bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 1 2 3 “Haplopappus platylepis (Asteraceae) resin: an adhesive trap for pest control of 4 crawling arthropods, with antimicrobial potential” 5 6 Cristian A. Villagra1*¶, Verónica Macias-Marabolí1&, Constanza Schapheer2&, Jorge 7 Bórquez 3&, Mario J. Simirgiotis4&, Javier Echeverría5, 8 Alejandro Urzúa5*¶. 9 1Instituto Marcia González-Teuber5, de Entomología, Universidad Metropolitana de Ciencias de la Educación, 10 Santiago, Chile. 11 2 12 Conservación de la Naturaleza, Universidad de Chile. Av. Santa Rosa 11315, La Pintana, 13 Santiago, Chile 14 3Laboratorio 15 Básicas, Universidad de Antofagasta, Chile. 16 4Instituto 17 5Laboratorio 18 Santiago de Chile, Chile. Laboratorio de Sistemática y Evolución de Plantas, Departamento de Silvicultura y de Productos Naturales, Departamento de Química, Facultad de Ciencias de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Chile. de Química Ecológica, Facultad de Química y Biología, Universidad de 19 20 *Corresponding authors 21 cristian.villagra@umce.cl, alejandro.urzua@usach.cl 22 ¶ These authors are Joint Senior Authors 23 & These authors contributed equally to this work 1 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 24 2 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 26 Abstract 27 The use of plant secondary metabolites has been incorporated as key part of integrated 28 pest management and as an alternative to the use of pesticides. This may even be more 29 relevant regarding domiciliary pest insects, capable of vectoring pathogens to humans. In 30 these environments control its more difficult due to its possible effect on non-target 31 organisms and human health. Here we evaluated the use of the resinous exudate of 32 Chile’s endemic bush Haplopappus platylepis (Asteraceae) as a sticky trap for crawling 33 pest insects. We used Blatta orientalis Linneus (oriental cockroach), a cosmopolitan 34 synanthropic pest, as test organism. We compared effectiveness on cockroach-trapping of 35 H. platylepis’ resin versus a commercially available sticky trap, and analyzed these two 36 sticky substances using UHPLC-DAD-MS and GC-MS. We found that H. platylepis 37 resin was as effective as the commercial adhesive on trapping B. orientalis. Plant 38 resinous exudate was composed by a mixture of flavonoids, labdane diterpenoids and 39 unsatured fatty acids oxylipins, which are known for their antimicrobial and antioxidant 40 properties. In contrast, the commercial sticky trap was rich in 1-bromohexadecane and 2- 41 clorociclohexanol, which have been described as allergens and as potentially toxic to 42 humans. Considering these findings, we suggest the use of the resinous extract of H. 43 platylepis as an effective adhesive trapping method against pest cockroaches and possibly 44 other crawling synanthropic arthropods cohabiting with humans. We highlight the 45 importance of novel, non-toxic and eco-friendly products as strategies to be applied in the 46 management of insect pests. 47 Keywords: synanthropic pest, integrated pest management, labdane terpenoids 48 antimicrobial properties. 3 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 49 Introduction 50 Synthetic insecticides are controversial as they may represent a potential risk for human 51 health and non-target organisms, beside its contribution to air and soil pollution [1–3]. 52 Furthermore, controlling effects on pests can be rapidly ameliorated due to the evolution 53 of resistance on target organisms [4–6]. This is especially concerning in the case of 54 synanthropic arthropods related to vector-borne and zoonotic diseases inhabiting 55 household, food storage facilities and hospitals [7,8]. These pests are hard to control due 56 to their proximity to human-used spaces, restricting even more the use of various 57 chemical control methods [9,10]. 58 This is the case of several crawling pest arthropods including arachnids such as ticks 59 [11,12], and insects belonging to: Hemiptera, like bedbugs [13] and triatomines [14] and 60 Blattodea: such as pest cockroaches [15,16]. Synanthropic cockroaches [17] such as 61 Periplaneta americana (Blattidae), Blattella germanica (Ectobiidae) and Blatta orientalis 62 (Blattidae) have evolved associated to human-modified environments and usually act as 63 vectors of allergens and diverse pathogenous microorganisms responsible for human 64 diseases [18–21]. Thus, these insects represent a serious threat for human health [22]. 65 66 The use of insecticides for the control of these insects has been extremely 67 difficult, as cockroaches may become resistant to commonly-used chemical compounds 68 [6]. Moreover, many insecticides at sublethal doses, are repellent to cockroaches and they 69 are capable to avoid its contact [23]. In addition, some studies have shown that the use of 70 pesticide against cockroach infestation paradoxically increases the level of the cockroach 71 allergens Bla g 1 and Bla g 2, and possibly other allergens [24,25]. For example, adults of 4 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 72 B. germanica exposed to sub-lethal doses of the pesticide boric acid increase the 73 production of the major allergen of Bla g 2 [25], which can lead to significant health 74 problems, including asthma, eczemas skin reactions and allergic rhinitis [26]. 75 Furthermore, it has been demonstrated the evolution of antibiotic resistance in pathogenic 76 strains carried by P. americana and B. germanica collected from domiciliary and 77 intensive care hospital facilities [27–30]. 78 Therefore, in order to avoid the development of resistances either in the animal or 79 their microbial counterparts, control strategies must combine the suppression of both 80 crawling arthropod vectors and its associated pathogens. This approach must also 81 consider current concerns on the safe use of pesticides for controlling difficult insect 82 pests, especially regarding inhabited and food storaging places [31,32]. In this work we 83 studied the chemical composition of the resinous exudate of a Chilean endemic shrub 84 Haplopappus platylepis Phil. (Asteraceae), focusing with particular interest on the 85 presence of antimicrobial potential compounds. Coupled with this, we studied if adhesive 86 extracts of this secretion can be used for the control of pest crawling arthropods, testing 87 its adhesive function against the cosmopolitan pest cockroach Blatta orientalis Linnaeus, 88 1758 (Blattodea: Blattidae). 89 90 The use of plant-derived substances, capable of repelling and/or killing 91 synanthropic pests, has been shown in several studies as an effective alternative to 92 insecticides [33–35]. Among these, plant resins have demonstrated to be effective not 93 only against several arthropods [36], but also in the combat against pathogenic 94 microorganisms [37,38]. Moreover, the use of sticky traps could represent a more 5 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 95 restrictible pesticide format in comparison with air-borne product, where spray drift 96 unwanted consequences on human health have been reported [39]. 97 In addition, adhesive traps can be displayed in refuge areas where airborne products can 98 not easily reach [40], and reduce pest insects mechanically by catching them [41]. 99 Moreover, these collected insects allow pest density monitoring [42]. This latter is a 100 guide during decision-making for the most appropriate control measurement [43]. 101 Considering the above-mentioned information, adhesive plant secretions such as resinous 102 extractions may arise as suitable candidate for safe pest control of house pest and 103 zoonotic vector insect [44]. 104 Haplopappus platylepis, also known as “Devil’s Lollipop”, produces an adhesive 105 resinous secretion covering its leaves and forming a natural sticky trap over floral buds 106 [45]. This plant belongs to an asteraceous lineage presenting copious resin production 107 with known antibacterial and antifungal properties, widely distributed in north and central 108 Chile [38,46–48]. Previously, under field conditions, we showed that H. platylepis’ sticky 109 exudate was capable of trapping several groups of insects that were fatally adhered 110 during its blooming season [45]. In this study, we evaluated the potential use of H. 111 platylepis inflorescence’s sticky exudate as an alternative adhesive trap for pest crawling 112 insects. For these propose we tested it, in laboratory bioassays, on a common global 113 household pest: the oriental cockroach B. orientalis. We compared its effectiveness on 114 adhering pest cockroaches in relation to a commercial adhesive trap (Eco-opción®). In 115 addition, we analyzed and compared the chemical composition of the sticky exudate of 116 H. platylepis and the commercial adhesive trap using UHPLC-DAD-MS (ultra-high- 117 performance liquid chromatography-diode array detector- mass spectrometry) and GC- 6 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 118 MS (gas chromatography-mass spectrometry). Finally, we reviewed for bioactivity of 119 compounds detected in both natural and commercial adhesives, in order to assess both 120 their potential toxicity and harmful effects for humans, as well as any additional 121 biological properties, especially focusing against pathogenic microorganisms. 122 123 Materials and methods 124 Plant material and trap extractions 125 Plant specimens of Haplopappus platylepis Phil. (Asteraceae) were determined following 126 Klingenberg’s monography for Haplopappus genus [49]. Floral buds of devil’s lollypop 127 were collected during March 2016 at Los Molles, Provincia de Petorca, V Region de 128 Valparaíso, Chile (32°14'07.0"S71°31'24"W) and at Punta Hueso, Pichidangui, Provincia 129 de Choapa, IV Region de Coquimbo, Chile (32°10'27"S 71°31'21"W). Samples were 130 preserved until analysis at -10° C. Voucher specimens (SGO 166498) were deposited in 131 the Herbarium of the “Museo Nacional de Historia Natural” (MCCN), Santiago, Chile. 132 The sticky exudate of H. platylepis was obtained by dipping fresh plant material (300 133 g) in cold CH2Cl2 (8 L) for 48 h, following Urzúa 2004's method[50]. The resulting 134 extract was filtered through a cotton layer and concentrated to a sticky residue (36 g, 135 12%) Commercial adhesive trap used was Eco-Opción® (Anasac Corporation, Santiago, 136 Chile), sticky trap offered for the control of cursorial domiciliary pest such as ants, 137 cockroaches and spiders. Each unit brings four 29.6x23.3 cm cardboard sticky traps with 138 a total adhesive surface of 11x13 cm. The adhesive mixture from the cardboard was 139 removed with a spatula and followed above-mentioned procedure for extraction. Extracts 7 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 140 of both natural (H. platylepis inflorescence’s resin) and commercial sticky traps were 141 kept under 4°C for further chemical analyses (see below). 142 143 Insects 144 Oriental cockroaches used in this work were obtained from a population maintained in 145 our laboratory since year 2014. Further specimens used for this study were collected 146 from locations in San Miguel, Santiago, Metropolitan Region, Chile (33°29'54''S 147 70°38'42''W). For taxonomic identification a general key for cosmopolitan and pest 148 cockroaches present in Chile was used [51]. Insects were kept in captivity under 149 laboratory conditions (20°-25°C and 40%-50% humidity) in 120x50x15 cm plastic 150 rearing boxes, fed with dog food (MasterDog Adult ®) and water ad libitum, at Instituto 151 de Entomología, UMCE. Blatta orientalis from both sexes were used for sticky-trapping 152 bioassays (with body lengths among 5 to 25 mm, measured dorsally from head to last 153 abdominal segments). 154 155 Trapping bioassays 156 Two treatments and one control were defined for the experiment. Treatments 157 corresponded to cardboard surfaces (40x13cm) painted either with H. platylepis resinous 158 exudate or with the commercial trap’s adhesive. For control, a cardboard surface 159 (40x13cm) with no adhesive mixture added was used. Each of these options was 160 presented individually in the experimental arena. For this, the cardboard section was 161 placed in the center of the horizontal space inside the arena, fixing its position with 162 double-contact tape (Fig. 1). For each replicate 10 individuals from different sizes 8 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 163 (measured as explained above) were placed in the experimental arena habituation area 164 (Fig. 1), a subdivision of the box from where insect were released without contact them 165 directly. For each trial we lifted the opening section of the habituation area and gave light 166 pulses (10s) during three instances of the experiment: 0, 180 and 360s. At each of these 167 pulses cockroaches tended to leave the habituation area and run to the other extreme of 168 the box crossing the cardboard section. Total time of each test was 6min. After this 169 period, for each treatment and control the number of individuals found attached to the 170 cardboard was counted. Trapped insects were ultimately sacrificed by applying cold 171 temperature (-10 ºC). For each of these alternatives we repeated this test 10 times. Before 172 using the experimental arena for each trial, this was cleaned with ethanol (95%), distilled 173 water and dried in order to remove any chemical cue. The response variable was the 174 proportion of insects trapped in each trial for each treatment. As data did not meet the 175 criterion of normality distribution (Hammer, 1999), it was analyzed with a non- 176 parametric analysis of variance Kruskal-Wallis followed by post hoc Mann Whitney test. 177 In order to determine if H. platylepis inflorescence’s resin and the commercial sticky trap 178 are equally efficient trapping cockroaches of different sizes (seven ranges: from 5 to 7; 8 179 to 10; 11 to 13; 14 to 16; 17 a 19; 20 to 22 and 23 to 25mm), insect proportion per range, 180 captured in both traps, was compared. This was analyzed by using a Chi square test for 181 two proportions [53]. All analyses were done with the PAST Paleontological Statistic, 182 version 3.15. 183 184 Fig. 1. Bioassay setup A. Experimental arena: a. Background pattern. b. Treatment Area, 185 c. Darkened walls, d: Hatch e: Habituation cubicle. f: Arena’s door. B. Sticky trap made 9 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 186 with H. platylepis resin (upper picture) and Eco-opción® adhesive (lower picture). 187 Trapped roaches are highlighted with arrows. 10 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 189 Chemicals 190 UHPLC-MS solvents, LC-MS formic acid and reagent grade chloroform were from 191 Merck (Santiago, Chile). Ultrapure water was obtained from a Millipore water 192 purification system (Milli-Q Merck Millipore, Chile). HPLC standards, (kaempferol, 193 quercetin, isorhamnetin, eriodictyol, luteolin, apigenin, naringenin, all standards with 194 purity higher than 95 % by HPLC) were purchased either from Sigma Aldrich (Saint 195 Louis, Mo, USA), ChromaDex (Santa Ana, CA, USA), or Extrasynthèse (Genay, 196 France). 197 198 UHPLC-DAD-MS analyses 199 Chemical resinous components were analyzed by using ultra-high-performance liquid 200 chromatography-diode array detector-tandem mass spectrometry (UHPLC-DAD-MS). 201 UHPLC-DAD-MS analysis was performed using a Thermo Scientific Dionex Ultimate 202 3000 UHPLC system hyphenated with a Thermo Q exactive focus machine as it was 203 reported by Simirgiotis et al. (2016). 5 mg of the resinous exudate were dissolved in 2 204 mL of methanol and filtered with a PTFE filter for a final injection of 10 µL into the 205 instrument. Measurements were done as previously reported by Simirgiotis et al. (2016). 206 The generation of molecular formulas was performed using high resolution accurate mass 207 analysis (HRAM) and matching with the isotopic pattern. Lastly, analyses were 208 confirmed using MS/MS data and comparing the fragments found with the literature. 209 210 LC and MS parameters 11 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 211 Liquid chromatography was performed using an UHPLC C18 column (Acclaim, 150 mm 212 × 4.6 mm ID, 2.5 µm, Thermo Fisher Scientific, Bremen, Germany) operated at 25 ◦C. 213 The detection wavelengths were 254, 280, 330 and 354 nm, and DAD was recorded from 214 200 to 800 nm for peak characterization. Mobile phases were 1 % formic aqueous 215 solution (A) and acetonitrile (B). The gradient program time (min, % B) was: (0.00, 5); 216 (5.00, 5); (10.00, 30); (15.00, 30); (20.00, 70); (25.00, 70); (35.00, 5) and 12 minutes for 217 column equilibration before each injection. The flow rate was 1.00 mL min−1, and the 218 injection volume was 10 µL. Standards and the resin extract dissolved in methanol were 219 kept at 10◦C during storage in the autosampler. The HESI II and Orbitrap spectrometer 220 parameters were optimized as previously reported [54]. 221 222 GC-MS analyses 223 Chemical composition of the commercial adhesive trap was analyzed by gas 224 chromatography-mass spectrometry (GC-MS). GC-MS analysis was performed using a 225 Thermo Scientific Trace GC Ultra linked to an ISQ quadrupole mass spectrometric 226 detector with an integrated data system (Xcalibur 2.0, Thermo Fisher Scientific Inc., 227 Waltham, MA, USA), equipped with a capillary column (Rtx-5 MS, film thickness 0.25 228 μm, 60 x 0.25 mm, Restek Corporation, Bellefonte, PA, USA) The operating conditions 229 were as follows: on-column injection; injector temperature, 250 °C; detector temperature, 230 280 °C; carrier gas, He at 1.25 mL/min; oven temperature program: 40 °C increase to 260 231 °C at 4 °C/min, and then 260 °C for 5 min. The mass spectra were obtained at an 232 ionization voltage of 70 eV. Recording conditions employed a scan time of 1.5 s and a 233 mass range of 40 to 400 amu. The identification of compounds in the chromatographic 12 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 234 profiles was achieved by comparison of their mass spectra with a library database 235 (NIST08, NIST, Gaithersburg, MD, USA) and by comparison of their calculated 236 retention indices with those reported in the literature [55] for the same type of column. 237 238 Results 239 Trapping bioassays 240 The proportion of insects found over the cardboards was statistically different among 241 treatments (H (X2) = 19.43, p < 0.001, Kruskal-Wallis, Fig. 2A). H. platylepis 242 inflorescence’s sticky exudate and the commercial sticky trap differed with statistical 243 significance from control clean cardboard (in both cases: U Mann-Whitney pairwise, p < 244 0.001). However, no differences were found in post hoc test for the total number of 245 insects attached on cardboards between the H. platylepis’ resin and the commercial sticky 246 trap (U Mann-Whitney pairwise, p = 0.691). When the proportion of cockroaches trapped 247 by H. platylepis’ sticky exudate and by the commercial sticky trap for each size range 248 was compared, no statistical differences were found between natural and commercial 249 sticky traps (X2 = 1.57, p = 0.211) (Fig. 2B). 250 251 Fig. 2. Cockroach adhesion results. A. Mean and 1SE for the proportion of B. orientalis 252 found over the cardboard (Y axis) painted with: H. platylepis resin (green), Eco-opción® 253 commercial adhesive (red) and control (clean cardboard, black) obtained from 10 254 replicates each (X axis). Different letters correspond to statistical differences after post 255 hoc test at p < 0,05. B. Proportion of cockroaches trapped (Y axis) by either H. platylepis 256 resin (light grey) or Eco-opción® commercial adhesive (dark grey) for each insect size 13 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 257 range (X axis). No statistical differences were found for each pair compared. 258 259 260 261 262 Chemical analyses 263 The data-dependent scan experiment was very useful for the identification of unknown 264 compounds since it provides high resolution and accurate mass product ion spectra from 265 precursor ions that are unknown beforehand within a single run. Combining data- 266 dependent scans and MSn experiments, phytochemicals were tentatively identified in H. 267 platylepis including simple phenolic acids flavones, flavanones, fatty acids, and labdane 268 diterpenoids. UHPLC Q-orbitrap mass spectrometry analysis of H. platylepis sticky 269 exudate showed the presence of twenty seven metabolites in the chromatograms (Fig. 3) 270 including: 7 flavonoids (peaks 5, 6, 8-10, 15 and 16), 3 phenolic acids (peaks 1-3), 8 fatty 271 acids (Peaks 4, 7, 13, 14, 18, 21, 22 and 25), and 9 labdane terpenoids (peaks 11, 12, 17, 272 19,20, 23, 24, 26, and 27). The detailed identification is explained below (Table 1, Figs. 4 273 and 1S). 274 Fig. 3: UHPLC chromatograms A. TIC (total ion current, negative mode) and B. UV at 275 280 nm, of H. platylepis resin. 276 Fig. 4: Proposed biogenetic relationships between labdane diterpenoids. 277 14 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 278 Table 1: High resolution UHPLC PDA-Q-orbitrap identification of metabolites in 279 Haplopappus platylepis resin. Peak # Retention UV max time (nm) (min) Tentative identification Elemental composition [M-H] Theoretical mass (m/z) Measured mass (m/z) Accuracy (δppm) MSn ions (δppm) 1 11.43 - 12-Hydroxyjasmonate C12H17O4- 225.11276 225.11313 4.27 2 12.93 - Dihydroxyphaseic acid C15H21O5- 281.13953 281.13945 -0.28 3 13.71 325 Ferulic acid C10H9O4- 193.05063 193.05040 -1.19 4 18.76 285 Trihydroxyoctadecaenoic acid C18H33O5- 329.23335 329.23367 0.97 5 19.05 255, 354 7,3'-dimethoxyquercetin C17H13O7- 329.06668 329.06702 1.03 6 19.26 287 Hesperetin C16H13O6- 301.07176 301.07199 0.76 7 19.38 285 Trihydroxyoctadecadienoic acid C18H31O5- 327.21770 327.21799 0.89 8 19.56 287 5,3’5’-trihydroxy-3,7,4’trimethoxyflavanone C17H15O7- 331.08261 331.08233 1.22 9 20.02 255-354 5,3’-dihydroxy-3,7,4’trimethoxyflavone C18H15O8- 343.08233 343.08267 1.25 313.03580 (C16H9O7-, [M-OCH3CH3] 10 20.04 255-354 7, 3’, 5’trimethoxymyricetin C18H15O8- 359.07724 359.07748 0.58 285.04031 (C15H9O6-, kaempferol) 11 20.07 289 Dehydropinifolic acid C20H33O4- 337.23843 337.23886 1.28 12 20.10 289 Pinifolic acid (labd-8(20)en-15,18-dioic acid) C20H31O4- 335.22278 335.22287 0.98 13 21.13 305 Trihydroxyheneicosahexaen oic acid C21H29O5- 361.20205 361.20242 1.02 14 21.36 303 dihydroxyeicosapentaenoic acid C20H29O4- 333.20713 333.20740 0.90 15 21.71 255-354 3,7-dimethoxyquercetin C17H13O7- 329.06668 329.06705 1.12 16 21.96 255-354 3,5, dihydroxy-3’,4’,7trimethoxyflavone C18H15O8- 343.08233 343.08273 1.17 15 160.84154, 135.04446 273.18622 (C18H25O2-); [M- - (CO2 CH3 - H)] 313.03467 (C16H9O7-, (M- -OCH3 CH3) bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 17 22.12 - (epi) Pinifolic acid C20H31O4- 335.22278 335.22287 0.54 317.21219 (C20H29O3- ; [M- - H2O]; 273.18652 (C18H25O2-) 18 22.87 302 Tetrahydroxytetracohexaeno ic acid C24H35O6- 419.24423 419.24391 3.37 319.22806 19 22.92 289 18-hydroxy-8(17)en-15labdanoic acid C20H33O3- 321.24377 321.24377 0.00 20 23.94 289 Dehydropinifolic acid isomer C20H33O4- 337.23843 337.23886 1.28 21 24.25 308 Hydroxyeicosapentaenoic acid C20H29O3- 317.21222 317.21255 1.04 22 22.87 303 Hydroxyeicosatetraenoic acid C20H31O3- 319.22787 319.22821 1.07 23 25.40 289 13-en-Pinifolic acid methyl ester C21H31O4- 347.22278 347.22311 1.04 24 25.78 289 Pinifolic acid methyl ester C21H33O4- 349.23843 349.23880 1.06 25 25.88 306 Trihydroxydocosahexaenoic acid C22H31O5- 375.21770 375.21823 1.41 26 25.99 289 18-acetyl-13,8 (17)dien-15labdanoic acid C22H33O4- 361.23843 361.23877 0.94 27 26.56 289 18-acetyl-8(17)en-15labdanoic acid C22H35O4- 363.25408 363.25443 1.13 273.18616 (C18H25O2-); 239.26134 (C16H31O-) 321.24319 (C18H25O2-; M- - H2O) 280 281 Flavonoids 282 Peak 15 with a [M-H]- ion at m/z 329.06705 was identified 3,7-dimethoxyquercetin 283 (C17H13O7-) and peak 5 with an ion at m/z 329.06702 as its isomer: 7,3'- 284 dimethoxyquercetin (Table 1). Peak 9 with a [M-H]- ion at m/z 343.08276 was identified 285 as the trimethoxylated flavonoid 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (C18H15O8-), 286 while peak 10 with a [M-H]- ion at m/z 359.07745 as 7,3’,5’-trimethoxymyricetin 287 (C18H15O8-). Peak 16 with a pseudomolecular ion at m/z 343.08273 was identified as 3,5- 288 dihydroxy-3’,4’,7-trimethoxyflavone (C18H15O8-). The flavanone hesperetin, peak 6, have 16 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 289 been previously reported as main component in extracts of several Nolana species by 290 some of us (Simirgiotis, et al., 2015) and its HR-MS (C16H13O6-) and UV data matched 291 the one obtained in our chromatograms (m/z: 301.07176). Another flavanone, peak 8 with 292 a [M-H]- ion at m/z 331.08261 was identified as 5,3’,5’-trihydroxy-3,7,4’- 293 trimethoxyflavanone (C17H15O7-). 294 295 Phenolic acids 296 The examination of the chromatograms revealed the presence of 3 phenolic acids: 297 dihydroxyphaseic acid (peak 2, ion at m/z 281.13945, C15H21O5-) [56], ferulic acid (peak 298 1, m/z 193.05040) and 12-hydroxy jasmonate (peak 3, m/z 225.11313) [57]. 299 300 Fatty acids 301 Several peaks were tentatively identified as the dietary antioxidant polyhydroxylated 302 unsaturated fatty acids known as oxylipins [58,59], antioxidant fatty acids. Peak 4 with a 303 [M-H]- ion at m/z 329.23367 was identified as trihydroxy-octadecenoic acid (C18H33O5-), 304 and peak 7 as its diene derivative (C18H31O5-), as previously reported by some of us from 305 Keule fruits [59]. Peak 13 with a pseudomolecular ion at m/z 361.20242 was identified as 306 trihydroxyheneicosahexaenoic acid (C21H29O5-). Peak 14 with a [M-H]- ion at m/z 307 333.20743 was identified as a dihydroxyeicosapentaenoic acid (C20H29O4-) while peak 18 308 with a [M-H]- ion at m/z 419.24391 was identified as dihydroxytetracosatrienoic acid 309 (C24H35O6-) [58]. Peak 21 and 22 were identified as hydroxyeicosapentaenoic acid and 310 hydroxyeicosatetraenoic acid (C20H29O3-) and (C20H31O3-), respectively. Finally, peak 25 17 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 311 with a [M-H]- ion at m/z 375.21823 was identified as trihydroxydocosahexaenoic acid 312 (C22H31O5-). 313 314 Labdane terpenoids 315 Labdane terpenoids corresponded to derivatives of pinifolic acid (labd-8(20)-en-15,18- 316 dioic acid, peak 12, C20H36O3) [60] most of them reported for the first time in this 317 species. Thus, peak 11 with a [M-H]- ion at m/z 337.23886 was identified as its 318 hydrogenated derivative of dehydropinifolic acid (C20H33O4-) and peak 17 with a [M-H]- 319 ion at m/z 335.22296 as an isomer of pinifolic acid (C20H31O4-), probably the epimer at C- 320 4 of the latter. Peak 24 was identified as pinifolic acid methyl ester (C21H33O4-) and peak 321 23 as its derivative 13-en-pinifolic acid methyl ester (C21H31O4-). Peak 20 with a [M-H]- 322 ion at m/z 337,23886 was identified as pinifolic acid derivative (C20H33O4-). Three 323 compounds were identified as labdanoic acid derivatives [61]. Thus, peak 19 with a [M- 324 H]- ion at m/z 321.24377 was identified as 18-hydroxy-8(17)en-15-labdanoic acid 325 (C20H33O3-), Peak 27 with a [M-H]- ion at m/z 363.25449 was identified as 18-acetyl- 326 8(17)en-15-labdanoic acid (C22H35O4-) and peak 26 as its diene derivative (C22H33O4-) 327 (Fig. 4). 328 329 Components identified in the commercial sticky trap 330 GC-MS identified only two compound in the commercial sticky trap as: 1- 331 bromohexadecane and 2-chlorocyclohexanol. 332 333 Discussion 18 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 334 The aim of this study was to compare the effectiveness of a natural sticky trap against a 335 commercial one in capturing cockroaches by adhesion. In addition, the chemical 336 composition of both traps was analyzed in order to estimate potential harmful effects for 337 humans as well as potential antimicrobial chemical compounds. Our results provide 338 evidence that the natural sticky trap of H. platylepis was as effective as the commercial 339 one on trapping pest cockroaches. Considerable differences, however, were found in the 340 chemical composition between the natural and the commercial trap. Whereas the former 341 was rich in plant-derived antimicrobial compounds, the latter was rich in halogenated 342 compounds, whose potential toxic effects for humans have been previously reported. 343 The H. platylepis sticky exudate seems to offer multiple benefits in relation to its 344 use for controlling synanthropic pest crawling insect, such as cockroaches. First, because 345 of its stickiness, it resulted as effective as the commercial trap for capturing cursorial 346 insects, and second, due to its chemical composition rich in antibacterial compounds [62], 347 it shows a further potential for controlling pest arthropod-borne transmitted pathogens. 348 As far as we know, most of the compounds identified for H. platylepis resin are reported 349 for the first time in this species. Antibacterial properties of H. platylepis sticky exudate 350 can be associated with the phytochemical families detected in the mixture [62]. For 351 instance, flavonoids have shown a wide-sprectrum of inhibitory activity against a variety 352 of human pathogens, including antibiotic-resistant Gram-positive and Gram-negative 353 bacteria, viruses and fungus [62–66]. Labdane diterpenoids are also well known as 354 antimicrobials [67,68]. It has been proved that the presence of a carboxylic acid in the C- 355 15 position, which acted as a hydrogen-bond donor (HBD), is essential for the 356 antibacterial activity of ent-labdanes [64]. Furthermore, derivatives of pinifolic acid, 19 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 357 which were characterized in the H. platylepis sticky exudate, showed this main structural 358 characteristic of labdanes. In addition, pinifolic acid has been previously reported as an 359 effective compound in the treatment of leishmaniasis [69], a global insect-borne disease 360 related to trypanosomes [70]. Long-chain polyunsaturated fatty acids, which were also 361 abundant in H. platylepis resin, including oxylipins, have been widely tested for its 362 antimicrobial activity [71–75]. Therefore, further functions of chemical compounds 363 found in H. platylepis’ resinous exudate expand the potential value of this plant-derived 364 adhesive to act as a control against various vectoring-disease scenarios. 365 Synanthropic crawling arthropods are usual carriers of several human pathogens 366 [76]. In the case of B. orientalis, it has been described to bear several human pathogenic 367 bacteria genera such as Mycobacteria, Klebsiella, Staphylococcus, Escherichia and 368 Enterobacter [77,78]. Therefore, the occurrence of compounds with anti-microbial 369 functions in the sticky exudate of H. platylepis may synergistically contribute as an 370 integrative pest control method, not only directly affecting the insect pests but also its 371 associated pathogenic microorganisms. The commercial sticky trap, in contrast, is poor in 372 its chemical composition and lacks antimicrobial compounds. 1-Bromohexadecane (1) 373 and 2-chlorocyclohexanol (2) were the only two compounds identified on the commercial 374 trap. Both are known as halogenated compounds. Based on Globally Harmonized System 375 of Classification and Labeling of Chemicals (GHS), both are characterized as irritant for 376 humans, due to the fact that these compounds induce skin corrosion (category 2), 377 respiratory tract irritation (category 3) as well as severe eye irritation (category 2A) 378 (European Chemical Agency- ECHA, 2017). This chemical profile suggests that this 379 commercial trap would not be innocuous for human health; nevertheless, it is 20 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 380 commercially offered as an eco-friendly option. Our results highly suggest that H. 381 platylepis sticky exudate may be a suitable alternative for controlling synanthropic 382 crawling insects, including cockroaches, at low cost and with additional benefits such as 383 potential antimicrobial properties. These virtues of H. platylepis sticky exudate trap fit 384 the current needs and trends in pest control, where several methodologies must be 385 integrated in order to generate novel alternatives in consideration of human and 386 environmental health [79]. Further research is needed in order to test this adhesive resin 387 in other formats for insect trapping as well as to evaluate its effectiveness against other 388 pest insects. For instance, resinous materials have been considered among the updated 389 alternatives for controlling domiciliary termites [44]. 390 391 Conclusions 392 Results here demonstrated that devil’s lollypop resin is a natural source of terpenoids and 393 flavonoids with potential applications as insecticide and antibacterial. Using UHPLC- 394 DAD-MS we have identified 27 secondary metabolites in H. platylepis’ resin. Most of 395 which, as far as we know, are reported here for the first time. Many of these compounds 396 are flavones, flavanones, phenolic acids, fatty acids, and labdane terpenoids. This 397 chemical knowledge may be helpful for further research on H. platylepis and its 398 applications in biomedicine and pest and pathogens control industry. In conclusion, this 399 plant is a rich source of phenolic and clerodane compounds with insecticide and 400 antibacterial activity that may be used as an effective biocontrol agent against zoonotic 401 crawling insects and their associate microorganisms . 402 21 bioRxiv preprint doi: https://doi.org/10.1101/328237. this version posted May 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Haplopappus platylepis resin for pest control 403 Supporting Information 404 Fig. A.1: Full HR-MS spectra and structures of compounds 3 (a), 9 (b), 10 (c), 12 (d), 14 405 (e), 22 (f), 23 (g), 26 (h) and 27 (i). 406 407 Acknowledgments 408 We thank Catherine Cabello and Angel Olguín for help during fieldwork and laboratory 409 work. 410 411 Funding 412 This research was funded by FONDECYT Iniciación No. 11100109 and CONICYT 413 Inserción No. 79100013 granted to Cristian Villagra, RSG N° 21286-2 to Constanza 414 Schapheer, Proyecto Fortalecimiento USACH USA1799_UA253010, Universidad de 415 Santiago de Chile granted to Alejandro Urzúa, Javier Echeverria and Marcia Gonzalez, 416 and CONICYT PAI/ACADEMIA No. 79160109 to Javier Echeverria. 417 418 419 References 420 1. Nicolopoulou-Stamati P, Maipas S, Kotampasi C, Stamatis P, Hens L. Chemical 421 Pesticides and Human Health: The Urgent Need for a New Concept in Agriculture. 422 Front Public Heal. 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