayed nine dsRNAs targeting iap gene fragments spanning conserved domains in nine insects and found that the dsIAPs work well in target species, but did not show significant knockdown of iap gene expression and apoptosis in cells derived from nontarget insects or mortality in nontarget insects. This is, in contrast, to reports on the cross‐species activity of dsRNA targeting the D. melanogaster iap gene in two dipteran insects (Powell et al., 2017). Interestingly, among the three stink bug species, the targets of dsIAPs showed some sequence similarity but this did not result in a significant knockdown of the iap gene expression or mortality in nontarget stink bug species. Based on the number of siRNA matches with the iap gene fragments in nontarget stink bug species, we expected some knockdown of iap gene and mortality, but this was not the case. Further research is needed to understand the relationship between the sequence similarity of siRNA targets and knockdown efficiency and mortality. Most of the published studies on dsRNA in nontarget species, especially those that are phylogenetically distant from the target insect, reported no significant effects (Bachman et al., 2013; Pan et al., 2016, 2017). However, a few studies using highly conserved target genes such as the vATPaseA, ribosomal protein (rpl19) gene, and ecdysone receptor (EcR) reported some effect on target gene knockdown and mortality. For example, dsRNA targeting WCRW vATPaseA induced mortality in CPB at a concentration 10× higher than the LC50 in WCRW due to >80% sequence identity of vATPaseA gene between WCRW and CPB (Baum et al., 2007). Also, the same dsRNA adversely affected two species of ladybird beetles by inducing knockdown of orthologs and affecting survival at dsRNA concentrations at orders of magnitude higher than expected to occur in the field (Haller, Widmer, Siegfried, Zhuo, & Romeis, 2019). The WCR vATPaseA dsRNA showed 34 and 6 siRNA matches, respectively, with those in Coccinella septempunctata and Coccinella bipunctata (Haller et al., 2019). Similarly, dsRNA targeting Spodoptera exigua EcR gene induced mortality in Helicoverpa armigera due to 89% sequence identify of EcR between these two insect species (Zhu et al., 2012). Also, dsRNA targeting the Bactrocera minax rpl19 gene induced knockdown of this gene ortholog in a closely related species, Bactrocera dorsalis due to >90% sequence similarity between these genes in the two insect species tested (Chen, Zheng, Zheng, & Zhang, 2015). The dsIAP tested did not show high sequence similarity among the orthologs of insect species used in our studies and this may be one of the reasons for no major nontarget effects detected. It would be interesting to test the cross‐species effects of dsIAPs targeting regions of iap genes that are highly conserved (e.g., BIR domain) among the species tested. Work is in progress to test this possibility. The results reported here lay a foundation toward designing dsRNAs for controlling only one target species or multiple related species, but further work is needed to establish guidelines for designing dsRNAs for targeting single or multiple species of insect pests.

Supplementary Material

SI

ACKNOWLEDGMENTS

Supported by grants from the National Institutes of Health (GM070559‐14 and 1R21AI131427‐02), the National Science Foundation (Industry/University Cooperative Research Centers, the Center for Arthropod Management Technologies under Grant IIP‐1821936), the National Institute of Food and Agriculture, USDA, HATCH under 2353057000 and the Agriculture and Food Research Initiative Competitive Grant no. 2019‐67013‐29351. We thank Mrs. Smitha George from Palli lab for help with the injection of dsRNAs into RFB larvae.

Funding information

National Institutes of Health, Grant/Award Numbers: GM070559‐14, 1R21AI131427; USDA, HATCH, Grant/Award Number: 2353057000; USDA, Agriculture and Food Research Initiative Competitive, Grant/Award Number: 2019‐67013‐29351

Footnotes

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section.

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