Dietary Rnai Toxicity Assay Exhibits Differential Responses to Ingested

Research Article

Received: 29 February 2020 Revised: 21 April 2020 Accepted article published: 13 May 2020 Published online in Wiley Online Library: 2 June 2020

(wileyonlinelibrary.com) DOI 10.1002/ps.5894 Dietary RNAi toxicity assay exhibits differential responses to ingested dsRNAs among lady beetles Huipeng Pan,a,b Xiaowei Yang,b Jörg Romeis,c Blair D Siegfriedd and Xuguo Zhoub*

Abstract

Background: Most recently, major federal regulatory agencies deregulated an in planta RNA interference (RNAi) trait against a devastating corn pest, the western corn rootworm Diabrotica virgifera virgifera, in the United States and Canada. The impact of double-stranded RNA (dsRNA) plant-incorporated protectants (PIPs) and dietary RNAi to non-target organisms, however, still needs further investigation. In this study, we assessed the potential risks of a Diabrotica virgifera virgifera active dsRNA to a group of predatory biological control agents, including Hippodamia convergens, Harmonia axyridis, Coleomegilla maculata, and Coccinella septempunctata. The overarching hypothesis is that the insecticidal dsRNA targeting Diabrotica virgifera virgifera has no or negligible adverse effect on lady beetles. Results: A 400-bp fragment with the highest sequence similarity between target and tested species was selected as the template for dsRNA synthesis. For the dietary RNAi toxicity assay, newly hatched ﬁrst instar larvae were administered with v-ATPase A dsRNAs designed from Diabrotica virgifera virgifera and the four lady beetles, respectively. A dsRNA from ⊎-glucuronidase (GUS), a plant gene, and H2O were served as the negative controls. The endpoint included both sub-organismal (gene expression), and organismal (survival rate, development time, pupa and adult weight) measurements. The results from dietary RNAi toxicity assay demonstrate signiﬁcantly impacts of Diabrotica virgifera virgifera-active dsRNAs on lady beetles under the worst- case scenario at both transcriptional and phenotypic level. Interestingly, substantial differences among the four lady beetle species were observed toward the ingested exogenous dsRNAs. Conclusion: Such differential response to dietary RNAi may shed light on the mechanisms underlying the mode-of-action of RNAi-based biopesticides. © 2020 Society of Chemical Industry Supporting information may be found in the online version of this article.

Keywords: lady beetle; dsRNA; dietary RNAi; differential response; ecological risk assessment1

1 INTRODUCTION Agriculture (USDA) in 2015 (https://www.aphis.usda.gov/brs/ Since its discovery near the end of the 20th century, the aphisdocs/13_29001p_det.pdf) and the US Environmental Protec- post-transcriptional gene silencing of RNA interference (RNAi) tion Agency (EPA) in 2017 (https://www.epa.gov/pesticide- has had a tremendous impact on biological research.1 RNAi is a registration/epa-registers-innovative-tool-control-corn-rootworm). naturally occurring, innate immune response common to eukary- Stacking a herbicide tolerance trait, an insecticidal Bacillus otes to fend off viruses and genomic parasites, such as transpos- able elements. The discovery of RNAi mechanisms in insects and recent advances in functional genomics facilitate the develop- * Correspondence to: X Zhou, Department of Entomology, University of Ken- ment of a new wave of biopesticides with an innovative mode tucky, S-225 Agricultural Science Center North, Lexington, KY 40546-0091, 2-5 of action (MOA). RNAi-based gene suppression has been USA, E-mail: [email protected] applied in several genetically engineered (GE) crops approved for cultivation and for use as food and feed.6-8 In the past decade, a Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou, China the potential application of in planta RNAi in the integrated pest 9–18 management has been extensively investigated. b Department of Entomology, University of Kentucky, Lexington, KY, USA Recently, SmartStax PRO maize carrying event MON87411 has received regulatory approval in Canada (2016) and the United c Agroscope, Research Division Agroecology and Environment, Zurich, States (2017) to control the western corn rootworm, Diabrotica vir- Switzerland

3606 19 gifera virgifera. Speciﬁcally, MON 87411, the ﬁrst commercial d Department of Entomology and Nematology, University of Florida, Gainesville, RNAi GE event, was deregulated by the US Department of FL, USA

Pest Manag Sci 2020; 76: 3606–3614 www.soci.org © 2020 Society of Chemical Industry Ecological risk assessment of dietary RNAi to lady beetles www.soci.org thuringiensis (Bt) trait, and a RNAi trait targeting Diabrotica virgifera (De Geer), and the seven-spotted lady beetle Coccinella septem- virgifera Snf7, a housekeeping gene encoding the SNF7 subunit of punctata Linnaeus were used to test our risk hypothesis due to the ESCRT-III complex, SmartStax PRO maize provided a viable their ecological functions and services. Lady beetles primarily option and enhanced durability of Bt traits for rootworm control.19,20 prey on aphids but also feed on thrips, spider mites, small insect Although double-stranded RNA (dsRNA) plant-incorporated protec- larvae and eggs, and honeydew secreted by aphids and other tants (PIPs) have received regulatory approval, commercialization sap-sucking arthropods, which make them accessible to dsRNA and sales have lagged. This is not only due to the technical chal- PIPs. Lady beetles can also consume pollen, nectar, and petals 33 lenges and regulatory hurdles to bringing the new technology from directly, all of which increases their risks of exposure to PIPs. Hip- the benchtop to the tabletop,15,21–23 or potential environmental risks podamia convergens was purchased from a commercial vender in associated with the technology, but also other factors, including California (High Sierra Ladybugs, Mariposa, CA, USA; http://www. investment behavior (investors’ lacking of interests in the long-term biconet.com/biocontrol/ladybug.html). Harmonia axyridis, Coleo- investment), patent blocking, consumer perception, market receptiv- megilla maculata, and Coccinella septempunctata were collected ity, cultural influences, as well as emerging competing technologies, from the North Farm of the University of Kentucky, Lexington, such as genome editing. KY, USA. Lady beetle larvae and adults were maintained in a ± ° The possibility of exploiting dsRNA expression in GE plants to growth chamber at 23 0.5 C, 16 h light/8 h dark (16L:8D) pho- ± control pests has necessitated the collection of data for ecological toperiod, and 60 1% relative humidity, and provisioned with risk assessment (ERA). Evaluation of the impacts of GE plants on pea aphids, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae). non-target organisms (NTOs) is a key component. Baum et al.9 Acyrthosiphon pisum clones were maintained on fava bean, Vicia – ° found that the dsRNA PIPs targeting Diabrotica virgifera virgifera faba L. (Fabales: Fabaceae), seedlings in a glasshouse at 20 28 C. v-ATPase A and E significantly reduced the survival of the spotted cucumber beetle, Diabrotica undecimpunctata howwadi Barber, 2.2 Dietary RNAi toxicity assay for predatory lady beetles and the Colorado potato beetle, Leptinotarsa decemlineata (Say) 2.2.1 Cloning and sequencing of v-ATPase A (both Coleoptera: Chrysomelidae). Without prior knowledge, the Diabrotica virgifera virgifera v-ATPase A sequence was obtained ERA model for Bt crops provides a foundation for the develop- from a de novo transcriptome assembly of cDNAs prepared from fi 34 ment of ERA framework for RNAi GE crops.24 It is a tiered approach eggs, rst instars, and the midguts of third instars. The full- which focuses on the effects of dsRNA PIPs on threatened and length cDNA of Hippodamia convergens v-ATPase A was cloned endangered species and organisms with ecological functions by a combination of regular polymerase chain reaction (PCR) – 35 fi beneficial to agriculture.15,24 26 Typically, tier I assessments are using degenerate primers, and rapid ampli cation of cDNA ends (RACE) PCR using a SMARTer RACE cDNA amplification kit carried out under the worst-case scenarios using laboratory pro- 0 0 cedures, including the administration of purified active ingredi- (Clontech Laboratories, Mountain View, CA, USA). The 3 - and 5 - ents in artificial diets. The US EPA recommends a 10× exposure full RACE core set (Clontech Laboratories) was used to obtain full factor margin.26,27 However, dsRNA-associated products present length v-ATPase A from Harmonia axyridis, Coleomegilla maculata, additional hazard and risk assessment challenges because of their and Coccinella septempunctata, respectively. distinct MOA. Therefore, other toxicological endpoints should be Total RNA was extracted from one adult pair per lady beetle spe- measured.24,25 The current Bt crop risk assessments use mortality cies using TRIzol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA μ as a primary endpoint measurement. However, due to our limited was synthesized from 2.0 g total RNA using an M-MLV reverse μ knowledge concerning the MOAs of in planta RNAi,24 the chronic transcription kit (Invitrogen). The 50 L PCR reaction mixture con- μ × 2+ μ and sublethal effects of dsRNA PIPs on the life history traits of the tained 10 Lof5 PCR Buffer (Mg Plus), 1 L of dNTP mix -1 μ μ -1 test organisms should be evaluated.15,22,24,28 (10 mmol L per nucleotide), 5 L of each primer (10 mol L μ μ −l The overall goal of this study was to assess the potential risks of a each), and 0.25 L of Go Taq (5 u L ) (Promega, Madison, WI, ° Diabrotica virgifera virgifera-active dsRNA toward an array of cole- USA). The PCR was run as follows: one cycle of 94 C for 3 min; ° ° ° opteran biological control agents. Previous studies suggest that 35 cycles of 94 C for 30 s, 59 C for 45 s; 72 C for 1 min; and a fi ° dsRNAs can have a specific activity spectrum, i.e. non-target species nal cycle of 72 C for 10 min. Amplicons of the expected size fi taxonomically closely related to the target organism may be sus- were puri ed and cloned into a pCR4-TOPO vector (Invitrogen) fi ceptible to dsRNA PIPs.9,28,29 Lady beetles (Coleoptera: Coccinelli- for sequencing con rmation. The full length v-ATPase A cDNAs dae) are predatory biological control agents sharing the same of Hippodamia convergens, Harmonia axyridis, Coleomegilla macu- insect order and ecological niche with the target insect pest, Dia- lata, and Coccinella septempunctata have been deposited in Gen- brotica virgifera virgifera, and have been used extensively in the Bank with accession numbers MK192778, MK192779, MK192780, ecological risk assessment of Bt crops.30–32 Here, our overarching and MK192781, respectively. hypothesis is that this novel dsRNA PIPs have no or negligible effects on predatory lady beetles. To examine this risk hypothesis, 2.2.2 Target region selection and bioinformatics analysis we (i) developed a standardized dietary RNAi toxicity assay for Pairwise sequence alignments were carried out between target predatory lady beetles; (ii) evaluated the impacts of exogenous insect pest, Diabrotica virgifera virgifera, and each of the lady bee- 36 dsRNAs on lady beetle gene expression and life history traits; and tle species using MUSCLE. To create the worst-case scenario, a (iii) proposed a strategy for ERA of transgenic RNAi crops on NTOs. 400 nt fragment of v-ATPase A with the highest sequence similarity between Diabrotica virgifera virgifera, and the non-target lady beetles was selected as the target region to synthesize dsRNAs 2 MATERIALS AND METHODS (Fig. 1(A, C)). Specifically, when we counted the number of con- 2.1 Lady beetles served (exactly matched) nucleotides per 400 bp along the v- In this study, the convergent lady beetle Hippodamia convergens ATPase A open reading frames (ORFs), we used a sliding window Guérin-Meneville, the multicolored Asian lady beetle Harmonia approach with a 400 bp window length and 1 bp step length. 3607 axyridis (Pallas), the pink lady beetle Coleomegilla maculata As a result, the region between nucleotide position 704–1103 of

Figure 1 The v-ATPase A region for dsRNA synthesis. (A) Schematic drawing of n-mer matches within v-ATPase A between lady beetle and Diabrotica virgifera virgifera. Distribution and location of identical nucleotide sequence matches (n-mers) embedded in the ORF and selected 400 bp region of v-ATPase A are mapped in the primary structures, and the sequence similarities (%) are in bold. (B) Number of 21mer matches between the target pest Diabrotica virgifera virgifera and each lady beetle species within the selected v-ATPase A 400 bp region and ORF, respectively. (C) Number of consensus nucleotides per 400 bp along the v-ATPase A ORFs of the target pest Diabrotica virgifera virgifera and the four surrogate lady beetle species. The arrow indicates the regions between nucleotide position 697 and 1096 (400 bp in length) within lady beetle v-ATPase A have the highest number of conserved nucleotides (255) with Diabrotica virgifera virgifera v-ATPase A. [Color ﬁgure can be viewed at wileyonlinelibrary.com]

Diabrotica virgifera virgifera and 697–1096 of the four surrogate control, while a 560 bp ⊎-glucuronidase (GUS) fragment was a neg- lady beetle species had the highest number of matches ative control. GUS was cloned into a pBTA2 vector and amplified (Fig. 1(C)). An in-house Perl script was used to determine the by PCR using gene-specific primers (Table S5). PCR was carried number of 21mer sequences in each pairwise alignment. The out in a 50 μL reaction mix, as described previously. The resultant script searches for any instances of n continuous positions in PCR product was used as a template to generate dsRNAs with a T7 any of the sequences in the alignment. The 21mer matches in MEGAscript kit (Ambion, Austin, TX, USA). The synthesized dsRNAs the entire ORF and the selected 400 bp were counted, respec- were suspended in nuclease-free water (H2O), quantified with a tively (Fig. 1(B)). The 21mer sequence and the start and end posi- NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, tions of Diabrotica virgifera virgifera and the lady beetles are Waltham, MA, USA), and stored at −20 °C. illustrated in Supporting Information Tables S1–S4 (n-mer contiguous matches in the selected 400 bp region between lady beetles 2.2.4 In vivo dietary RNAi toxicity assay and Diabrotica virgifera virgifera are highlighted in yellow). Dietary RNAi toxicity assays were conducted in small plastic Petri dishes (5 cm diameter, 1.3 cm height) with one insect per dish. For 2.2.3 Synthesis of dsRNAs each lady beetle species, newly hatched first instars were fed with Specific primers containing a T7 promoter sequence were used to Diabrotica virgifera virgifera v-ATPase A dsRNA (dsDVV), lady beetle generate dsRNAs targeting lady beetle v-ATPase A (Table S5). The dsRNA (dsHC or dsHA or dsCM or dsCS), GUS dsRNA (dsGUS), and 3608 conserved 400 bp regions are shown in Fig. 1(A). A 400 bp Diabro- H2O. Specifically, 2 μL droplet of 15% w/v sugar solution contain- tica virgifera virgifera v-ATPase A fragment was used as a positive ing 4 μg μL−1 dsRNA was supplied to the individual first instars

(~24 h within hatching) daily for 2 days. First instars typically con- When the homogeneity of the ANOVA was < 0.05, a non- sumed approximately 16 μg dsRNA for the first 2 days, and then parametric test (K independent samples) was used. One-way they were switched to the pea aphids ad libitum for the remaining ANOVA was also used to compare v-ATPase A expression levels assay days. Assays were conducted in a climate chamber at 23 in each lady beetle species across all treatments and at each time ± 0.5 °C, 14L:10D photoperiod, and 60 ± 1% relative humidity. point. Means were compared with Tukey’s test at P < 0.05. Pro- The endpoints measured both organismal and sub-organismal portional data were arcsine square root-transformed before anal- impacts. ysis. SPSS v. 21.0 (IBM Corp., Armonk, NY, USA) was used for all For organismal impacts, mortality and developmental time statistical analyses. were checked twice a day (9 a.m. and 9 p.m.). For Hippodamia convergens and Harmonia axyridis, pupa and adult weights were recorded. For Coleomegilla maculata, the development time from 3 RESULTS the first to third instar was measured. For Coccinella septempunc- 3.1 Molecular cloning of v-ATPase A tata, all the individuals died before they reached the third instar Degenerate primers were used to obtain the starting sequence of stage in the dsDVV treatment. Therefore, only the development v-ATPase A from Hippodamia convergens, Harmonia axyridis, Coleo- time from the first to second instar was recorded and analyzed. megilla maculata, and Coccinella septempunctata. RT-PCR and There were three independent runs, including 30 technical repli- RACE were used to amplify the entire coding sequence of the cates per treatment per run. For sub-organismal impacts, five indi- v-ATPase A from the lady beetles. The lady beetle v-ATPase A had viduals were collected per lady beetle species at days 0, 3, 5, 7, and an ORF of 1842 bp and encoded a 614-amino acid protein. Pair- 9. All samples were flash-frozen in liquid nitrogen and stored at wise comparisons indicated that v-ATPase A from the Diabrotica −80 °C for the subsequent reverse transcriptase-quantitative virgifera virgifera nucleotide sequence shares 80.68, 81.15, 78.66, polymerase chain reaction (RT-qPCR) analysis. For each time and 80.70% identity with Hippodamia convergens, Harmonia axyr- point, there were four biological replicates for Hippodamia conver- idis, Coleomegilla maculata, and Coccinella septempunctata, gens and three for the other lady beetle species. respectively (Fig. 1(A)). A ~400 bp region of v-ATPase A with the highest number of conserved nucleotides among the tested spe- 2.3 Reverse transcriptase-quantitative polymerase chain cies was selected as a template for dsRNA synthesis (Fig. 1(C)). reaction (RT-qPCR) analysis Sequence similarity of the 400-bp region between Diabrotica Total RNA was extracted with TRIzol (Invitrogen). First-strand virgifera virgifera and lady beetles was 83.71, 84.71, 82.71, and cDNA was prepared from 1.0 μg total RNA with the M-MLV reverse 84.63%, respectively, for Hippodamia convergens, Harmonia axyri- transcription kit (Invitrogen). Gene-specific primers (Table S5) dis, Coleomegilla maculata, and Coccinella septempunctata. were used in a 15 μL PCR reaction mixture, which consisted of 5.25 μL of double distilled H2O, 7.5 μLof2× SYBR Green Master- 3.2 Bioinformatics analysis Mix (Bio-Rad Laboratories, Hercules, CA, USA), 4 μM of each spe- We evaluated the number of 19 to 25 nt identical contiguous cific primer, and 1.0 μLoffirst-strand cDNA template. The RT- matches in each pairwise alignment of v-ATPase A between Dia- qPCR program included an initial denaturation for 3 min at 95 ° brotica virgifera virgifera and the lady beetles (Fig. 1(A, B), C followed by 40 cycles of denaturation at 95 °C for 10 s, anneal- Tables S1–S4). Coccinella septempunctata has more 19–25-nt con- ing for 30 s at 55 °C, and extension for 30 s at 72 °C. For the melt- tiguous matches with Diabrotica virgifera virgifera than the other ing curve analysis, a dissociation step cycle (55 °C for 10 s then three species (Fig. 1, Table 1). 0.5 °C for 10 s until 95 °C) was added. The reactions were run in triplicate in 96-well Microseal PCR plates (Bio-Rad Laboratories). 3.3 Temporal profiling of v-ATPase A expression Reference genes of Actin and CypA,35 rp49 and GAPDH,37 EF1A A standardized dietary RNAi toxicity assay was established for and Actin,38 EF1A and Actin39 were used to normalize gene expres- predatory lady beetles. The v-ATPase A expression in Hippodamia sions in Hippodamia convergens, Harmonia axyridis, Coleomegilla convergens was significantly downregulated by days −ΔΔCt maculata, and Coccinella septempunctata, respectively. 2 3(F3,12 = 16.003, P < 0.0001) and 5 (F3,12 = 5.407, P = 0.014) in 40 method was used to quantify v-ATPase A expression. dsDVV and dsHC treatments relative to dsGUS and H2O controls, respectively. At days 3 and 5, expression levels under dsHC treat- 2.4 Statistical analysis ment were reduced by ~82.6% and ~75.5% relative to H2O con- One-way analysis of variance (ANOVA) was used to compare the trol, respectively. Gene expression was also significantly life history traits of each lady beetle species across all treatments. suppressed under dsHC but not dsDVV treatment at day

Table 1 The number of n-mer matches within v-ATPase A between lady beetles and Diabrotica virgifera virgifera

n-mer matches (nt)a

Species 19 20 21 22 23 24 25

Hippodamia convergens 29 (17) 24 (15) 19 (13) 16 (11) 13 (9) 10 (7) 8 (6) Harmonia axyridis 24 (22) 21 (20) 18 (18) 16 (16) 14 (14) 12 (12) 11 (11) Coleomegilla maculate 29 (27) 25 (24) 21 (21) 18 (18) 15 (15) 12 (12) 10 (10) Coccinella septempunctata 50 (40) 45 (37) 40 (34) 36 (31) 32 (28) 28 (25) 24 (22)

The number of identical nucleotide sequence matches within the open reading frame (400 bp region) of v-ATPase A between each lady beetle and 3609 Diabrotica virgifera virgifera was calculated using a customized bioinformatics tool.

7(F3,12 = 13.905, P < 0.0001) (Fig. 2(A)). At that time, the expres- For Coccinella septempunctata, v-ATPase A expression was signif- sion level was reduced by ~53.1% relative to H2O control. icantly downregulated at days 3 (F3,8 = 40.447, P < 0.0001) and For Harmonia axyridis, v-ATPase A expression was significantly 5(F3,8 = 27.240, P = 0.0001) under dsDVV and dsCS treatments suppressed (≤ 36%) at day 3 under dsDVV and dsHA treatments compared to dsGUS and H2O treatments. Under dsDVV and dsCS relative to H2O control (F3,8 = 11.298, P = 0.003). The v-ATPase A treatments at day 3, expression levels were reduced by 90% and fi expression under dsDVV treatment at day 5 was signi cantly 68% relative to H2O control, respectively. Gene expression lower than that of dsHA treatment at the same time point efficiency was highly suppressed under dsDVV treatment. The (F3,8 = 11.619, P = 0.003) (Fig. 2(B)). v-ATPase A expression level was significantly lower at day 5 than For Coleomegilla maculata, v-ATPase A expression was signif- that for dsCS treatment at the same time point. At day 5, v-ATPase icantly downregulated at days 3 (F3,8 = 8.241, P = 0.008) and A expressions under dsDVV and dsCS treatments were reduced by 5(F3,8 = 9.826, P = 0.005) under dsDVV and dsCM treatments 92% and 63% relative to the controls, respectively (Fig. 2(D)). compared to dsGUS and H2Otreatments.Atday3,v-ATPase A expression under dsDVV and dsCM treatments were reduced 3.4 In vivo dietary RNAi toxicity assay by ≤ 66% relative to the H2O control. At day 5, under dsCM The survival rates of Hippodamia convergens (F3,8 = 27.505, treatment, v-ATPase A expression was reduced by 70% relative P < 0.0001), Harmonia axyridis (F3,12 = 10.163, P = 0.001), Coleo- to the H2O control. Expression levels at days 7 (F3,8 = 10.943, megilla maculata (F3,8 = 23.121, P < 0.0001), and Coccinella sep- P = 0.003) and 9 (F3,8 = 5.901, P = 0.020) remained significantly tempunctata (F3,8 = 1529.017, P < 0.0001) were significantly suppressed under dsCM treatment and were lower than other reduced by exposure to the species-specific and Diabrotica virgi- treatments. At days 7 and 9, under dsCM treatment, v-ATPase fera virgifera dsRNAs relative to H2O and dsGUS controls. For Hip- A expression remained 72% and 55% lower than H2O control, podamia convergens, the survival rates under dsDVV and dsHC respectively (Fig. 2(C)). treatments were reduced by 73% and 83% relative to H2O control,

Figure 2 Temporal expression profiles of v-ATPase A in lady beetles ingested exogenous dsRNAs. The v-ATPase A expression profiles were documented on days 0, 3, 5, 7, and 9 for Hippodamia convergens (A), Harmonia axyridis (B), Coleomegilla maculata (C), and Coccinella septempunctata (D). Results are expressed as the relative expression of v-ATPase A transcripts normalized to the reference genes (see Table S5 for details). The v-ATPase A transcript levels 3610 in the newly emerged (day 0), untreated larvae of each species were set to 1. Values are means + standard error. Different letters indicate significant differences among treatments (P < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3 Phenotypic impact of dietary RNAi measured by survival rates. Here, we documented the survival rate of Hippodamia convergens (A), Harmonia axyridis (B), Coleomegilla maculata (C), and Coccinella septempunctata (D) after ingested dsRNAs. Values are means + standard error. Different letters indicate signiﬁcant differences among treatments (P < 0.05). [Color ﬁgure can be viewed at wileyonlinelibrary.com]

respectively (Fig. 3(A)). For Coleomegilla maculata, the survival For Coleomegilla maculata, the development time from the first rates under dsDVV and dsCM treatments were reduced by 45% to third instar (χ2 = 14.634, df = 3, P = 0.002) was significantly and 48% relative to H2O, respectively (Fig. 3(C)). All Coccinella sep- reduced under dsGUS treatment than the others (Fig. 4(C)). All tempunctata individuals were dead under dsDVV treatment and Coccinella septempunctata individuals died before they reached the survival rate under dsCS treatment was reduced by 73% rela- the third instar under dsDVV treatment, therefore, only the devel- tive to H2O control (Fig. 3(D)). For Harmonia axyridis, the survival opment time from the first to second instar was documented rates were reduced by only ~10% relative to H2O control (Fig. 3 and analyzed. These individuals had the fastest and slowest devel- (B)). As such, the four lady beetle species displayed significantly opment time under dsCS and H2O treatments, respectively different responses to exogenously applied dsRNAs. (F3, 255 = 53.630, P < 0.0001) (Fig. 4(D)). Hippodamia convergens developed faster under H2O and dsGUS treatments than dsDVV and dsHC treatments (χ2 = 30.984, df = 3, P < 0.0001) (Fig. 4(A)). In contrast, pupa weight (χ2 = 3.976, df = 3, 4 DISCUSSION P < 0.264) and adult weight (χ2 = 2.750, df = 3, P < 0.432) did not 4.1 Development of a standardized dietary RNAi toxicity significantly differ across treatments (Fig. 4(E, G)). assay for predatory lady beetles Harmonia axyridis had the fastest and slowest development All lady beetle species tested were affected by their respective times under H2O and dsHA treatments, respectively species-specific v-ATPase A dsRNAs. Nevertheless, their sensitivi- (χ2 = 51.287, df = 3, P < 0.0001) (Fig. 4(B)). Pupa weight ties varied. All four lady beetle species were sensitive to dsDVV, (F3,386 = 14.763, P < 0.0001) and adult weight (F3,386 = 15.322, v-ATPase A dsRNA derived from Diabrotica virgifera virgifera. Simi- P < 0.0001) were both affected by the treatments (Fig. 4 larly, dsDVV also adversely affected Adalia bipunctata and Cocci- (F, H)). Harmonia axyridis had the highest and lowest adult nella septempunctata as indicated by a significantly (yet and pupa weights under dsHA/dsDVV and dsGUS treatments, marginally) prolonged developmental time for Adalia bipunctata 3611 respectively. and a significantly reduced survival rate for Coccinella

Figure 4 Phenotypic impact of dietary RNAi measured by life history traits. Here, the endpoint measurements included the development time (A–D), pupa weight (E and F), and adult weight (G and H). See text for treatment details. Values are means + standard error. Different letters indicate signiﬁcant differences among treatments (P < 0.05). [Color ﬁgure can be viewed at wileyonlinelibrary.com]

septempunctata.41 The dsDVV, however, has minimal or negligible unintended gene silencing could occur and result in negative impacts on other NTOs, including larvae and adult honey bees, consequences.46 In this study, bioinformatics analysis show that Apis mellifera, a collembolan, Sinella curviseta, and the monarch open reading frame/400 bp dsRNA region of v-ATPase A from Hip- butterfly larvae, Danaus plexippus, at both transcriptional and podamia convergens, Harmonia axyridis, Coleomegilla maculata, phenotypic levels.42–44 Our results are consistent with previous and Coccinella septempunctata had 20/13, 19/18, 22/21, and studies, which suggested that the dsRNA activity spectrum is nar- 40/34 21-nt contiguous (21mer) matches, respectively, with Dia- row and NTO species taxonomically related to the target organ- brotica virgifera virgifera, suggesting that in silico analysis can ism are relatively more susceptible to it. Therefore, phylogenetic complement empirical testing, especially for the risk assessment analyses may effectively predict possible adverse effects of dsRNA in non-model surrogate species. In comparison to conventional exposure.9,28,29 pest management strategies, dsRNA PIPs offer a control alterna- tive with much reduced risks of non-target effects because its 4.2 Impacts of exogenous dsRNAs on lady beetle gene cross activity is largely dependent on the sequence identify expression and life history traits (e.g. 21mer matches) rather than similarity. The advent of the In our temporal documentation of dietary RNAi toxicity, dsDVV Genomic Era has provided an unprecedented opportunity to inte- had stronger gene silencing effects at some time points relative grate in silico analysis with empirical testing to comprehensively to species-specific dsRNAs, including Harmonia axyridis and Cocci- assess potential risks associated with novel biotechnologies using nella septempunctata. These results are consistent with the corre- a much broader pool of surrogate species. Improved genomics sponding mortality data, which showed a higher morality in resources allow us to refine our dsRNA design to minimize un- Harmonia axyridis and Coccinella septempunctata when exposed intended effects. Specifically, non-conserved regions, which are to dsDVV than species-specific dsRNA treatments. In the case of not shared by the target pest and NTOs, and contain no or mini- Harmonia axyridis, slight but significant difference in gene expres- mal 21mer matches, should be selected to synthesize dsRNAs to sion between dsDVV and dsHA treatments at day 5 translated into exclude potential non-target effects. no effect at the phenotypic level (mortality). While for Coccinella The effectiveness of dsRNA may vary among lady beetle individ- septempunctata, gene expression difference between treatments uals or generations, depending on their physiological state and of dsDVV and dsCS at day 5 were substantial (2–5 fold), and not genetic background. Chu et al. documented differential effects of surprisingly, led to significant differences in mortality. Such a phe- RNAi treatments targeting DvRS5, a cysteine protease gene, among nomenon is not without precedent. Hamilton et al.45 documented three Diabrotica virgifera virgifera field populations.47 Individuals the role of glutathione-S-transferases in ovarian cancer cell lines’ within those populations may be naturally more or less resistant resistance to cisplatin, the first metal (platinum)-based agent for to gene silencing.46 In addition, RNAi does not work uniformly cancer therapy. The cell line with primary resistance to cisplatin among all beetles. Ingested dsRNA is significantly more effective exhibited a substantially stronger cross-resistance to CBDCA, a cis- in L. decemlineata and Diabrotica virgifera virgifera than the red flour platin analog. Hamilton et al.45 speculated a hierarchy of mecha- beetle, Tribolium castaneum,9,29,48 suggesting a wide range of envi- – nisms governing the cross-resistance was different from the ronmental uptake mechanisms among species and taxa.49 53 ones underlying the primary resistance. As for Coccinella septem- Although all four lady beetle species in this study were provisioned punctata, future research is warranted to elucidate the possible with equal quantity of dsRNA, Harmonia axyridis was particularly causes. recalcitrant to dietary RNAi, in comparison to Hippodamia conver- A recent study demonstrated that at least three 21-nt contigu- gens, Coleomegilla maculata,andCoccinella septempunctata. Har- ous sequences were required for efficacy against orthologs from monia axyridis relies on an intracellular obligate microsporidian 3612 other organisms.28 If there is sufficient homology between the parasite, Nosema thompsoni, in its hemocytes to wipe out indige- housekeeping gene in the target pest and that in a NTO, nous lady beetle competitors.54 Recently, Xu et al.55 documented

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