Counts of uniquely mapping reads were generated through HTSeq for all 35 RNA-seq datasets . Multimapping reads were excluded from the analysis except for the tandem gene expression analysis. Differential gene expression analysis was performed using the DESeq2 pipeline across fruit developmental stages with three biological replicates per developmental stage . Gene expression values were derived by calculating the fragments per kilobase per million reads mapped values using the standard formula for FPKM /gene length in kilobases. To construct the gene co-expression network, genes that were not expressed or very weakly expressed in 30 or more conditions were first excluded from the analysis. The count data was then transformed into variance stabilized values using the variance stabilizing transformation function in DEseq. Pairwise correlations of gene expression were calculated using Pearson correlation coefficient and mutual rank using scripts available for download from the project’s data repository. MR scores were transformed to network edge weights using geometric decay function e−; five different co-expression networks were constructed with x set to 5, 10, 25, 50, and 100, respectively. Edges with PCC <0.6 or edge weight <0.01 were excluded. For each network, modules of coexpressed genes were detected using ClusterONE v1.0 using default parameters, blueberry grow pot and modules with P value > 0.1 or quality score <0.2 were excluded. The results from all co-expression networks were then combined by collapsing modules into meta modules of non-overlapping gene sets.
Total antioxidant capacity of tissues from the fruit developmental panel was analyzed using the ORAC assay. Briefly, ∼20–30 mg of frozen ground fruit tissue was measured for tissue samples prior to extraction. Sample extractions were performed on ground tissue using 1.8 mL of ice cold 50% acetone. Samples were vortexed and then put on a shaker for 5 minutes at room temperature. Samples were then centrifuged at 4◦C for 15 minutes . The ORAC assay was performed in a 96-well black microplate using the FLUOstar OPTIMA microplate reader . Each reaction well contained 150 μL of 0.08 μM fluorescein and 25 μL of 75 mM phosphate buffer , Trolox standards , or diluted sample extracts. For blueberry tissue samples, 1:80–1:20 dilutions were used. Upon loading all appropriate wells, the 96-well microplate was put into the microplate reader and incubated for 10 minutes at 37◦C. Following incubation, 25 μL of 150 mM AAPH was added to each well, and fluorescence measurements began immediately. Fluorescence measurements were taken for 90 seconds per cycle for 70 cycles until the fluorescent probe signal was completely quenched. The area under the fluorescence decay curve was calculated for each well. The total antioxidant capacity of a sample was calculated by subtracting the AUC from the blank curve from the AUC of the sample curve to obtain the net AUC. Using Trolox of a known concentration, a standard curve was generated , and the total antioxidant capacity of each sample was calculated as Trolox equivalents. Each sample was run twice for two technical replicates. The coefficient of variation between technical replicates was required to be less than 0.20. Biological replicates were run for all tissues in the fruit developmental series.Berries from ”Draper” were collected as described above.
Approximately 100 mg of each frozen ground sample was resuspended in extraction solvent in a 2 mL tube . Ground tissue was immediately mixed thoroughly to prevent thawing during extraction and to prevent metabolism of analytes by enzymes in the samples. All tubes were spun down for 10 minutes at 13,000 × g to pellet protein and other insoluble material. Then, 1 mL of supernatant was transferred to an autosampler vial. Anthocyanin content was evaluated by LC-MS as follows: 5 uL of sample extract were separated using a 10 minute gradient on a Waters Acquity HSS-T3 UPLC column on a Waters Acquity UPLC system interfaced with a Waters Xevo G2-XS quadrupole time-of-flight mass spectrometer . Column temperature was maintained at 40◦C, and the flow rate was 0.3 mL/min with starting conditions of 100% solvent A and 0% solvent B . The gradient was as follows: hold at 100% A for 0.5 minutes, ramp to 50% B at 6 minutes, then ramp to 99% B at 6.5 minutes, hold at 99% B to 8.5 minutes, return to 100% A at 8.51 minutes, and hold at 100% A until 10 minutes. Mass spectra were acquired in positive ion mode electrospray ionization over m/z 50–1500 in continuum mode using a data-independent MSE method that acquires data under both low and high collision energy conditions with the high collision energy setting using a ramp from 20–80 V. Capillary voltage was 3 kV, desolvation temperature was 350◦C, source temperature was 100◦C, cone gas flow was 25 L/hr, and desolvation gas flow was 600 L/hr. Correction for mass drift was performed using continuous infusion of the lock mass compound leucine encephalin. Anthocyanins and other related flavonoids were identified based on accurate mass and fragmentation pattern. Peak areas were determined using Quanlynx within the Masslynx software package . Relative anthocyanin content was calculated for each sample using the formula: reported peak area of the compound/peak area of internal standard/weight of extracted tissue .Insect repellents have been used since antiquity to fend off disease-transmitting mosquitoes and other arthropods. They developed gradually from smoke generated by burning plants and topical applications of essential oils into repellent substances, including those isolated from plants and a broad-spectrum synthetic repellent DEET , which was discovered in the early 1940s from a screening of more than 7000 compounds . Thereafter, other synthetic repellents have been developed, including IR3535, aminopropionate and picaridin piperidine-1-carboxylate, but DEET remains the most widely used repellent substance worldwide , particularly in the United States of America. Repellents work primarily as spatial and contact repellents. Mosquitoes attracted to and flying towards vertebrate hosts may make oriented movements away from the source upon approaching chemically treated skin surfaces. In this case, the chemical is a repellent sensu stricto . Because the repellent is acting from a distance, it may be referred to as a spatial repellent . When mosquitoes land on a chemically treated skin thus making contact before starting increasing locomotion activity or taking off, the chemical is called a contact repellent, which is sometimes referred to as excitorepellent, irritant, or contact irritant . From a strict mechanistic viewpoint, these two groups of compounds should be named non-contact and contact disengagent for spatial and contact repellents, respectively. It is now known that at least Culex and Aedes mosquitoes smell DEET . More importantly, it has been demonstrated that an odorant receptor from the Southern house mosquito, Cx. quinquefasciatus, CquiOR136, is essential for reception of DEET as a noncontact disengagent . Recently, it has been demonstrated that as a contact disengagent, DEET is detected by sensilla on the tarsal segments of the legs of the yellow fever mosquito Aedes aegypti , but the receptors remain elusive. Lastly, it has been suggested that DEET merely masks the reception of human emanations by Anopheles coluzzii , thus reducing the attractiveness of the host. Although its modes of action remain a matter of considerable debate, DEET is a gold standard repellent. It also has many “off-label” properties that do not directly affect human mosquito interactions. For example, DEET is a feeding deterrent , hydroponic bucket but if this were the primary mode of action, it would have little value in epidemiology.
The great value of repellents is that they reduce biting rates, which represents a second order parameter in vector capacity . Another property that may have a value in epidemiology, albeit not by decreasing vector capacity, is the deterrent effect of DEET on oviposition, as first observed for Ae. aegypti . While using the Xenopus oocyte recording system to deorphanize odorant receptors involved in the reception of oviposition attractants, we observed that DEET elicited outward currents in our preparations, in contrast to oviposition attractants and other compounds that generated inward currents. We have now systematically investigated this phenomenon using different ORs from three different species of mosquitoes. Here, we report that DEET, IR3535, and picaridin elicit outward currents on OR involved in the reception of mosquito oviposition attractants in the Southern house mosquito, Cx. quinquefasciatus, and orthologues from the yellow fever and malaria mosquitoes. Dose dependent outward currents were also observed with compounds in a panel that included plant-derived and plant-inspired repellents. Like DEET, IR3535 and picaridin , plant-inspired compounds, elicited robust inward current in the DEET receptor, CquiOR136, and showed repellency activity.In vitro transcription, oocytes microinjection, and electrophysiology were performed as previously described . Briefly, in vitro transcription of cRNAs was performed by using an mMESSAGE mMACHINE T7 kit , according to the manufacturer’s protocol. Plasmids were linearized with NheI, SphI, or PstI, and capped cRNAs were transcribed using T7 or SP6 RNA polymerase. cRNA samples were purified with LiCl precipitation solution and resuspended in nuclease-free water at a concentration of 200 μg/mL and stored at −80°C in aliquots. RNA concentrations were determined by UV spectrophotometry. cRNA samples were microinjected into stage V or VI Xenopus laevis oocytes . A two-electrode voltage clamp was used to detect currents. Oocytes were placed in a perfusion chamber and challenged with test compounds. Odorant-induced currents were amplified with an OC-725C amplifier , the voltage held at −80 mV, low-pass filtered at 50 Hz and digitized at 1 kHz. Data acquisition and analysis were carried out with Digidata 1440A and pClamp10 software .In short, two 50-mL Dudley bubbling tubes painted internally with a black hobby and craft enamel were held in a wooden board , 17 cm apart from each end and 15 cm from the bottom. The board was attached to the frame of an aluminum collapsible field cage . Two small openings were made 1 cm above each Dudley tube to hold two syringe needles to deliver CO2. To minimize handling of mosquitoes, test females had been kept inside collapsible field cages since the latest pupal stage. These female cages had their cover premodified for behavioral studies. A red cardstock was placed internally at one face of the cage, openings were made in the cardboard and in the cage cover so the cage could be attached to the wooden board with the two Dudley tubes and CO2 needles projecting inside the mosquito cage 6 and 3 cm, respectively. Additionally, windows were made on the top and the opposite end of the red cardstock for manipulations during the assays and a video camera connection, respectively. The two cages were connected at least 2 h prior to bioassays. At least 10 min before the assays, water at 28°C started to be circulated with a Lauda’s Ecoline water bath, and CO2 at 50 mL/min was delivered from a gas tank just at the time of the behavioral observations. Sample rings were prepared from strips of filter papers 25 cm-long and 4-cm wide and hung on the cardstock wall by insect pins to make a circle around the Dudley tubes. Cotton rolls were loaded with 100 μl of defibrillated sheep blood purchased from UC Davis VetMed shop and placed between a Dudley tube and CO2 needle. For each run, one paper ring was loaded with 200 μL of hexane and the other with 200 μL of test repellent in hexane. The solvent was evaporated for 1-2 min, blood-impregnated cotton plugs and filter paper rings were placed on the arena, CO2 was started, and the assays recorded with an infrared camera . During the assay, the arena was inspected with a flashlight with a red filter. After 5 min, the number of females that have landed and continued to feed on each side of the arena was recorded. Insects were gently removed from the cotton rolls and the assays re-initiated after rotation of sample and control. Thus, repellence for each set of test mosquitoes was measured with the filter paper impregnated with the same sample at least once on the left and once on the right side of the arena.To revisit our earlier observation of repellent-induced outward currents on OR sensitive to oviposition attractants, we challenged CquiOR21/CquiOrco-expressing oocytes with DEET and then skatole. CquiOR21, formerly known as CquiOR10 , is narrowly tuned to the oviposition attractant skatole . CquiOR21/CquiOrcoexpressing oocytes generated robust inward currents when stimulated with 10 μM skatole, whereas 1 mM DEET elicited outward currents . These outward currents were dose-dependent and were not observed when oocytes were injected only with CquiOrco cRNA or CquiOR21 cRNA .