THE SUBLETHAL EFFECTS OF NEUROTOXIC INSECTICIDES ON THE BASIC BEHAVIOURS OF AGRICULTURALLY IMPORTANT CARABID BEETLES NEUROTOKSILISTE INSEKTITSIIDIDE SUBLETAALNE TOIME PÕLLUMAJANDUSLIKULT OLULISTE JOOKSIKLASTE PÕHIKÄITUMISTELE ENE TOOMING A Thesis for applying for the degree of Doctor of Philosophy in Agriculture Väitekiri filosoofiadoktori kraadi taotlemiseks põllumajanduse erialal Tartu 2017 Eesti Maaülikooli doktoritööd Doctoral Theses of the Estonian University of Life Sciences THE SUBLETHAL EFFECTS OF NEUROTOXIC INSECTICIDES ON THE BASIC BEHAVIOURS OF AGRICULTURALLY IMPORTANT CARABID BEETLES NEUROTOKSILISTE INSEKTITSIIDIDE SUBLETAALNE TOIME PÕLLUMAJANDUSLIKULT OLULISTE JOOKSIKLASTE PÕHIKÄITUMISTELE ENE TOOMING A Thesis for applying for the degree of Doctor of Philosophy in Agriculture Väitekiri Filosoofiadoktori kraadi taotlemiseks põllumajanduse erialal Tartu 2017 Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences According to verdict No 6-14/15-6, the Doctoral Committee of Agricultural and Natural Sciences of the Estonian University of Life Sciences has accepted the thesis for the defence for the degree of Doctor of Philosophy in Agriculture. Opponent: Prof. Pietro Brandmayr Department of Biology, Ecology and Earth Science; University of Calabria, Italy Supervisors: Dr. Enno Merivee Estonian University of Life Sciences, Estonia Dr. Anne Must Estonian University of Life Sciences, Estonia Reviewers: Prof. Indrikis Krams University of Tartu, University of Daugavpils Defence of this thesis: Estonian University of Life Sciences, room D239, Kreutzwaldi 5, Tartu on December 11, 2017 at 14.15. The English language was edited by Prof. Ingrid Helvi Williams and the Estonian by Dr. Luule Metspalu. Publication of this thesis is supported by the Estonian University of Life Sciences, Chair of Plant Health. © Ene Tooming, 2017 ISSN 2382-7076 ISBN 987-9949-629-04-6 (trükis) ISBN 978-9949-629-05-3 (pdf) CONTENTS LIST OF ORIGINAL PUBLICATIONS ......................................... 7 ABBREVIATIONS ........................................................................... 8 1. INTRODUCTION ................................................................... 9 2. REVIEW OF THE LITERATURE .......................................... 11 2.1. Neurotoxic insecticide use in agriculture .......................... 11 2.2. Deleterious effects of neurotoxic insecticides on non-target beneficial insects ............................................................... 12 2.3. Basic behaviours as valuable ecotoxicological biomarkers .. 13 2.4. The main routes of exposure to pesticides ........................ 14 2.5. The model carabid of the study ........................................ 14 3. HYPOTHESES AND AIMS OF THE STUDY ...................... 15 4. MATERIALS AND METHODS ............................................. 16 4.1. Test beetles ....................................................................... 16 4.2. Insecticide treatments ....................................................... 16 4.2.1. Fastac® 50EC (BASF) ............................................. 16 4.2.2. Actara® 25WG (Novartis) ....................................... 17 4.3. Behavioural experiments .................................................. 17 4.3.1. Video tracking ........................................................ 17 4.3.2. CFCR (III) ............................................................ 18 4.4. Data management and statistical analyses ......................... 19 5. RESULTS ................................................................................. 20 5.1. The sublethal effects of α-cyp on the GMA and LA of the carabid beetle P. assimilis ................................................... 20 5.2. The sublethal effects of α-cyp on the behavioural thermo- regulation of P. assimilis .................................................... 21 5.2.1. The proportion of the insecticide treated beetles killed by overheating on the experimental thermal mosaic arena 21 5.2.2. The effect of α-cyp exposure on the LA of the beetles 21 5.2.3. The effect of α-cyp on the tendency of the beetles to hide in shelters ................................................................. 22 5.2.4. The effect of α-cyp exposure on time spent by the beetles at noxious high temperatures ................................ 22 5.3. The sublethal effects of thiamethoxam on the LA and CFCR .............................................................................. 23 5.3.1. Effect of sublethal doses of thiamethoxam on the LA rhythms ............................................................................ 23 5.3.2. Effect on the LA, in terms of the distances moved .. 24 5.3.3. Effect on the CFCR ............................................... 24 6. DISCUSSION ......................................................................... 26 7. CONCLUSIONS .................................................................... 32 REFERENCES ............................................................................... 33 SUMMARY IN ESTONIAN .......................................................... 42 ACKNOWLEDGEMENTS ........................................................... 47 ORIGINAL PUBLICATIONS ....................................................... 49 CURRICULUM VITAE ................................................................. 82 ELULOOKIRJELDUS ................................................................... 84 LIST OF PUBLICATIONS ............................................................ 86 6 LIST OF ORIGINAL PUBLICATIONS This thesis is a review of the following papers, which are referred to by Roman numerals in the text. The papers are reproduced by kind permis- sion of the publishers. I Tooming, E., Merivee, E., Must, A., Sibul, I., Williams I., 2014. Sub-lethal effects of the neurotoxic pyrethroid insecticide Fastac 50EC on the general motor and locomotor activities of the non-tar- geted beneficial carabid beetle Platynus assimilis (Coleoptera: Carabi- dae). Pest Management Science, 70, 959–966. II Merivee, E., Tooming, E., Must, A., Sibul, I., Williams I., 2015. Low doses of the common alpha-cypermethrin insecticide affect behavioural thermoregulation of the non-targeted beneficial carabid beetle Platynus assimilis (Coleoptera:Carabidae). Ecotoxicology and Environmental Safety, 120, 286–294. III Tooming, E., Merivee, E., Must, A., Merivee, M-I., Sibul, I., Nurme, K., Williams, I., 2017. Behavioural effects of the neonicotinoid insecticide thiamethoxam on the predatory insect Platynus assimilis. Ecotoxicology, 26 (7), 902–913. Table 1. The contributions of the authors to the papers: Paper Idea and study Laboratory Data Manuscript design work analysis preparation I ET, EM, AM EM, ET, AM EM, ET, AM All II EM EM, ET, M EM, ET, AM All III EM, AM, ET EM, ET, AM, EM, ET, AM, All KN, MIM KN, MIM ET – Ene Tooming; EM – Enno Merivee; AM – Anne Must; IS – Ivar Sibul, IHW – Ingrid Helvi Williams; MIM – Marten-Ingmar Merivee; KN – Karin Nurme; All – all authors of the papers. 7 ABBREVIATIONS α-cyp alpha-cypermethrin CFCR clean food consumption rate FSA food source area GMA general motor activity IPM integrated pest management LA locomotor activity LD50 lethal dose (to 50% of test animals) MFRC maximum field recommended concentration RH relative humidity 8 1. INTRODUCTION There is a need in agriculture to achieve more sustainable use of insecti- cides with the least possible disruption to agro-ecosystems. The applica- tion of pyrethroid and neonicotinoid insecticides in agriculture is exten- sive and due to their high bio-efficacy as well as their broad-spectrum range of action, they are still the best means to control most harmful arthropod pests (Desneux et al., 2007; Garcia, 2011). Pyrethroids with their contact mode of action affect both the peripheral and central nerv- ous systems of an insect, stimulating nerve cells to produce repetitive discharges, causing paralysis (known as insect ‘knockdown’) and possible insect death (Ray, Fry, 2006; Clark, Garcia, 2011; Symington, 2012). Compared to pyrethroids, neonicotinoid insecticides are systemic, which means, after treatment, they distribute through all plant parts and thus are particularly effective against sucking insects. Similarly to pyrethroids, the active ingredients in neurotoxic neonicotinoid insecticides affect the central nervous system of insects, by inducing continuous nervous excita- tion at low doses, receptor blockage, cell energy exhaustion, paralysis as well as death at higher doses (Goulson, 2013; Simon-Delso et al., 2015). Unfortunately, non-target, beneficial organisms, such as pollinators, par- asitoids and predatory arthropods are inevitably exposed to insecticide treatment. The routes of exposure to pesticides can be topical, residual or even dietary (Kunkel et al., 2001; Mauchline et al., 2004; van Toor, 2006) and depending on pesticide dose/concentration, the effect can be lethal or sublethal. The sublethal effects are defined as “effects on individuals that survive exposure to a pesticide” (Desneux et al., 2007). In addition to direct mortality, the sub-lethal effects of insecticides can cause changes in their physiology and behaviour (Thompson, 2003; Goulson, 2013, Pisa et al., 2015) negatively affecting their population dynamics. In behavioural ecotoxicology, locomotion reflects the physiological, neu- rological, anatomical and metabolic status of an individual (Baatrup, Bay- ley, 1993, 1998; Bayley, 2002; Desneux et al., 2007). Insecticides affect