Efficient RNAi knockdown at 20 °C in Aedes albopictus (Diptera: Culicidae)

Abstract

RNA interference (RNAi) is a conserved cellular pathway found in nearly all eukaryotes that leads to the silencing of target mRNAs. Using RNAi as a mechanism to knockdown specific genes has enabled functional and reverse genetics studies in a wide range of eukaryotes. Previous work suggests that RNAi is inhibited at lower temperatures, potentially limiting the possibility to perform knockdown studies on ecologically relevant phenotypes that are only expressed at low temperatures. To determine whether RNAi is inhibited at low temperatures in Aedes albopictus (Skuse), we injected mosquitoes reared at 20 ± 1 °C, with dsRNA targeting yellow-g2 and compared knockdown efficacy to mosquitoes injected at 26.5 ± 1 °C. Our results demonstrate efficient knockdown at both temperatures, thereby establishing the feasibility of RNAi for functional genetic studies in A. albopictus at low temperatures.

Introduction

RNA interference (RNAi) is a conserved cellular pathway that leads to the degradation and transcriptional repression of specific mRNA targets (Wilson and Doudna 2013). The RNAi pathway has been identified in nearly all eukaryotes and likely evolved as part of the innate antiviral immune response (Shabalina and Koonin 2008). The pathway is typically triggered by double-stranded RNA (dsRNA), which can originate exogenously (e.g., via viral infection) or endogenously (e.g., via expression of repetitive or transposable elements) (Wilson and Doudna 2013, Zhu and Palli 2020). The dsRNA precursor is processed into short, single-stranded RNA fragments, which bind to a protein-silencing complex that then cleaves and degrades the complementary mRNA target. Importantly, this mechanism of sequence-specific gene silencing can be experimentally induced by the exogenous introduction of dsRNA, enabling functional and reverse genetics studies in a wide range of eukaryotes. For example, in mosquitoes, experimental RNAi has been used to determine the function of genes involved in development, morphogenesis, olfaction, blood feeding, reproduction, and insecticide resistance (Airs and Bartholomay 2017, Homem and Davies 2018). Furthermore, because RNAi is the primary mosquito antiviral response, studies of this pathway can identify mosquito host factors that influence viral infection and provide insight into antiviral mechanisms (Blair 2011, Olson and Blair 2015).

Multiple types of evidence indicate the RNAi pathway is sensitive to temperature (Samuel et al. 2016, Bellone and Failloux 2020). One line of evidence is a general trend between lower temperatures and higher viral infection rates, primarily in Aedes and Culex mosquitoes. For example, the infection rate and viral titer of Western equine encephalitis virus in Culex tarsalis reared at 18 and 25 °C were significantly higher than that of mosquitoes reared at 32 °C (Kramer et al. 1983). Similarly, increased infection and transmission of chikungunya (CHIKV) virus have been detected at lower temperatures in both Aedes albopictus (Skuse) and Aedes aegypti (Westbrook et al. 2010, Adelman et al. 2013, Heitmann et al. 2018). In a separate line of evidence, Adelman et al. (2013) compared RNAi-mediated silencing at 18 °C relative to 28 °C in A. aegypti. They observed inhibition of gene silencing at the mRNA and phenotypic level at 18 °C relative to 28 °C. Taken together, these diverse results suggest that lower temperatures may inhibit the RNAi pathway.

Inhibition of RNAi at low temperatures implies a potential limitation to knocking down genes that affect phenotypes that are only expressed at low temperatures, such as cold tolerance or photoperiodic diapause. Photoperiodic diapause is an anticipatory form of dormancy necessary for a wide range of temperate insects to survive extended periods of unfavorable conditions such as temperate winters (Saunders 2012). Short photoperiods act as an anticipatory, token cue to induce photoperiodic diapause, although low temperatures can also impact the induction and duration of this form of dormancy. Therefore, to test the functional role of genes involved in photoperiodic diapause regulation, it is necessary to perform experiments at ecologically relevant, low temperatures. However, it is unknown if low-temperature inhibition of RNAi is a widespread phenomenon across species and target genes. With the exception of RNAi in Culex pipiens (Sim and Denlinger 2008, Meuti et al. 2015), the majority of RNAi studies in mosquitoes are performed between 25 and 28 °C (see studies reviewed in Homem and Davies 2018).

The objective of this study was to determine whether RNAi efficiency is reduced in A. albopictus at 20 ± 1 °C, a temperature commonly used to study diapause in this species (Armbruster 2016). We injected mosquitoes reared at 26.5 ± 1 and 20 ± 1 °C, with dsRNA targeting yellow-g2 (AalY-g2), a gene in the yellow family. Expression of AalY-g2 is ovary specific and important for eggshell formation. Previous work at 27 ± 0.5 °C by Noh et al. (2020) demonstrated a strong effect of AalY-g2 knockdown via both qPCR and a discernable crescent-shaped egg phenotype (Noh et al. 2020). Here, we demonstrate with qPCR and phenotypic results that RNAi-mediated knockdown of AalY-g2 is efficient at both 26.5 ± 1 and 20 ± 1 °C.

Methods

Mosquito Rearing

A lab colony of A. albopictus was established with pupae and larvae collected from an auto-salvage yard located in Manassas, VA, in 2018. Animals were reared under standard laboratory conditions at 21 °C, 16 h light:8 h dark for 12 subsequent generations (Batz et al. 2020). For the experimental generation, F12 eggs were moved to an incubator (Precision 815, Thermo Scientific), and mosquitoes were reared at either 26.5 ± 1 or 20 ± 1 °C. Eggs were stimulated to hatch in a 5.5-liter container containing 2 liters of deionized water and 1 ml of a larval food slurry consisting of 1-liter deionized water, 120-g dog food (Nutro Ultra Small Breed Puppy, Nutro Products Inc., Franklin, TN, USA), and 40-g frozen brine shrimp (Sally’s Frozen Brine Shrimp, San Francisco Bay Brand, Newark, CA, USA) (Armbruster and Conn 2006). Larvae were reared at a density of 200 larvae per 2-liter deionized water in 5.5-liter bins. Larvae were transferred to clean water and fed 1 ml of larval food slurry 3 times weekly. Pupae were transferred into 2-liter adult cages provisioned with organic raisins (Newman’s Own, Westport, CT, USA) to allow ad libitum sugar feeding. Four to 10 days after pupation, raisins were removed from the adult cages for 24 h, and then adult females were allowed to blood feed on a human host. The Georgetown University Institutional Review Board has determined that mosquito blood feeding is not human research and does not require IRB approval; however, the blood feeding protocol has been approved by the Georgetown University Office of Health and Safety.

dsRNA Synthesis

Ten F12 A. albopictus females from a Manassas, VA, colony were reared at 26.5 ± 1 °C and flash frozen in liquid nitrogen 48 h post-bloodmeal. Ovaries were dissected into RNAlater (Invitrogen, Waltham, MA, USA) and pooled into a single sample. RNA was extracted by homogenizing pooled ovaries in 1-ml TRIzol (Invitrogen, Waltham, MA, USA) and performing a phenol-chloroform extraction followed by an isopropanol precipitation according to the manufacturer’s directions. One microgram of RNA was converted to cDNA using Bio-Rad’s iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Then, cDNA was used as a template to amplify a 457 bp region of Aaly-g2 (NCBI gene ID: 109403201). See Supplementary Table S1 and Supplementary Methods for primers and PCR cycling parameters used to produce a template for dsRNA synthesis. dsRNA was synthesized using the T7 RiboMAX Express RNAi System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

RNAi Injections

Two hours after blood feeding, females were lightly anesthetized with CO₂ for 10 s and placed on ice for 10 min. While on ice, fully engorged females were injected intrathoracically using a Nanoject II (Drummond Scientific, Broomall, PA, USA) with 0.138 µl of 7.25 ng/µl dsRNA (total 1 µg dsRNA) targeting either the ovary-specific yellow gene, AalY-g2, or dsRNA targeting Bgal as a negative control. A total of 6 biological replicate cages were established for each injection treatment (dsBgal and dsAaly-g2) to collect animals for qRT-PCR (described below) and to collect eggs for phenotypic evaluation. Additionally, 6 biological replicate cages were established for noninjected control mosquitoes to collect eggs for phenotypic evaluation. Noninjected control mosquitoes were anesthetized and placed on ice for the same duration of time as injected mosquitoes. Following injection, dead mosquitoes were removed from cages and counted to evaluate survivorship.

qRT-PCR

Six biological replicates per injection treatment (dsBgal and dsAalY-g2) at each temperature regimen (26.5 ± 1 and 20 ± 1 °C) were used to evaluate knockdown via qRT-PCR. Injected females were flash frozen in liquid nitrogen 71.5 h post-blood meal if reared at 26.5 ± 1 °C or 122.75 h post-blood meal if reared at 20 ± 1 °C. These time points for qRT-PCR were chosen to correspond with a distinct peak of AalY-g2 expression as determined by preliminary experiments.

To obtain RNA, ovaries from 6 to 14 females per biological replicate were dissected into RNAlater, and RNA was extracted as described above. Primer sequences to amplify the reference gene ribosomal protein S6 (AalRpS6, accession number: AF154066) and Aaly-g2 were developed and validated by Noh et al. (2020) (Supplementary Table 1). The 2^−ΔCt method (Livak and Schmittgen 2001) was used to determine the relative mRNA abundance (i.e., fold change) of Aaly-g2 (see Supplementary text for additional details). Comparison of the average relative expression between dsBgal and dsAalY-g2 samples was performed with a Wilcoxon-rank-sum test. Analyses were performed independently for the 2 temperature treatments.

EGG Collection

After establishing replicate cages of blood-fed injected and noninjected females, each cage was provided a brown oviposition cup half-filled with deionized water and lined with an unbleached paper towel. Papers with eggs were removed from the oviposition cups 4–8 days after blood feeding and were gently air dried 48 h later and stored in a sealed Tupperware container. Eggs were imaged with a Leica Stereoscope (model # MVX10) 48–96 h after collection for phenotypic assessment of knockdown.

Results

The survivorship of injected and noninjected adults was consistently >90%. AalY-g2 transcript abundance was significantly lower in the ovaries of blood-fed adult females injected with dsAalY-g2 relative to adult females injected with dsBgal at both 26.5 ± 1 °C (Wilcoxon test, W = 0, n = 12, P-value = 0.002) and 20 ± 1 °C (Wilcoxon test, W = 0, n = 12, P-value = 0.002). The average reduction in AalY-g2 transcript levels was 90.57% at 26.5 ± 1 °C and 95.96% at 20 ± 1 °C (Fig. 1). Additionally, eggs that were collected from dsAalY-g2-injected females showed a crescent-shaped phenotype relative to eggs produced by uninjected and dsBgal-injected females at both 26.5 and 20 ± 1 °C (Fig. 2), similar to the results documented in Noh et al. (2020).

Fig. 1.

Injection of dsAalY-g2 results in knockdown of AalY-g2 at both 26.5 and 20 ± 1 °C. dsAalY-g2 or dsBgal (control) was injected into adult females, and transcript levels of AalY-g2 relative to that of Aedes albopictus ribosomal protein S6 (AalRpS6) were assessed by qRT-PCR from dissected ovaries as described in the Methods section. Data are shown as the mean ± 1 SE. **P < 0.01.

Fig. 2.

Injection of dsAalY-g2 results in a crescent-shaped egg phenotype at both 26.5 ± 1 and 20 ± 1 °C. Images show representative egg phenotypes produced by female Aedes albopictus that were either uninjected, injected with dsBgal, or injected with dsAalY-g2.

Discussion

Multiple studies have provided evidence that low temperatures are associated with increased viral susceptibility in Aedes and Culex mosquitoes (Kramer et al. 1983, Turell 1993, Westbrook et al. 2010, Adelman et al. 2013). Furthermore, Adelman et al. (2013) observed normal processing of dsRNA but inhibition of gene silencing at the mRNA level at low temperatures (18 °C) in the yellow fever mosquito, A. aegypti. Similarly, studies in Drosophila and Arabidopsis have shown reduced efficiency of RNAi-mediated gene silencing at low temperatures (22 and 15 °C, respectively) (Fortier and Belote 2000, Szittya et al. 2003). Nevertheless, it is important to note that studies in the mosquito, C. pipiens, and in the grapevine plant, Vitis vinifera, have successfully used RNAi to knockdown genes at different low temperatures (18 and 4 °C, respectively) (Sim and Denlinger 2008, Romon et al. 2013, Meuti et al. 2015). Given most RNAi studies in mosquitoes are performed at temperatures above 25 °C, it remains unknown whether the inhibitory effect of low temperature on RNAi efficiency is widespread.

We tested the effect of temperature on the efficacy of RNAi by targeting a previously characterized gene, yellow-g2 (AalY-g2), in A. albopictus. Our results indicate a reduction of the AalY-g2 transcript by 96% at 20 ± 1 °C, which is comparable to the reduction achieved at 26.5 ± 1 °C (91%; Fig. 1). Thus, our results demonstrate that for yellow-g2, RNAi can be utilized as a functional genomics tool in A. albopictus at lower temperatures. These results correspond with the successful RNAi studies in C. pipiens performed at 18 °C, supporting the general feasibility of using RNAi at lower temperatures in at least some mosquito species.

We selected a low temperature of 20 ± 1 °C because it represents a temperature commonly used to study diapause in A. albopictus (Armbruster 2016). Our results, therefore, establish the feasibility of using an RNAi-knockdown approach to test the function of putative diapause genes in A. albopictus. Our results also support the feasibility of using RNAi at low or fluctuating temperatures for other purposes, including field-based studies and bioinsecticide strategies. We suggest a possible explanation for why RNAi was effective in C. pipiens and A. albopictus is that the populations used in these studies were temperate in origin, whereas the populations used in previous studies where RNAi was not effective at low temperatures were tropical or subtropical in origin and/or maintained in the lab for many generations under benign conditions (Fortier and Belote 2000, Szittya et al. 2003, Adelman et al. 2013). Given the complexity of interactions between viruses, the host RNAi immune pathway, and temperature, it will be valuable to evaluate RNAi efficiency across a wider range of temperatures in a broader set of insect species.

Funding

This research was funded by the National Institutes of Health (5R01AI132409) and funds from the Davis Family Endowment.

Author Contributions

Mara Heilig (Conceptualization [Equal], Data curation, Formal analysis [Equal], Investigation, Methodology [Equal], Visualization, Writing—original draft, Writing—review & editing [Equal]), and Peter Armbruster (Conceptualization [Equal], Formal analysis [Equal], Funding acquisition, Methodology [Equal], Project administration, Resources , Supervision, Validation [Equal], Writing—review & editing [Equal])