Total RNA samples were extracted from one thousand 4-7 day-old Culex female antennae with TRIzol reagent . Antennal cDNA was synthesized from 1 μg of antennal total RNA from each species using iScript cDNA synthesis kit according to the manufacturer’s instructions . Total RNA was extracted from Aedes mosquitoes provided by Dr. Anthon J. Cornel. This colony was established in 2016 from eggs that were laid by females collected in BG-sentinel traps in the City of Clovis, California . Aedes mosquitoes were kept at 25 ± 2 °C, 75 ± 5% relative humidity, and under a photoperiod of 12:12h. The bio-assay arena was modified from our surface-landing assay initially designed to mimic a human arm without odors or humidity. CO2 at 50 mL/min was added to activate female mosquitoes, and blood was provided as both an attractant and a reward. 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 the handling of mosquitoes, test females were kept inside collapsible field cages since the latest pupal stage. These female cages had their cover modified for behavioral studies. A red card stock was placed internally at one face of the cage,pot with drainage holes and openings were made in the cardboard and 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 card stock for manipulations during the assays and a video camera connection, respectively. The mosquito cage housing 30–50 test females was connected to the platform holding the Dudley tubes at least 2h before bio-assays. At least 10 min before the assays, water at 38 °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 card stock wall by insect pins to make a circle around the Dudley tubes. Cotton rolls were loaded with 100 μL of defibrinated sheep blood purchased from the University of California, Davis, VetMed shop and placed between a Dudley tube and a CO2 needle. For each run one paper ring was loaded with 200 μL of hexane and the other with 200 μL tested compounds at a certain concentration in hexane. The solvent was evaporated for 1–2 min, blood-impregnated cotton plugs, and filter paper rings were placed in the arena, CO2 was started, and the assays were recorded with a camcorder equipped with Super NightShot Plus infrared system . During the assay, the arena was inspected with a flashlight whose lens was covered with a red filter. After 5 min, the number of females that landed and continued to feed on each side of the arena was recorded. Insects were gently removed from the cotton rolls, and the assays were reinitiated after rotation of sample and control. Thus, repellency 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. After three runs, filter paper strips and cotton plugs were disposed of, and new loads were prepared.
We cloned the cDNAs for CquiOR2, CquiOR4, CquiOR5, CquiOR84, CquiOR85, AaegOR14, and AaegOR15, which are clustered along CquiOR1 . Then, each OR was separately coexpressed along with its coreceptor in Xenopus oocytes for deorphanization. We used a panel of odorants containing oviposition attractants, repellents, plant-derived compounds and other physiologically or behaviorally relevant compounds. We expected that the odorant profiles for these receptors would resemble that of CquiOR1 , but found marked differences. For example, three floral compounds elicited robust currents on CquiOR4/CquiOrco-expressing oocytes. Specifically, these oocytes were very sensitive to 2-phenylethanol, phenethyl formate, and, propionate . To obtain more information about the sensitivity of the CquiOR4 + CquiOrco receptor, we performed concentration-response analyses for the three best ligands. Currents elicited by 2- phenylethanol were already saturated at the normal screening dose of 1 mM. Thus, we performed these analyses with concentrations in the range of 0.1 μM to 0.1 mM . 2- Phenylethanol was indeed the most potent of the compounds in our panel, activating CquiOR4 + CquiOrco with EC50 of 28 nM. CquiOR5 + CquiOrco receptor showed a quite different profile, with the three best ligands being linalool, p-methane-3,8-diol and linalool oxide . Although the responses were not as robust as those elicited by the best ligands for CquiOR4 + CquiOrco, they were dose-dependent . Linalool activated CquiOR5/CquiOrco-expressing oocytes with an EC50 of 574 nM. By contrast, no compounds in our panel elicited relevant currents in CquiOR2/CquiOrco-expressing oocytes, except for isopentyl acetate . On the other hand, CquiOR84/CquiOrco-expressing oocytes responded with large currents when challenged with N–3-methyl-benzamide or PMD .
Similar responses were recorded with CquiOR85 + CquiOrco receptor. The two receptors from Ae. aegypti in the cluster, ie, AaegOR14 and AaegOR15, gave remarkably different responses in terms of profile and sensitivity. AaegOR14/AaegOrcoexpression oocytes elicited only weak currents when challenged with 4-methylphenol and 4- ethylphenol . By contrast, AaegOR15/AaegOrco-expressing oocytes generated dose-dependent, robust currents when challenged with phenethyl propionate, phenethyl formate, and acetophenone . As far as the two major ligands are concerned, AaegOR15 profile resembles that of CquiOR4 + CquiOrco.The best ligands for CquiOR4 and CquiOR5 have been previously reported to have repellency activity, particularly the commercially available PMD and 2-phenylethanol . To get a better insight into the possible role of these receptors in repellency behavior, we performed qPCR analyses. We surmised that transcript levels of the receptor for repellents and host attractants would decrease after a blood meal because blood-fed mosquitoes cease host finding. By contrast, transcripts of receptors involved in the reception of oviposition attractants would increase given that gravid females use their olfactory system to locate suitable sites for oviposition. Transcript levels of CquiOR4 decreased significantly after a blood meal . Additionally, this receptor was highly enriched in female antennae, with only basal levels in maxillary palps, proboscis, and legs thus further supporting a possible role in hosting finding or repellency activity. By contrast, transcript levels of CquiOR5 did not change significantly after a blood meal . Moreover, this receptor is not specific to or enriched in female antennae. We found high transcript levels in the proboscis, maxillary palps, and legs .We performed RNAi experiments to test whether reducing transcript levels of CquiOR4 would affect repellency activity. Similarly, we tested CquiOR5 vis-à-vis the best ligands. First, we compared the transcript levels of these genes in water-,large pot with drainage β-galactosidase-dsRNAand CquiOR4-dsRNA-injected mosquitoes. Transcript levels of CquiOR4 decreased significantly in knockdown mosquitoes . Similarly, CquiOR5-dsRNA-treated mosquitoes had significantly lower transcripts of CquiOR5 than mosquitoes injected with water or β- galactosidase-dsRNA. Next, RNAi-treated mosquitoes were used to compare repellency activity. Because RNAi treatment reduced transcript levels only by ca. 60%, it is important to test repellency at low doses, otherwise, a possible link between reception and behavior may be overlooked. When tested at 0.01%, protection conferred by 2-phenylethanol, albeit low, was significantly reduced . At a higher dose , 2- phenylethanol-elicited protection is lower in CquiOR4-dsRNA-treated than in β- galactosidase-dsRNA-treated mosquitoes, but there was no significant difference . By contrast, DEET-elicited repellency in these two groups of mosquitoes was not significantly different . Measuring linalool-elicited repellency activity at 0.1% showed a non-significant reduction of protection in CquiOR5-dsRNA-treated mosquitoes as compared to β-galactosidase-dsRNAtreated mosquitoes . Likewise, protection by PMD was also reduced, but not significantly different . There was no significant difference in repellency elicited by DEET . Attempts to test repellency at lower doses were unrewarding. At 0.05% linalool, 56 ± 5.1% of mosquitoes responded to the control side of the arena, whereas 44 ± 5% responded to treatment . Likewise, PMD at 5% provided no protection: control, 51.7 ± 2%; treatment, 48.2 ± 2%. Thus, we were unable to test the effect of RNAi treatment on repellency activity at lower than 0.1% dose. In conclusion, repellency activity mediated by linalool and PMD might involve multiple receptors. Here, we show that PMD activates not only CquiOR5 but also CquiOR84/85.
Previously, we showed that PMD activated a DEET receptor in the Southern house mosquito, CquiOR136 . The reduction in protection observed with knockdown mosquitoes, albeit not statistically significant, suggests that CquiOR5 might be one of the receptors mediating linalool and PMD repellency activity. On the other hand, we cannot rule out the possibility that other receptors are involved in a combinatorial code reception of 2-phenylethanol, but the significant reduction in protection in CquiOR4- dsRNA-treated mosquitoes suggests it may play a significant part in 2-phenylethanolmediated repellency activity.The 2020–2025 Dietary Guidelines for Americans encourages the intake of a variety of plant-based foods including nuts and berries. With the goal of increasing current knowledge on nuts and berries, as well as addressing research challenges and opportunities, the Nuts and Berries Conference: Pathways to Oxidant Defense, Vascular Function, and Gut Microbiome Changes was held on 5 to 6 May, 2022 at the University of California, Davis. Tree nuts and berries were selected as the focus of the conference for their unique composition, bioactivity, and multitude of associated health-promoting qualities. With over 50 different edible nut species and hundreds of berry varietals, the following were selected for the purpose of the conference and this review: walnuts, almonds, hazelnuts, cashews, pecans, pistachios, strawberries, blueberries, raspberries, and blackberries. Tree nuts and berries are significant commodities in the United States. The total value of tree nuts grown in California in 2021 was estimated at $8.961 billion. The total value of berries grown in California in 2021 was approximately $3.667 billion. With over two-thirds of US tree nuts and berries grown in California, the agricultural land-grant institution of the University of California, Davis was the appropriate location to convene this conference of leading researchers, registered dietitians, community partners, and industry representatives. Regular tree nut and berry consumption is associated with a decreased risk for the development of cardiovascular disease along with favorable effects on brain and gut health. Tree nuts provide protein and fiber and monounsaturated and polyunsaturated fatty acids, along with vitamins, minerals, and bio-active carotenoids, phytosterols, phenolics and flavonoids, and lignan and tannins, such as the condensed proanthocyanidins and hydrolysable ellagitannins. Berries are also a significant source of fiber and vitamin C, along with bio-active carotenoids, phenolics, including proanthocyanins and ellagitannins, and anthocyanins that provide berry color. Moreover, berries provide flavan-3-ols in quantities up to 37 mg/100 g serving , which would contribute to a recently proposed daily recommended intake level of 400 to 600 mg/d. Although research results to date have been promising, mechanisms of action in general, and for vascular and gut health specifically, have yet to be fully defined. More data are needed that can be generalized to diverse population groups as well as for modeling of precision nutrition recommendations. This paper will review the progress and challenges of current nut and berry research and suggest future directions for the field.Many different study designs have been used to assess the effects of nuts and berries on cardiometabolic health. A summary of the past 5 y of studies on nuts and berries on outcome measures of cardiovascular and gut health is presented in Tables 4, 5, 6 7, 8, 9 and Tables 10, 11, 12, 13, respectively. Eligible studies consisted of clinical human trials in children, adolescents, and adults published within the last 5 y , exploring associations between the consumption of nuts and berries and associated biomarkers of interest. Two long-term intervention trials, the PREDIMED and the COcoa Supplement and Multivitamin Outcomes Study , published in 2018 and 2022, respectively, provide examples of study designs that could be useful for future planning. The PREDIMED dietary intervention trial provides the strongest evidence to date that incorporation of nuts into a healthy Mediterranean dietary pattern in individuals ages 55 to 80 y old for 4.8 y can reduce risk of cardiovascular events by 28%.