Drought-stressed tomato plants trigger bottom–up effects on the Invasive Tetranychus evansi

19 p.-5 fig.-3 tab.Climate change will bring more drought periods that will have an impact on the irrigation practices of some crops like tomato, from standard water regime to deficit irrigation. This will promote changes in plant metabolism and alter their interactions with biotic stressors. We have tested if mild or moderate drought-stressed tomato plants (simulating deficit irrigation)have an effect on the biological traits of the invasive tomato red spider mite, Tetranychus evansi. Our data reveal that T evansi caused more leaf damage to drought-stressed tomato plants ( 1.5 fold for both drought scenarios). Mite performance was also enhanced, as revealed by significant increases of eggs laid ( 2 fold) at 4 days post infestation (dpi), and of mobile forms ( 2 fold and 1.5 fold for moderate and mild drought, respectively) at 10 dpi.The levels of several essential amino acids (histidine, isoleucine, leucine, tyrosine, valine) and free sugars in tomato leaves were significantly induced by drought in combination with mites. The non-essential amino acid proline was also strongly induced, stimulating mite feeding and egg laying when added to tomato leaf disks at levels equivalent to that estimated on drought-infested tomato plants at 10 dpi. Tomato plant defense proteins were also affected by drought and/or mite infestation, but T. evansi was capable of circumventing their potential adverse effects. Altogether, our data indicate that significant increases of available free sugars and essential amino acids, jointly with their phagostimulant effect, created a favorable environment for a better T. evansi performance on drought-stressed tomato leaves. Thus, drought-stressed tomato plants, even at mild levels, may be more prone to T evansi outbreaks in a climate change scenario, which might negatively affect tomato production on area-wide scales.This work was funded by an INIA grant:GENOMITE, Proposal No 618105 FACCE Era Net Plus-Food security, Agriculture, Climate Change (new generation sustainable tools to control emerging mite pests under climate change).Peer reviewe

Inheritance, Fitness Cost, and Management of Lambda-Cyhalothrin Resistance in a Laboratory-Selected Strain of Ceratitis capitata (Wiedemann)

The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered one of the most destructive and economically damaging pests of citrus and other fruit crops worldwide. Current control practices in Spain rely on the use of insecticides (mainly lambda-cyhalothrin, spinosad, and deltamethrin) and the release of sterile males. However, the sustainability of medfly control programs is threatened by reports of resistance to lambda-cyhalothrin in field populations. In this work, we used a laboratory-selected lambda-cyhalothrin-resistant strain to study key factors required for devising effective insecticide resistance management strategies. Specifically, we have (1) determined that the inheritance of resistance is autosomic (non-associated to the sexual chromosome), completely dominant (a single copy of the gene is enough to confer resistance), and polygenic (controlled by more than one gene); (2) observed that resistant individuals present fitness alterations in regard to biological parameters (lower survival in the first growth stages, a slower developmental time, and higher adults’ weight and longevity); and (3) shown under laboratory conditions that the alternation of lambda-cyhalothrin with spinosad helped delay the development of resistance. Taken together, our results indicate that it would be advisable to encourage the rotation of these insecticides to manage the resistance problem

Digestive proteases in bodies and faeces of the two-spotted spider mite, Tetranychus urticae

31 p.-3 fig.-3 tab.-2 fig. supl.-1 tab. supl.Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.This work was supported by a Grant from CSIC (Grant CSIC-201040E049 to F.O), Ministerio de Ciencia e Innovación (Grant AGL11-23650 to I.D.) and the Government of Canada through Genome Canada and the Ontario Genomics Institute (Grant OGI-046 to V.G.), and the Ontario Research Fund-Global Leadership in Genomics and Life Sciences (Grant GL2-01-035 to V.G.).Peer reviewe

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

31 p.-11 fig.-2 tab.+ Erratum (2 p.) Papanikolaou, Alexie et al.Background: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control.Results: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A highquality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT.Conclusions: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolutionSupport of this project was provided by the U.S. Department of Agriculture(USDA), Agricultural Research Service (ARS), Animal and Plant Health Inspection Service (APHIS), and National Institute of Food and Agriculture(NIFA)-Biotechnology Risk Assessment Grants Program (grant #2011-39211-30769 to AMH) for funding the initial phase of this project, and to the National Institutes of Health (NIH)-National Human Genome Research Institute (NHGRI) for funding the medfly genome sequencing, assembly and Maker 2.0 automated annotation as part of the i5K 30 genome pilot project (grant #U54 HG003273 to RAG). The NIH Intramural Research Program, National Library of Medicine funded the NCBI Gnomon annotation and the USDA-National Agricultural Library (NAL) provided support for the WebApollo curation website, with support for manual curation training (to MM-T) provided by NIGMS (grant #5R01GM080203),NHGRI (grant #5R01HG004483), and the U.S. Department of Energy(contract #DE-AC02-05CH11231). Support was provided for: toxin metabolism and insecticide resistance gene studies from MINECO,Spain (AGL2013-42632-R to FO and PH-C); microRNAs, horizontal gene transfer and bacterial contaminant studies from the European Social Fund and National Strategic Reference Framework-THALES (MIS375869 to KB, GT, AGH, and KM) and the U.S. National Science Foundation(DEB 1257053 to JHW); cuticle protein gene studies from USDA-NIFA(grant #2016-67012-24652 to AJR); sex-determination studies from L.R. Campania (grant 5/02, 2008 to GS); male reproduction and sexual differentiation studies from the FAO/IAEA (Technical Contract No: 16966 to GGa) and Cariplo IMPROVE (to FS); and programmed cell death gene studies and genomic data analysis (to MFS) from the Emmy Noether program, DFG(SCHE 1833/1-1) and the LOEWE Center for Insect Biotechnology & Bioresources grant of the Hessen State Ministry of Higher Education, Research and the Arts(HMWK), Germany and from the USDA-NIFA-Biotechnology Risk Assessment Grants Program (grant #2015-33522-24094 to AMH).Peer reviewe

Early Molecular Responses of Tomato to Combined Moderate Water Stress and Tomato Red Spider Mite Tetranychus evansi Attack

Interaction between plants and their environment is changing as a consequence of the climate change and global warming, increasing the performance and dispersal of some pest species which become invasive species. Tetranychus evansi also known as the tomato red spider mite, is an invasive species which has been reported to increase its performance when feeding in the tomato cultivar Moneymaker (MM) under water deficit conditions. In order to clarify the underlying molecular events involved, we examined early plant molecular changes occurring on MM during T. evansi infestation alone or in combination with moderate drought stress. Hormonal profiling of MM plants showed an increase in abscisic acid (ABA) levels in drought-stressed plants while salicylic acid (SA) levels were higher in drought-stressed plants infested with T. evansi, indicating that SA is involved in the regulation of plant responses to this stress combination. Changes in the expression of ABA-dependent DREB2, NCED1, and RAB18 genes confirmed the presence of drought-dependent molecular responses in tomato plants and indicated that these responses could be modulated by the tomato red spider mite. Tomato metabolic profiling identified 42 differentially altered compounds produced by T. evansi attack, moderate drought stress, and/or their combination, reinforcing the idea of putative manipulation of tomato plant responses by tomato red spider mite. Altogether, these results indicate that the tomato red spider mite acts modulating plant responses to moderate drought stress by interfering with the ABA and SA hormonal responses, providing new insights into the early events occurring on plant biotic and abiotic stress interaction

Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice

Functional characterization and fitness cost of spinosad-resistant alleles in Ceratitis capitata

[EN] The sustainability of control programs for the Mediterranean fruit fly, Ceratitis capitata, for citrus crops in Spain has been threatened by the development of resistance to malathion and lambda-cyhalothrin in recent years. Spinosad is widely used without apparent loss of efficacy. However, a highly resistant strain, JW-100s, has been obtained after laboratory selection. Spinosad resistance in JW-100s has been associated with different mutant alleles of the alpha 6 subunit of the nicotinic acetylcholine receptor (Cc alpha 6) including an isoform-specific truncation allele, Cc alpha 6(3aQ68*). Using the GAL4 > UAS system in Drosophila melanogaster to demonstrate expression of this truncated alpha 6 subunit, in a d alpha 6 loss-of-function genetic background, does not rescue susceptibility to spinosad, while the expression of Cc alpha 6 wild-type isoforms does. We have also generated C. capitata isolines from JW-100s homozygous for: (1) the Cc alpha 6(3aQ68*Delta 3b-4) allele, which contains the mutation 3aQ68*, and (2) the Cc alpha 6(3aQ68*-K352*) allele, which contains the mutations 3aQ68* and K352*. Neither of these produce complete Cc alpha 6 transcripts. The frequency of resistant alleles declined when in competition with individuals carrying the wild-type allele. Through extensive testing of both biological and behavioral fitness traits, we identified a reduced ability of Cc alpha 6(3aQ68*Delta 3b-4) males to detect the parapheromone and to mate with females carrying the Cc alpha 6(3aQ68*-K352*) allele in competition experiments. Thus, not only the potential for spontaneous resistant mutations to arise in Cc alpha 6 but also their fitness costs must be considered when planning resistance management strategies for C. capitata.This work received financial support from CICYT (AGL2016-76516-R). The Spanish MINECO granted A. Guillem-Amat a predoc (BES-C-2014-068937) and a mobility (EEBB-I-16-11336) fellowships. We gratefully acknowledge Maria Torne (Dow Agro-Science Iberica) for providing technical grade spinosad, Charles Robin (University of Melbourne) for assisting with bureaucratic issues with the Australian Government, Tinna Yang (University of Melbourne) for the keeping and shipping of the flies and Sandra Vacas (Universitat Politecnica de Valencia) for the scientific advice on electroantennography.Guillem-Amat, A.; Ureña, E.; López-Errasquín, E.; Navarro-Llopis, V.; Batterham, P.; Sánchez, L.; Perry, T.... (2020). Functional characterization and fitness cost of spinosad-resistant alleles in Ceratitis capitata. Journal of Pest Science. 93(3):1043-1058. https://doi.org/10.1007/s10340-020-01205-xS10431058933Abbas N, Mansoor MM, Shad SA et al (2014) Fitness cost and realized heritability of resistance to spinosad in Chrysoperla carnea (Neuroptera: Chrysopidae). Bull Entomol Res 104:707–715. https://doi.org/10.1017/S0007485314000522Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267. https://doi.org/10.1093/jee/18.2.265aAnstead CA, Korhonen PK, Young ND et al (2015) Lucilia cuprina genome unlocks parasitic fly biology to underpin future interventions. Nat Commun 6:1–11. https://doi.org/10.1038/ncomms8344Arouri R, Le Goff G, Hemden H et al (2015) Resistance to lambda-cyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain. Pest Manag Sci 71:1281–1291. https://doi.org/10.1002/ps.3924Bao WX, Narai Y, Nakano A et al (2014) Spinosad resistance of melon thrips, Thrips palmi, is conferred by G275E mutation in α6 subunit of nicotinic acetylcholine receptor and cytochrome P450 detoxification. Pestic Biochem Physiol 112:51–55. https://doi.org/10.1016/j.pestbp.2014.04.013Baxter SW, Chen M, Dawson A et al (2010) Mis-spliced transcripts of nicotinic acetylcholine receptor α6 are associated with field evolved spinosad resistance in Plutella xylostella (L.). PLoS Genet. https://doi.org/10.1371/journal.pgen.1000802Berger M, Puinean AM, Randall E et al (2016) Insecticide resistance mediated by an exon skipping event. Mol Ecol 25:5692–5704. https://doi.org/10.1111/mec.13882Bielza P, Quinto V, Fernandez E et al (2007) Genetics of spinosad resistance in Frankliniella occidentalis (Thysanoptera: Thripidae). J Econ Entomol 100:916–920. https://doi.org/10.1603/0022-0493(2007)100%5b916:gosrif%5d2.0.co;2Bielza P, Quinto V, Gravalos C et al (2008a) Lack of fitness costs of insecticide resistance in the western flower thrips (Thysanoptera: Thripidae). J Econ Entomol. https://doi.org/10.1603/0022-0493(2008)101%5b499:lofcoi%5d2.0.co;2Bielza P, Quinto V, Grávalos C et al (2008b) Stability of spinosad resistance in Frankliniella occidentalis (Pergande) under laboratory conditions. Bull Entomol Res 98:355–359. https://doi.org/10.1017/S0007485308005658Bischof J, Maeda RK, Hediger M et al (2007) An optimized transgenesis system for Drosophila using germ-line-specific C31 integrases. Proc Natl Acad Sci 104:3312–3317. https://doi.org/10.1073/pnas.0611511104Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:289–295. https://doi.org/10.1101/lm.1331809Campos MR, Rodrigues ARS, Silva WM et al (2014) Spinosad and the tomato borer Tuta absoluta: a bioinsecticide, an invasive pest threat, and high insecticide resistance. PLoS ONE. https://doi.org/10.1371/journal.pone.0103235Cossé AA, Todd JL, Millar JG et al (1995) Electroantennographic and coupled gas chromatographic-electroantennographic responses of the mediterranean fruit fly, Ceratitis capitata, to male-produced volatiles and mango odor. J Chem Ecol 21:1823–1836Engebrecht J, Brent R, Kaderbhai MA (1991) Minipreps of plasmid DNA. Curr Protoc Mol Biol. https://doi.org/10.1002/0471142727.mb0106s15Fayyazuddin A, Zaheer MA, Hiesinger PR, Bellen HJ (2006) The nicotinic acetylcholine receptor Da7 is required for an escape behavior in Drosophila. PLoS Biol 4:0420–0431. https://doi.org/10.1371/journal.pbio.0040063Ferguson JS (2004) Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad. J Econ Entomol 97:112–119. https://doi.org/10.1603/0022-0493-97.1.112Ffrench-Constant RH, Bass C (2017) Does resistance really carry a fitness cost? Curr Opin Insect Sci 21:39–46. https://doi.org/10.1016/j.cois.2017.04.011Geng C, Watson GB, Sparks TC (2013) Nicotinic acetylcholine receptors as spinosyn targets for insect pest management, 1st edn. Elsevier, AmsterdamHsu JC, Feng HT, Wu WJ et al (2012) Truncated transcripts of nicotinic acetylcholine subunit gene Bdα6 are associated with spinosad resistance in Bactrocera dorsalis. Insect Biochem Mol Biol 42:806–815. https://doi.org/10.1016/j.ibmb.2012.07.010IRAC (2019) Arthropod pesticide resistance database. https://www.pesticideresistance.org/index.php. Accessed 16 May 2019Jang EB, Light DM, Binder RG et al (1994) Attraction of female mediterranean fruit flies to the five major components of male-produced pheromone in a laboratory flight tunnel. J Chem Ecol 20:9–20. https://doi.org/10.1007/BF02065987Jin Y, Tian N, Cao J et al (2007) RNA editing and alternative splicing of the insect nAChR subunit alpha6 transcript: evolutionary conservation, divergence and regulation. BMC Evol Biol 7:1–12. https://doi.org/10.1186/1471-2148-7-98Jones AK, Raymond-Delpech V, Thany SH et al (2006) The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera. Genome Res 16:1422–1430. https://doi.org/10.1101/gr.4549206Khan HAA, Akram W, Shad SA (2014) Genetics, cross-resistance and mechanism of resistance to spinosad in a field strain of Musca domestica L. (Diptera: Muscidae). Acta Trop 130:148–154. https://doi.org/10.1016/j.actatropica.2013.11.006Li ZM, Liu SS, Liu YQ, Ye GY (2007) Temperature-related fitness costs of resistance to spinosad in the diamondback moth, Plutella xylostella (Lepidoptera: Plutelidae). Bull Entomol Res 97:627–635. https://doi.org/10.1017/S0007485307005366Li X, Wan Y, Yuan G et al (2017) Fitness trade-off associated with spinosad resistance in Frankliniella occidentalis (Thysanoptera: Thripidae). J Econ Entomol 110:1755–1763. https://doi.org/10.1093/jee/tox122Magaña C, Hernandez-Crespo P, Ortego F, Castañera P (2007) Resistance to malathion in field populations of Ceratitis capitata. J Econ Entomol 100:1836–1843. https://doi.org/10.1603/0022-0493(2007)100%5b1836:rtmifp%5d2.0.co;2Magaña C, Hernández-Crespo P, Brun-Barale A et al (2008) Mechanisms of resistance to malathion in the medfly Ceratitis capitata. Insect Biochem Mol Biol 38:756–762. https://doi.org/10.1016/j.ibmb.2008.05.001MAPA (2019) Ministerio de Agricultura, Pesca y Alimentación. https://www.mapa.gob.es/es/. Accessed 12 Jun 2019Navarro-Llopis V, Primo J, Vacas S (2015) Bait station devices can improve mass trapping performance for the control of the Mediterranean fruit fly. Pest Manag Sci 71:923–927. https://doi.org/10.1002/ps.3864Okuma DM, Bernardi D, Horikoshi RJ et al (2018) Inheritance and fitness costs of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to spinosad in Brazil. Pest Manag Sci 74:1441–1448. https://doi.org/10.1002/ps.4829Perry T, Batterham P (2018) Harnessing model organisms to study insecticide resistance. Curr Opin Insect Sci 27:61–67. https://doi.org/10.1016/j.cois.2018.03.005Perry T, McKenzie JA, Batterham P (2007) A D α6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad. Insect Biochem Mol Biol 37:184–188. https://doi.org/10.1016/j.ibmb.2006.11.009Perry T, Batterham P, Daborn PJ (2011) The biology of insecticidal activity and resistance. Insect Biochem Mol Biol 41:411–422. https://doi.org/10.1016/j.ibmb.2011.03.003Perry T, Somers J, Yang YT, Batterham P (2015) Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor: spinosyn interaction. Insect Biochem Mol Biol 64:106–115. https://doi.org/10.1016/j.ibmb.2015.01.017Puinean AM, Lansdell SJ, Collins T et al (2013) A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis. J Neurochem 124:590–601. https://doi.org/10.1111/jnc.12029Raymond M, Berticat C, Weill M et al (2001) Insecticide resistance in the mosquito Culex pipiens: what have we learned about adaptation? Genetica 112–113:287–296. https://doi.org/10.1023/A:1013300108134Reddy PVR, Rashmi MA (2016) Sterile insect technique (SIT) as a component of area-wide integrated management of fruit flies: status and scope. Pest Manag Hortic Ecosyst 22:1–11. https://doi.org/10.1097/01.ede.0000100289.82156.8bRehan A, Freed S (2014) Selection, mechanism, cross resistance and stability of spinosad resistance in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Crop Prot 56:10–15. https://doi.org/10.1016/j.cropro.2013.10.013Rehan A, Freed S (2015) Fitness cost of methoxyfenozide and the effects of its sublethal doses on development, reproduction, and survival of spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Neotrop Entomol 44:513–520. https://doi.org/10.1007/s13744-015-0306-5Rinkevich FD, Scott JG (2009) Transcriptional diversity and allelic variation in nicotinic acetylcholine receptor subunits of the red flour beetle, Tribolium castaneum. Insect Mol Biol 18:233–242. https://doi.org/10.1111/j.1365-2583.2009.00873.xRinkevich FD, Chen M, Shelton AM, Scott JG (2010) Transcripts of the nicotinic acetylcholine receptor subunit gene Pxyla6 with premature stop codons are associated with spinosad resistance in diamondback moth, Plutella xylostella. Invertebr Neurosci 10:25–33. https://doi.org/10.1007/s10158-010-0102-1Robertson JL, Preisler HK (1992) Pesticide bioassays with arthropods. CRC Press, Boca RatonSalgado VL, Sparks TC (2005) 6.5—the spinosyns: chemistry, biochemistry, mode of action, and resistance. In: Comprehensive molecular insect science. pp 137–173Sattelle DB, Jones AK, Sattelle BM et al (2005) Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster. BioEssays 27:366–376. https://doi.org/10.1002/bies.20207Sayyed AH, Saeed S, Noor-Ul-Ane M, Crickmore N (2008) Genetic, biochemical, and physiological characterization of spinosad resistance in Plutella xylostella (Lepidoptera: Plutellidae). J Econ Entomol 101:1658–1666. https://doi.org/10.1603/0022-0493Shao YM, Dong K, Zhang CX (2007) The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori. BMC Genom 8:1–10. https://doi.org/10.1186/1471-2164-8-324Shi M, Yue Z, Kuryatov A et al (2014) Identification of redeye, a new sleep-regulating protein whose expression is modulated by sleep amount. Elife 2014:1–17. https://doi.org/10.7554/eLife.01473Silva WM, Berger M, Bass C et al (2016) Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Pestic Biochem Physiol 131:1–8. https://doi.org/10.1016/j.pestbp.2016.02.006Somers J, Nguyen J, Lumb C et al (2015) In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad. Insect Biochem Mol Biol 64:116–127. https://doi.org/10.1016/j.ibmb.2015.01.018Somers J, Luong HNB, Batterham P, Perry T (2017) Deletion of the nicotinic acetylcholine receptor subunit gene Dα1 confers insecticide resistance, but at what cost? Fly (Austin) 12:46–54. https://doi.org/10.1080/19336934.2017.1396399Ureña E, Guillem-Amat A, Couso-Ferrer F et al (2019) Multiple mutations in the nicotinic acetylcholine receptor Ccα6 gene associated with resistance to spinosad in medfly. Sci Rep 9:2961. https://doi.org/10.1038/s41598-019-38681-wVontas J, Hernández-Crespo P, Margaritopoulos JT et al (2011) Insecticide resistance in Tephritid flies. Pestic Biochem Physiol 100:199–205. https://doi.org/10.1016/j.pestbp.2011.04.004Wang D, Qiu X, Wang H et al (2010) Reduced fitness associated with spinosad resistance in Helicoverpa armigera. Phytoparasitica 38:103–110. https://doi.org/10.1007/s12600-009-0077-9Wang J, Wang X, Lansdell SJ et al (2016) A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella. Insect Biochem Mol Biol 71:29–36. https://doi.org/10.1016/j.ibmb.2016.02.001Watson GB, Chouinard SW, Cook KR et al (2010) A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression. Insect Biochem Mol Biol 40:376–384. https://doi.org/10.1016/j.ibmb.2009.11.004Wu M, Robinson JE, Joiner WJ (2014) SLEEPLESS is a bifunctional regulator of excitability and cholinergic synaptic transmission. Curr Biol 24:621–629. https://doi.org/10.1016/j.cub.2014.02.026Wyss CF, Young HP, Shukla J, Roe RM (2003) Biology and genetics of a laboratory strain of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae), highly resistant to spinosad. Crop Prot 22:307–314. https://doi.org/10.1016/S0261-2194(02)00153-

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.Instituto de Salud Carlos III, Grant/Award Number: PI12/0058; National Institutes of Health, Grant/Award Number: ZIA DE000721-12; Ministerio de Ciencia e Innovación, Grant/Award Numbers: SAF2009-11175, SAF2010-14990-R, SAF2016-77233-R; Ministerio de Economía y Competitividad, Grant/Award Numbers: BES-2011-047063, BES-2017-080782; Severo Ochoa Programme for Centres of Excellence in R&D, Grant/Award Number: SEV-2017-0723 and cofinanced by the European Regional Development Fund; Generalitat Valenciana, Grant/Award Number: GRISOLIA/2008/02

Estudio de la herencia y mapeo de la resistencia de Ceratitis capitata (Wiedemann, 1824) (Diptera, Tephritidae) al insecticida λ-cihalotrina

La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann, 1824), es una de las principales plagas de cítricos y otros frutales en España. Las actuales estrategias de control, basadas mayoritariamente en el uso de insecticidas, están viendo amenazada su eficacia debido a la aparición de resistencia en poblaciones de campo. Recientemente se ha descrito resistencia metabólica a lambda-cihalotrina mediada por P450s. Con el fin de estudiar el tipo de herencia de la resistencia a lambda-cihalotrina, se realizaron cruzamientos recíprocos entre una línea de laboratorio resistente a este insecticida y una línea susceptible, se obtuvieron la generación F1 y la F2 y se realizó el retrocruce de la F1 con el parental susceptible. Los resultados muestran que la herencia de la resistencia es completamente dominante y autosómica, y que no se ajusta a un modelo mendeliano monogénico. Asimismo, se está realizando un mapeo genético de la resistencia a lambda-cihalotrina a partir de los individuos ensayados en el estudio de la herencia, con el propósito de identificar marcadores moleculares que nos permitan la detección precoz y el seguimiento de la resistencia en campo, facilitando así el control de la plaga. Por último, mediante el simulacro en laboratorio de distintos escenarios de tratamiento se ha podido determinar que la rotación de lambda-cihalotrina con spinosad resulta eficaz para el manejo de la resistencia a lambda-cihalotrina

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature

[Abstract] Aim: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. Methods: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). Results: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. Conclusion: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.Ministerio de Ciencia e Innovación (España); RT2018-099995-B100Ministerio de Ciencia e Innovación (España); AEI/10.13039/501100011033Generalitat Valenciana; PROMETEO/2021/031Ministerio de Asuntos Económicos y Transformación Digital (España); BES-2011-04706