Addressing the challenges of symbiont-mediated RNAi in aphids

Collection and analysis of aphid RNAi data

To gather the data for the comparison of aphid RNAi experiments, we read and manually parsed 86 scientific publications for information (Table S1). The aphid species tested, the target tested, the method of application of dsRNA, the length and concentration of the RNA, the type of RNA administered (e.g., double-stranded RNA or small hairpin RNA), the level of gene knockdown, and the phenotypic effect of knockdown were recorded for each experiment in these publications. To calculate the total numbers of unique and repeat experimental designs, an experimental design was described as follows. Each experiment was first categorized by whether it delivered dsRNA by injection, feeding, or topical application. For injection experiments, a method was defined by the number of injections, the number of gene targets, and the concentration of dsRNA injected. For feeding experiments, a method was defined by the material fed on, the number of gene targets, and the concentration of dsRNA in the feeding solution. For topical application, a method was defined by the number of targets and the concentration of dsRNA applied to the aphid. All data was analyzed using R (Version 4.0.0; R Core Team, 2020).

Maintaining bacterial and insect stocks

Serratia symbiotica CWBI-2.3^T strains were grown using Trypticase Soy Broth (TSB) and Trypticase Soy Agar (TSA) at room temperature. E. coli HT115-DE3 strains were grown in LB at 30 °C. Media were supplemented where indicated with 0.3 mM diaminopimelic acid (DAP), 100 µg/mL carbenicillin (Carb), 20 µg/mL chloramphenicol (Cam), or 60 µg/mL spectinomycin (Spec). Acyrthosiphon pisum LSR1, PA1, 5A, and Tucson were acquired from long-term stocks maintained in the lab of Nancy Moran (University of Texas at Austin, Austin, TX, USA) and were reared on Broad Windsor Vicia faba plants (Mountain Valley Seed Company, South Salt Lake, UT, USA). Aphid cup cages were maintained in Percival I-36LLVL incubators (Perry, IA, USA) at 20 °C with a long (16L:8D) photoperiod. Aphids reared under these conditions have guts that are essentially sterile, and they are colonized in laboratory experiments only with the bacteria that are added to their diets (Elston et al., 2021).

Preparation of dsRNA for injection and feeding experiments

A. pisum LSR1 RNA was extracted from a single aphid using the Zymo Quick RNA Miniprep Kit according to the manufacturer protocols (Zymo Research, Irvine, CA, USA). The RNA was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Extended dsRNA regions were amplified from the cDNA template using their respective “Long” primers from Table S2. This product served as the template for the second PCR with the “T7” primers to amplify the target dsRNA fragment and add overhangs for T7 transcription. The T7 PCR template was used to prepare dsRNA with the HiScribe T7 Quick High Yield RNA Synthesis Kit according to the manufacturer protocols (New England BioLabs, Ipswich, MA, USA). For the injection experiments, dsRNA was purified with the Zymo Quick RNA Miniprep Kit. For the feeding experiments, LiCl precipitation was performed (New England Biolabs, 2022). The concentration of dsRNA was measured using a nanodrop, and the length of the transcripts was confirmed by running samples on a 1% agarose gel (Nolan, Hands & Bustin, 2006).

Feeding aphids C002 dsRNA

For the feeding experiments, four genotypes of A. pisum (LSR1, PA1, 5A, and Tucson) were fed dsRNA in an artificial diet mix. Each diet mix contained 100 ng/µL dsRNA in a total of 100 µL Febvay diet + 1 µl yellow food dye (Gel Spice Company Inc., Bayonne, NJ, USA) (Febvay, Delobel & Rahbé, 1988; Elston et al., 2021). Sixty total 3–5-day old, second instar aphids were split into two feeding dishes per condition and fed for 18 h before being transferred to plants (20 aphids per plant, three plants per condition). Aphid mortality was monitored daily for a week and offspring were removed from the plants to prevent overcrowding.

Engineering HT115 and CWBI-2.3^T to express dsRNA

Construction of CWBI-2.3^T-GFP was described previously (Elston et al., 2021). HT115-GFP was built using pGRG36::PA1-GFP, a plasmid derived from pGRG-36 (Phillips & Cooper, 2021). To apply this system to HT115, we adapted methods from McKenzie & Craig (2006). The donor E. coli strain, MFDpir, and the recipient HT115 strain were first grown overnight, then washed twice with 145 mM NaCl saline solution, mixed to a 1:2 ratio of donor:recipient, and 50 µL of the mix was spot-plated on LB + DAP agar. After a day of growth, the spot was scraped up and washed with saline as before. Dilutions of these bacteria were plated on LB + Carb agar and grown at 30 °C to select for transconjugants that had acquired the plasmid. Then, colonies were picked and re-grown on LB + Cam agar at 37 °C to select for transposition of the GFP cassette into the chromosome and loss of the temperature-sensitive plasmid. The resulting colonies were picked and regrown at 37 °C in liquid LB with Cam or Carb to confirm loss of the plasmid and successful integration.

The design of our dsRNA expression vector is based on a system for smRNAi in honey bees (Leonard et al., 2020; Lariviere et al., 2022). In brief, the plasmid has a low to medium copy RSF1010-based origin of replication, spectinomycin resistance, and a dsRNA target sequence flanked by two identical inverted CP25 promoters. Each target for the RNAi experiments was first amplified from A. pisum LSR1 cDNA with “GGA” primers specified in Table S2, then cloned into the dsRNA site on the pBTK800 expression vector (Lariviere et al., 2022) with BsaI Golden Gate Assembly. The choice of amplified gene fragment was selected to replicate previous publications (Mutti et al., 2006; Vellichirammal et al., 2017; Chung et al., 2018). Assembled plasmids were conjugated or electroporated into CWBI-2.3^T-GFP and HT115-GFP for aphid feeding as described previously (Elston et al., 2021), except the dsNuc1-800 plasmid, which was tested in wild-type CWBI-2.3^T and HT115 hosts. Plasmids used in this study are shown in Table S3.

Symbiont-mediated RNAi targeting C002

Exponential phase cultures of HT115-GFP or CWBI-2.3^T-GFP with the indicated plasmids were grown in media with spectinomycin and then washed and resuspended in phosphate buffered saline (PBS) at an optical density of 1.0 at 600 nm (OD600). A. pisum LSR1 was reared to 5 days of age (late second instar) then fed on 98 μL of Febvay diet, plus 1 µL of resuspended bacterial cells and 1 µL of yellow food dye (Elston et al., 2021). Aphids were divided into 3–6 groups per condition and fed on the diet mix for 24 h, then transferred to the same number of respective plants. Their mortality was recorded daily for 1 week, and all dead aphids were crushed and plated on selective media as previously described (Elston et al., 2021) to confirm colonization. For the dsNuc1 co-inoculation experiments, 0.5 µL of the resuspended cell solution for each strain was added to the diet + food dye mixture.

Injection of EcR dsRNA

To prepare for injection of EcR dsRNA, colonies of A. pisum LSR1 were reared at low density (5–7 aphids per plant) for at least two generations to decrease colony stress and the numbers of alate adults used in the experiments. Then, 45 10-day-old adult aphids were injected with ~0.1 μL of 2 μg/μL EcR or non-targeting E2-Crimson dsRNA. The injected aphids were allowed to recover overnight in petri dishes, then placed onto fava beans with five aphids per plant. Every 24 h the adults were transferred to fresh plants for a total of 3 days (Vellichirammal et al., 2017). The offspring were then reared to the 4th instar and scored for wing bud development.

Symbiont-mediated RNAi targeting EcR

As described in the injection methods for EcR dsRNA, A. pisum LSR1 were first reared at a low density for at least two generations. CWBI-2.3^T-GFP with the dsEcR-800 or dsE2C-800 plasmid were grown and resuspended in PBS as described for the C002 experiments. Forty aphids per condition were fed overnight on a diet mix containing 98 μL of Febvay diet, 1 μL of blue food dye, and 1 μL of resuspended bacterial cells. After feeding, the aphids were transferred to plants and reared to adulthood (about 5 days post-feeding). At this point the aphids were screened for colonization with fluorescent CWBI-2.3^T bacteria on a blue light transilluminator. Successfully colonized aphids were moved to fresh plants (four aphids per plant) and these groups were transferred to new plants every 24 h for 3 consecutive days. After 24 h on the final plants, these colonized adult aphids were removed to prevent the accumulation of additional offspring. Offspring produced by the colonized adults were reared to the 4th instar and scored for the development of wing buds.

qPCR to assess gene knockdown in aphids

Bacteria were delivered to A. pisum LSR1 in artificial diet as described in the previous methods sections. For these experiments, five conditions were prepared: CWBI-2.3^T-GFP + BTK-NR, CWBI-2.3^T-GFP + dsE2C-800, CWBI-2.3^T-GFP + dsC002-800, CWBI-2.3^T-GFP + dsE2C-800 and CWBI-2.3^T + dsNuc1-800, and CWBI-2.3^T-GFP + dsC002-800 and CWBI-2.3^T + dsNuc1-800. Two sets of 20 aphids were treated to each condition; the aphids were then transferred to plants after 24 h of feeding. Five days after treatment, 6–10 aphids colonized with fluorescent bacteria from each condition were collected and crushed in DNA/RNA shield (Zymo Research, Irvine, CA, USA). Whole aphid RNA was purified using an RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) which includes an on-column DNA digestion step. Five hundred nanograms of each RNA sample were then reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). Primers for qPCR were designed using IDT’s Primerquest tool and are listed in Table S4. All qPCR reactions were prepared in triplicate in 384-well plates with SYBR Green reagents (Thermo Fisher, Waltham, MA, USA), and reactions were carried out using a ViiA-7 Real-Time PCR System (Thermo Fisher, Waltham, MA, USA).

Control qPCRs were run to determine the efficiency of each primer set and establish the most reliable reference genes for analysis. To determine primer efficiency, PCR products for each qPCR primer pair were synthesized, serially diluted, and run as the template to generate a standard curve. To choose the most reliable reference genes, we tested Beta-tubulin, GAPDH, NADH, Rpl32, and SDBH. We selected SDBH and Rpl32 to serve as references because they had the lowest variance between conditions. We calculated the gene expression ratio for each aphid target relative to the geometric mean of both reference genes by the Pfaffl method (Pfaffl, 2001). The amount of dsRNA production was determined by running the cDNA samples along with a standard curve for the gene of interest. The Cq values returned for the standard curve were then used to calculate the absolute copy number for each condition (Bustin, 2000).

Analysis of aphid RNAi experiments to select targets for knockdown

We wanted to select proven targets of RNA interference for testing our symbiont-mediated RNAi delivery system in aphids. To identify target genes expected to exhibit strong and reliable knockdown, we compiled a dataset of aphid RNAi results from 86 publications. We tabulated how RNA was administered, the effectiveness of gene knockdown, and other information for a total of 656 experiments (Table S1). By looking at the trends, we can get a sense of what is known about the reproducibility of different RNAi experimental designs in aphids (Fig. 1).

The main challenge in using this information to judge reliability is that there are few examples of the same gene being targeted using the same RNAi procedure, and even fewer cases in which experimental conditions have been replicated by different labs. Out of the 116 different experimental designs used for RNA delivery, 86 have been replicated in different papers, but only 22 of these have been repeated by different labs, and only two of these targeted the same gene (Fig. 1A). Most experiments with aphid RNAi are focused on aphid population management. Aphid mortality and reproduction are the phenotypes that are assayed most often (Fig. 1B). Experimental replication is also relatively low at the level of the genes targeted: out of 168 different gene targets, only 20 (12%) have appeared in more than one paper (Fig. 1C). The reported level of knockdown achieved via RNAi for these genes often varies greatly from study to study (Fig. 1D). Nevertheless, there are five targets (C002, hunchback, cathepsin-L, chitin synthase, and ecdysone receptor) for which the reported knockdown of gene expression by RNAi was more than 50%, on average, across RNAi experiments reported in at least three different publications.

The most highly tested target is the C002 gene, which was one of the first genes reported to be knocked down by RNAi in aphids (Mutti et al., 2006). This target has been tested by eight different laboratories and in multiple publications from some of these research groups (Mutti et al., 2008; Pitino et al., 2011; Zhang et al., 2013; Pitino & Hogenhout, 2013; Christiaens, Swevers & Smagghe, 2014; Zhang et al., 2015; Coleman et al., 2015; Wang et al., 2015; Yan et al., 2016; Ghodke et al., 2019; Ye et al., 2019a; Sun et al., 2019; Niu et al., 2019; Jacques et al., 2020) (Fig. 1C). Although there is high variability in the observed effectiveness of C002 knockdown (Fig. 1D), we decided that C002 would be the best benchmark for testing symbiont-mediated RNAi. Knockdown of C002 prevents aphid feeding and causes aphid mortality (Mutti et al., 2008), so we could examine both gene expression changes and this phenotype.

For a second target to test smRNAi we selected the ecdysone receptor (EcR). When EcR is knocked down it leads to an increase in the proportion of winged offspring birthed by the affected mother, which offers a phenotype to score that is different from the survival effect of C002 knockdown. RNAi results for EcR have been reported by two different labs in three papers using dsRNA delivery via feeding or injection (Christiaens, Swevers & Smagghe, 2014; Yan et al., 2016; Vellichirammal et al., 2017) (Fig. 1C), and the effectiveness of EcR knockdown appears to be similar to that observed for C002 (Fig. 1D).

Effects of feeding dsRNA targeting C002

The effectiveness of RNAi varies in different aphid species and genotypes. We wanted to reproduce prior studies and select an aphid with the largest RNAi response (Yoon et al., 2020), so we assayed whether we could achieve knockdown of C002 in four different genotypes of A. pisum: LSR1, PA1, Tucson, and 5A. The C002 dsRNA sequence was designed to match the sequence in a prior study (Mutti et al., 2006), and the non-targeting dsRNA sequence, dsE2C, matches a 412 bp fragment of the E2-Crimson (E2C) fluorescent reporter gene not present in the aphid genome. We fed each aphid line on artificial diet supplemented with 100 ng/µL dsRNA for 24 h, then transferred them to plants and monitored their mortality for 6 days. Overall, mortality rates were low and none of the biotypes showed a significant increase in mortality when they were treated with C002 dsRNA in our experiment vs when they were treated with the non-targeting E2C dsRNA control (Fig. 2). Out of the four biotypes tested, we selected the two with the largest effects when testing whether there was a significant difference associated with dsRNA feeding—5A (two-sided log-rank test, N_dsE2C = 40, N_dsC002 = 59, Z = 2.0, p = 0.16) and LSR1 (two-sided log-rank test, N_dsE2C = 60, N_dsC002 = 57, Z = 1.3, p = 0.26)—to see if smRNAi could achieve more effective results.

Symbiont-mediated RNAi against C002 does not consistently lead to an increase in aphid mortality

In order to engineer our strains to produce dsRNA, we built plasmids based on the design used for smRNAi in honey bees (Leonard et al., 2020; Lariviere et al., 2022). The dsC002-800 and dsE2C-800 plasmids were constructed with the same C002 and E2C target sequences used in the feeding experiment by cloning these sequences into pBTK800, a vector with two inward facing promoters that lead to expression of the insert as dsRNA. After building these constructs, we transformed them into versions of each of our bacteria that express green fluorescent protein (GFP) from a cassette integrated into their chromosomes, HT115-GFP and CWBI-2.3^T-GFP. We can easily identify aphids colonized with these strains because the fluorescent bacteria can be visualized inside the aphid gut on a blue light transilluminator (Elston et al., 2021).

We decided to first test smRNAi with HT115 E. coli because it has an RNase III mutation that increases accumulation of dsRNA. This mutation improves the effectiveness of RNAi in C. elegans (Dasgupta et al., 1998; Timmons, Court & Fire, 2001). We fed A. pisum on the dsRNA-expressing bacteria and monitored their mortality on fava beans for about 1 week (Fig. 3A). In our first trial with A. pisum LSR1, we observed a significant increase in aphid mortality in the C002 knockdown condition compared to the E2C knockdown control (two-sided log-rank test, N_dsE2C = 22, N_dsC002 = 17, Z = 9.8, p = 0.0018) (Fig. 3B). However, when we repeated this trial with a larger number of aphids that were also divided into more enclosures (six instead of three per condition), we no longer observed a significant change (log-rank test, N_dsE2C = 54, N_dsC002 = 59, Z = 0.33, p = 0.56) (Fig. 3C). We also did not observe a significant result in a smRNAi experiment in A. pisum 5A (two-sided log-rank test, N_dsE2C = 38, N_dsC002 = 38, Z = 1.8, p = 0.18), though the average effect was in the expected direction (Fig. 3D).

Due to the high overall aphid mortality that resulted from colonizing aphids with E. coli HT115, we chose to test this knockdown with S. symbiotica CWBI-2.3^T as well. Using the same experimental setup with three separate enclosures per condition, we delivered the dsRNA-expressing bacteria to A. pisum LSR1 and monitored their survival. Although the overall level of aphid mortality was lower with CWBI-2.3^T (Fig. 3D) than with HT115 (Fig. 3B, Trial two) in the non-targeting controls (two-sided log-rank test, N_HT115 = 54, N_CWBI = 55, Z = 45, p < 0.0001), we found the opposite result of what was expected for dsRNA expression. The non-targeting control condition with bacteria expressing E2C dsRNA had significantly higher mortality than the C002 condition (two-sided log-rank test, N_dsE2C = 55, N_dsC002 = 34, Z = 7.7, p = 0.0056) (Fig. 3D). Overall, the relatively small effect size and substantial variation in these results indicates that we did not achieve a reliable increase in aphid mortality using HT115 or CWBI-2.3^T as hosts for symbiont-mediated RNAi.

Additional targeting of aphid Nuclease1 for knockdown does not increase the phenotypic effect of C002 symbiont-mediated RNAi

To attempt to improve the effectiveness of smRNAi we chose to co-target an aphid nuclease, Nuc1 along with C002. This approach has been shown to increase the potency of RNAi, most likely by decreasing dsRNA degradation in the aphid gut (Chung et al., 2018; Yoon et al., 2020). We first tested this technique with E. coli HT115. We co-inoculated aphids with two strains, one expressing dsC002-800, and one expressing dsNuc1-800, a construct built to target the same 328 bp of the Nuc1 gene as that were used in a prior study (Chung et al., 2018), and monitored their mortality over 1 week. In our conditions, however, we did not see any improvement in RNAi effectiveness. Mortality in the HT115 double-target C002/Nuc1 condition was not significantly different than that observed for the E2C + Nuc1 control (two-sided log-rank test, N_{dsE2C + dsNuc1} = 71, N_{dsC002 + dsNuc1} = 49, Z = 0.0033, p = 0.95) (Fig. 4A).

To see if results would improve with the use of the native symbiont, we performed this same experiment using S. symbiotica CWBI-2.3^T. The first attempt at this technique yielded encouraging results: mortality of the aphids in the C002/Nuc1 condition was significantly increased compared to the control condition (two-sided log-rank test, N_{dsE2C + dsNuc1} = 57, N_{dsC002 + dsNuc1} = 39, Z = 20, p < 0.0001) (Fig. 4B). However, when we repeated this experiment under the same conditions, with increased numbers of aphids divided into five enclosures per condition instead of three, we saw the opposite of the expected result: aphids in the control condition died more than in the C002 condition (log-rank test, N_{dsE2C + dsNuc1} = 55, N_{dsC002 + dsNuc1} = 35, Z = 4.9, p = 0.027) (Fig. 4C). These phenotypic results demonstrate that even with the addition of Nuclease1 dsRNA, this technique is highly inconsistent.

Knocking down EcR with smRNAi does not change numbers of winged offspring

To test the generality of our results, we performed smRNAi using S. symbiotica against a second gene target, EcR. Knockdown of this gene is expected to have a non-lethal phenotype. When the expression of this protein decreases, the proportion of winged (alate) offspring birthed by the treated mother increases. We first performed a control experiment where we injected aphids with EcR dsRNA to assess whether we could recapitulate the results of experiments by another group, using the same 466 bp sequence of EcR as their experiments (Vellichirammal et al., 2017). As shown in Fig. 5A, we observed a significant change in the number of winged offspring on days 0, 1, and 2 post-injection (two-sided Fisher’s exact tests, N_dsE2C = 271, N_dsEcR = 273, odds ratio = 1.6, p = 0.011, N_dsE2C = 372, N_dsEcR = 307, odds ratio = 1.3, p = 0.090, N_dsE2C = 366, N_dsEcR = 380, odds ratio = 1.5, p = 0.0096, respectively), though the effect did not persist through day 3 (two-sided Fisher’s exact test, N_dsE2C = 660, N_dsEcR = 710, odds ratio = 1.1 p = 0.54). This mirrors the results of the original study, in which significant knockdown was only seen on the first day after injection (Vellichirammal et al., 2017).

Because we were able to achieve a significant phenotypic effect through dsRNA injection, we tested EcR knockdown with smRNAi. We designed our knockdown plasmid (dsEcR-800) to target the same 466 bp EcR sequence used in the injection test and transformed it into CWBI-2.3^T-GFP. Aphid stress plays a role in the production of winged offspring and seems to have been lower during this trial because the adults from both the on-target and non-targeting conditions had at most 1.1% winged offspring per day when we were monitoring them after colonization compared to the ~25–75% winged offspring that we observed in the injection experiment (Fig. 5B). This extremely low baseline, with almost no winged offspring at all in the smRNAi experiment, may have contributed to our inability to detect a significant effect from the EcR dsRNA treatment vs the non-targeting E2C dsRNA control (two-sided Fisher’s exact test, N_dsE2C = 121, N_dsEcR = 460, odds ratio = 0, p = 1.0). We are unsure whether the added delay waiting for aphids to be fully colonized by S. symbiotica, an effect from S. symbiotica colonization itself, the lack of trauma from microinjection, or other, unknown environmental factors that varied in this trial gave rise to this inconsistency.

qPCR to test for dsRNA expression and RNAi machinery upregulation

We performed qPCR to attempt to diagnose why the smRNAi system might be ineffective. To do so we set up a repeat of the C002/Nuc1 experiment with five conditions: aphids fed S. symbiotica containing an empty vector (pBTK-NR) or plasmids dsE2C, dsC002, dsE2C + dsNuc1, or dsC002 + dsNuc1. After 5 days we attempted to collect 10 colonized aphids from each condition for RNA extraction. However, there were few aphids with GFP+ guts in the dsC002, dsE2C + dsNuc1, and dsC002 + dsNuc1 conditions, so we only obtained seven, six, and three aphids from each, respectively. We did not further analyze the dsC002 + dsNuc1 condition due to its low sample size.

We first sought to confirm production of our dsRNA target sequences by S. symbiotica inside of the aphid. We cannot use qPCR to specifically verify production of C002 or Nuc1 dsRNA by engineered symbionts because the exact same sequences are also expressed as RNA by the aphid host, but this test is possible for the E2C non-targeting control. We used primers targeting E2C to estimate the copy number of dsRNA based on the amount of cDNA generated from 100 ng of total RNA. We were able to see clear expression of the dsRNA sequence in both the dsE2C and dsE2C + dsNuc1 conditions, although expression was lower in the paired condition (Fig. 6A). This difference could be due to unequal colonization by the dsE2C- and dsNuc1-producing bacteria that were co-inoculated in the paired condition.

For the treatments that included dsNuc1 or dsC002 we assayed the relative expression of the dsRNA construct compared to expression of the corresponding host gene using a set of primers that targets a region found in both the dsRNA sequence and the aphid’s mRNA for the gene (dsRNA + mRNA). The dsNuc1 construct causes an increase in the amount of the dsRNA + mRNA amplicon compared to the other control conditions, consistent with dsRNA expression from the symbiont (Fig. 6B). Expression of the dsC002 construct appears to be bimodally distributed (Fig. 6C). Three samples show an increased copy number for the dsRNA + mRNA amplicon, reflecting dsRNA production by the symbiont, while the other four samples have similar copy numbers to both control conditions which lack the dsC002 construct.

We next tested to see whether the RNAi pathway is upregulated by screening for changes in Dicer and Argonaute-2 (Ago-2). Dicer and Ago-2 were not significantly upregulated in the dsE2C condition (two-sided Mann–Whitney U tests, N_NR = 10, N_dsE2C = 10, U = 31, r = 0.32, p = 0.17, and N_NR = 10, N_dsE2C = 10, U = 64, r = 0.24, p = 0.32, respectively) (Figs. 6D and 6E). In the dsC002 condition, Dicer was significantly upregulated (two-sided Mann–Whitney U test, N_NR = 10, N_dsC002 = 7, U = 6, r = 0.69, p = 0.0031) (Fig. 6D), but Ago2 is only marginally significantly upregulated compared to the control (two-sided Mann–Whitney U test, N_NR = 10, N_dsC002 = 7, U = 15, r = 0.47, p = 0.055) (Fig. 6E). These results may indicate that the RNAi pathway has not been fully activated.

Finally, we tested whether smRNAi was effective at reducing C002 expression. For most of the dsC002 samples C002 expression is lower than in the no dsRNA (NR) treatment average. This effect is marginally significant (two-sided Mann–Whitney U test, N_NR = 10, N_dsC002 = 7, U = 51, r = 0.38, p = 0.13) (Fig. 6F). Based on our qPCR data, it appears that we were able to produce enough dsRNA to activate Dicer and Ago-2 in the dsC002 condition which may be lowering expression of C002. This effect is still very slight, however, and the dsE2C condition also shows a knockdown trend, though it is more variable, so the reduction is not statistically significant (two-sided Mann–Whitney U tests, N_NR = 10, N_dsE2C = 10, U = 64, r = 0.24, p = 0.32) (Fig. 6F). We also measured our ability to knock down expression of Nuc1, but it is complicated by the general upregulation of this gene expected in the presence of dsRNA. This upregulation is significant in the dsC002 condition, but not in the dsE2C condition (two-sided Mann–Whitney U tests, N_NR = 10, N_dsC002 = 7, U = 11, r = 0.57, p = 0.019, and N_NR = 10, N_dsE2C = 10, U = 40, r = 0.17, p = 0.48, respectively). For the dsE2C + dsNuc1 condition, we see marginally significant upregulation of Nuc1 (two-sided Mann–Whitney U test, N_NR = 10, N_{dsE2C + dsNuc1} = 6, U = 14, r = 0.43, p = 0.093), and the upregulation is higher than dsE2C alone. These results indicate that it may be possible to achieve knockdown of at least some aphid genes using smRNAi, but additional experiments are needed to understand where the system needs further optimization.