Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
(N/A)
Non Technical Summary
The circadian clock is an internal mechanism that allows organisms to predict and respond to rhythmic changes in their environment. Studies spanning various taxa have demonstrated that host biological clocks influence daily patterns in immune responses, disease severity, and disease transmission. Although much is known about rhythms in host-parasite interactions in mammals, equivalent understanding within plant-pest interactions is lacking. Comparable to human parasites, some plant pests exhibit clock-regulated rhythms in traits that allow them to successfully parasitize their hosts. Our research aims to characterize the influence of circadian clock regulated rhythms in gene expression, behavior, and performance in the bird cherry-oat aphid (Rhopalosiphum padi), a notorious pest of wheat and other grains worldwide. We aim to conduct a time-course transcriptome experiment in rhythmic and constant light regimes to determine the genes in R. padi which are regulated by the circadian clock. Next, we aim to use video tracking and traditional insect assays in rhythmic and constant light regimes to determine clock regulated rhythms in locomotory behavior, honeydew excretion, and reproduction. Finally, we aim to characterize the direct influence of the circadian clock on aphid behavior and performance by silencing circadian clock components using a nanoparticle-facilitated topical RNAi approach. The proposed project will allow for the development of skills in aphid behavioral assays, bioinformatics, applied insect molecular biology, as well as data analysis and science communication and will provide knowledge to aid in the development of innovative pest management strategies.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The first major goal of this project is to understand the diurnal and circadian transcriptome of the aphid Rhopalosiphum padi by performing RNA sequencing in both diurnal and constant light regimes on both plants and artificial diets. This project will uncover what transcripts show rhythmicity in the absence of environmental cues,which are thus directly regulated by the master circadian clock.The second major goal of this project is to understand how diurnal and circadian rhythms influence behaviors in R. padi. From locomotion to honeydew, to feeding behaviors. This project will utilize the Noldus suite of video tracking software and behavioral chambers as well as honeydew clocks to get a good picture of how the behavior of R. padi changes across the course of a 24h day in varying light regimes. Again, behavioral patterns that are consistent in the absence of environmental cues are thus directly regulated by the master circadian clock.The final major goal of this project is to directly correlate the master circadian clock with both the aphid transcriptome and aphid behavioral rhythms. This project will entail selectively silencing master circadian clock genes in the aphid using a nanoparticle facilitated RNAi approach followed by monitoring the previously studied transcriptome and behavioral rhythms. If silencing of clock genes causes a significant disruption in rhythms compared the control; then there will be good evidence that the transcriptome and behavioral rhythms are directly tied to the master circadian clock in the aphid brain.The final goal of this project is to understand how we can use the inherent properties of aphid rhythmicity and their master clocks to provide updated or novel sustainable pest management strategies.
Project Methods
Objective 1: Characterize the daily and circadian transcriptome of R. padi on wheat and on artificial diets.Design: Independent groups of 24 wheat plants (cv. Chinese spring) will be grown to the 3-leaf stage (Zadoks 1.13) in long-day conditions (16h light, 8h dark: LD). Using breathable cages, ten aphids will be confined onto the 2nd and 3rd leaf. After 24h, the adult aphids will be removed, leaving behind age-synchronized nymphs. After growing for 4d in LD conditions, one set of the plant-aphid cages will be placed into constant darkness (0h light, 24h darkness: DD). After an additional 48h in their respective light regimes, aphid sample collection over a 24h-period will begin at 0 zeitgeber-time (zt; German: lit. 'time giver'; i.e., the time at which the 16h light duration begins) and continue every four hours for 24h (Fig. 2A). At each timepoint, 10 aphids per plant-aphid cage will be collected into individual microcentrifuge tubes using an aspirator and immediately placed in liquid nitrogen. For the LD samples at timepoints 16zt and 20zt, aphids will be collected using a red LED headlamp to prevent disrupting the circadian clock. For DD samples, collection will take place using a red LED headlamp and growth chambers will only be opened once all lights in the room have been turned off. Total RNA will be extracted with the ZYMO Research - Quick RNA Kit following the manufacturer's instructions. Samples (n=48, x4 bio reps per timepoint) will be sent to Novogene for library preparation and paired-end mRNA sequencing. RNAseq data analysis will be performed on the Colorado University Research Computing Alpine high-performance cluster using the Tuxedo pipeline33. Rhythmicity analysis in each light treatment will be conducted using the R-package, MetaCycle34. Rhythmicity p-values of less than 0.05 will determine diurnal and circadian rhythmic transcripts in the LD and DD groups. Rhythmic transcripts will be clustered using the R-package mFuzz35 followed by GO term and KEGG pathway enrichment analysis. Promoter and E-box analysis of transcripts that show significant circadian rhythmicity will be conducted to determine circadian clock gene-mediated transcription using the biomaRt package36. To determine whether rhythms in R. padi gene expression are under the circadian clock's direct control, another set of age-synced plant-aphid cages will be set up as described above. Age-synced nymphs will be allowed to feed for 4d in LD, after which they will be moved onto artificial diets to complete development. One group of 24 diet-aphid cages will be allowed to develop in LD, while the other group will be placed into DD conditions. After an additional 48h in their respective light regimes, aphids will be sampled every 4h for 24h, and total RNA will be extracted as described above (Fig. 2A). qPCR followed by MetaCycle analysis will be performed on aphids feeding from artificial diet, verifying the rhythmicity of the top 15 transcripts with the lowest p-value and highest amplitude identified in the RNAseq MetaCycle analysis.Objective 2: Elucidate daily and circadian rhythms in key R. padi behaviors on wheat and artificial diets. (2A) Measure daily and circadian rhythms in locomotion and host finding on wheat and artificial diet. (2B) Measure daily and circadian rhythms in honeydew excretion and reproduction on wheat and artificial diet. Design: Aphids used in all behavioral experiments will be age-synced as described in obj. 1. (2A) Twenty individual age-synced aphids will be video recorded using an infrared (IR) sensitive camera at 12h intervals over a 24h-period on the 2nd leaf of wheat seedlings using custom acrylic chambers in LD and DD. Twenty individual age-synced aphids will be video recorded while confined in custom acrylic artificial diet sachets in LD and DD The 8h dark period will be illuminated with an IR-LED array to allow for the visualization of aphids in recordings. Video tracking and locomotion parameter calculation will be conducted automatically with the EthovisionXT software (Noldus; Wageningen, Netherlands). The distance traveled per aphid will be averaged and binned every 10 minutes. Rhythmicity analysis on locomotion data will be calculated using a cosine curve fitting approach with the package COSINOR using significance cutoffs as described in obj. 1. (2B) 20 individual age-synced aphids will be video recorded in LD and DD conditions on wheat seedlings and artificial diets as previously described. Flicking motions (R. padi behavior to discard honeydew), visible honeydew droplets, and nymphs will be quantified manually using the ObserverXT software (Noldus; Wageningen, Netherlands). Parameters will be averaged and binned every 10 minutes. Rhythmicity analysis on honeydew and reproduction parameters will be calculated using COSINOR.Objective 3: Functionally characterize the influence of the circadian clock in key R. padi behaviors.Design: dsRNA complementary to core clock genes timeless, period, clock, and cycle, as well as the clock output pathway genes insulin-like peptide-439-41 and pigment dispersing factor20,42 will be designed and generated using the eRNAi webtool (https://www.dkfz.de/signaling/e-rnai3/) and the Promega T7 RiboMAX™ Express RNAi System. R. padi transcripts will be silenced by the topical application of dsRNA and star-polycation nanocarrier (SPc) at a 1:1 mass ratio (Fig. 3). Four replicates of 20 age-synched aphids will be anesthetized using CO2 gas and have 0.5µL of dsRNA-SPc solutions at concentrations of either 0.25, 0.5, 1, 1.5, or 2µg/µL applied to their abdomens to determine the optimal dose for each dsRNA. Ten silenced aphids will be collected at 24h and 48h post-application, and qPCR performed to determine silencing efficiency. To determine the influence of circadian clock and clock output genes on R. padi gene expression, 4 replicates of 10 age-synced aphids will be treated with optimal doses of dsRNA-SPc complexes and confined on the 2nd and 3rd leaf of 3-leaf stage wheat seedlings for 24h. After 24h, aphid RNA will be extracted over 24h in both LD and DD conditions as previously described (Fig. 2B,2A). qPCR followed by MetaCycle analysis will then be performed for the top 5 transcripts that showed the lowest p-value and highest amplitude on both plants and artificial diets from objective 1. This assay will be repeated using dsRNA from each core clock and clock output member. To determine the influence of circadian clock and clock output genes on R. padi behaviors, 4 replicates of 10 age-synced aphids will be treated with optimal doses of dsRNA-SPc complexes and confined on wheat seedlings or artificial diets for 24h as described above. After 24h, aphids will be moved to custom acrylic chambers and video recorded on plants and artificial diets in LD and DD as previously described (Fig. 2B,2A). Locomotory, reproductive, and honeydew behavior data will be analyzed using EthovisionXT and ObserverXT software and undergo COSINOR analysis as previously described. These assays will be repeated using dsRNA for each core clock and clock output member.