I have included the entire article. For Figure 4 provide a breif overview of the

Question

I have included the entire article. For Figure 4 provide a breif overview of the experimnet done and describe the general trends/coclusions that could be made from this data in your own words.

Pingxi Xu'. Young-Moo Choo1. Alyssa De La Rosa2, and Walter S. Leal Department of Molecular and Cellular Biology. University of California, Davis, CA 95616 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved October 3, 2014 (received for review September 8, 2014) Insect repellents are important prophylactic tools for travelers and ti putative receptor along with coreceptor CquilR8a in the populations living in endemic areas of malaria, dengue, enceph- Xenopus oocytes. CquilR40a Cil8a-expressing oocytes did alitis, and other vector-borne diseases. DEET (N,N-diethyl-3-meth not generate detectable currents when challenged with DEET, ylbenzamide) is a 6-decade-old synthetic repellen considered the gold standard of mosquito repellents. Mosquitoes repellents, ethyl anthranilate, butyl anthranilate, and methyl use their sense of smell to detect DEET, but there are currently two N,N-dimethylanthranilate (3). hypotheses regarding its mode of action: activation of ionotropic To rule out a possible malfunction of the coreceptor CquilR8a, receptor IR40a vs. odorant receptor(s). Here, we demonstrate that we first recorded from a known Drosophila IR coreceptor system DEET, picaridin, insect repellent 3535, and p-menthan-3,8-diol ac- and then mismatched receptors and coreceptors. DmelIR84a tivate the odorant receptor Cqui0R1 36 of the southern house rnos- DmellR8a-expressing oocytes generated robust currents (253 ± t, which is still 200 compounds in our panel, or the newly identified mosquito ito, Culex quinquefasciatus. Electrophysiological and behavioral 26n assays showed that CquiR40a knockdown had no significant ef- to phenylacetaldehyde, in agreement with a previous report (6) fect on DEET detection and repellency. By contrast, reduction of Although CquilR40a coexpressed with DmellR8a remained CquiOR136 transcript levels led to a significant decrease in electro- silent, DmelIR84a coexpressed with CquilR8a responded to antennographic responses to DEET and a complete lack of repel- phenylacetaldehyde (717 ± 166 nA 1 mM), thus suggesting lency. Thus, direct activation of an odorant receptor, not an hat CquilR8a was indeed functional. It is worth mentioning. ionotropic receptor, is necessary for DEET reception and repellency however, that our findings, albeit inconclusive, are not entirely in Culex mosquitoes, Interestingly, methyl jasmonate, a repellent surprising, given that to date only a handful of IRs have been derived from the nonvolatile jasmonic acid in the signaling path deorphanized (6-8). way of plant defenses, elicited robust responses in CquioR136 We then changed our strategy and next used RNAi to reduce Cquiorco-expressing Xenopus oocytes, thus suggesting a possible A, 1 M; 1,616 ± 294 nA, 10 M; and 3,783 ± 159 nA, 100 M) CquilR40a transcript levels in adult female mosquitoes and ex between natural products with long insect-plant evolutionary amined the phenotypes by electrophysiology and behavic history and synthetic repellents. Our RNAi approach has been demonstrated to cause long- lasting transcript-level reductions (9). Thus, we injected three groups of female pupae with CquilR40a-double-strand RNA (dsRNA), water, or -galactosidase-dsRNA. As a result, transcript levels of CguilR4 in adults were significantly reduced in CquilR40a- picaridin | IR 3535 odorant receptor | Culex quinquefasciatus | nsect repellents have been used since ancient times as pro- dsRNA-injected mosquitoes compared with the two control groups phylactic agents against diseases transmitted by mosquitoes (Fig 1A) Given that the antennae of C quinquefasciatus house and other arthropods, including malaria, dengue fever, and en- cephalitis. They were developed from plant-based smoke or Significance extracts (essential oils) into formulations with a single active ingredient. DEET (N,N-diethyl-3-methylbenzamide), a synthetic compound developed more than 6 decades ago, is the most widely used substance. Unfortunately, people in endemic areas who need insect repellents the most cannot afford to use DEET daily, whereas a significant proportion of those who need and southern house can afford it, do not use DEET because of undesirable ties such as an unpleasant odor. Molecular modeling led to the development of insect repellent (IR) 3535 (1) and picaridin (2), but progress toward development of better and more affordable repellents has been slow, because DEET receptors in mosquitoesmay pave the way for the development of better and more are hitherto unknown. entomologists, neurobiologists, insect physiologists, and chemical ecologists for decades, and hitherto it was not known how and why it works. We have discovered an odorant receptor in the mosquito, which is essential for repellency proper unravelling how DEET works. Additionally, we have identified a link between this synthetic repellent and methyl jasmonate, thus suggesting that DEET might work by mimicking defensive compound(s) from plants. The discovery of a molecular target There are currently two hypotheses regarding DEET re ception. One school postulates that the widespread effet of Author contributioms: Px., Y MC ADLR, and W.SL. performed research: Px, Y.M.C DEET olfactory repellency is mediated by a well-conserved ADLR, and W.S.L. analyzed data; W.S.L. designed research; and W.S.L wrote the paper. ionotropic receptor, IR40a (3), whereas the other (4, 5) favors The authors dedare no conflict of interest. a pathway involving odorant receptor(s). Here we report that the This article is a PNAS Direct Submission. southern house mosquito Culex quinquefasciatus the PHAS open access option. receptor, CquiOR136, to detect DEET, picaridin, IR3535, menthan-3,8-diol (PMD), and a plant defense-signaling com Bank database (accession no. KM229531-8). uses an odan Freely available online through P Data deposition: The sequences reported in this paper have been deposited in the Gen 'P x, and Y-M.C, contributed equally to this work. Present address: United States Department of Agriculture-Agricultural Reseanch Service, Commodity Protection and Quality Research Unit, Davs, Parlier, CA 93648. Results and Discussion To test whether DEET olfactory repellency in the southern To whom correspondence should be addressed. Emaiaeucdavis.edu. house mosquito is mediated by IR40a (3), we cloned CquilR40a, the C quinquefasciatus ortholog of DmelIR40a (3), and expressed1417244111CSupplemental This article contains supporting information online at www.pnas.orglookupsupplidoi10. 16592-16597PNASNovember 18, 2014 | vol. 1no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1417244111

Explanation / Answer

CquiOR136.CquiOrco were the genes activated by various repellents such used to study the expression of numerous genes.

Further, Experiments were done to find out whether DEET, a more recent synthetic counter-part of picaridine and methyl jasmonate, could mimic plant defence compound that was being used by plants as a defence mechanism from a long time.

Strong response was oberserved when the two gene were expressed against jasmonate (used as repellent against southern house mosquito, as well as ticks), whereas a very weak signals were produced against jasmonic acid.

Next, Dose dependent curve was made using the above mentioned plant products and four important repellents (DEET, IR3535, PMD and picaridin) against CquiOR136.CquiOrco expressing oocytes.

According to the curve in fig. 4, Four repellents along with jasmonate showed almost same response in the beginning but at higher concentrations IR3535 showed most sensitivity, whereas, methyl jasmonate showed a bit higher sensitivity than DEET. jasmonic acid showed very less sensitivity even at high concentrations.

Also, the experiment showed that CquiOR136 was involved in forming receptors against the above mentioned repellent.

To conclude, the various repellents showed strong response in presence of CquiOR136, which means that it is the possible gene for expresssing receptors against the repellents, which increases according to the dose concentration. In addition, the plant derivative, methyl jasmonate also showed strong responses as a natural repellent. So, there can be other alternatives used by plants as defence to be used as repellents.

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