Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: Laboratory and Field Evaluation

Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: Laboratory and Field Evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: Laboratory and Field Evaluation Gustavo Frensch, Scheila R. M. Zaleski, Renan R. Schorr, Liliane G. Dantas, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1696409/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2023 Read the published version in Chemoecology → Version 1 posted 5 You are reading this latest preprint version Abstract Coniferous trees of the genus Pinus (Pinaceae) are under continuous threats by numerous herbivorous insect species and pathogens attacking nearly all parts and tissues of the plants. To defend themselves, pine trees produce large amounts of oleoresin that is accumulated in a highly developed network of specialized resin ducts, which are distributed in the wood, bark, and needles. Such defense reactions in pines can be induced by the attack of herbivores. The banded pine weevil, Pissodes castaneus (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of Pinus in Brazil, where it has been an invasive species since 2001. The female lays its eggs under the tree bark of trees and the larvae feed in the phloem of the trunk and branches, interrupting the sap circulation and eventually causing its death. In the present study, we conducted detailed GC–MS analyses of volatiles emitted by twigs of Pinus taeda L . We analyzed how the attack by P. castaneus males and females affects the volatile pattern emitted by the twigs. When comparing volatiles produced by healthy plants and by female- and male-attacked P. taeda , qualitative and quantitative differences were detected, as the decreased production of limonene, germacrene D and ( E )-caryophyllene and the increase of α-pinene. Laboratory bioassays showed that plants attacked by male and female P. castaneus were more attractive to the insects. Understanding about what compounds may attract or repel the insects may help in the development of more effective traps, as well as preventing stress to avoid infestation. banded pine weevil induced volatiles infochemicals loblolly pine Figures Figure 1 Figure 2 Figure 3 1. Introduction The banded pine weevil, Pissodes castaneus (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of Pinus (Pinaceae) in South America (Iede et al. 2004 ). It was detected in Brazil in 2001 in the county of São José dos Ausentes, Rio Grande do Sul, on Pinus taeda L and later it was detected in the states of Santa Catarina and Paraná (Iede et al. 2004 ). In 2002, 7.6% of the trees of a plantation of P. taeda grown in Cambará do Sul, Rio Grande do Sul, were attacked by P. castaneus while in São Joaquim, Santa Catarina, 16.5% of the trees were attacked (Iede et al. 2004 ). This insect represents a threat to Brazilian forest production, as it has the potential to cause economic losses, such as those that occurred in Uruguay where there was a mortality rate higher than 10% (Grez et al. 2000). More than 50% of trees in a stand can be attacked in one year (Cadahia et al. 1992 ). Conifers are long lived gymnosperms, which includes many species that successfully inhabit large areas of our planet. As raw material for many products (wood, paper, plastic, fuel, and many chemicals), their economic impact on our society is of great importance. In European countries such as Sweden, 83% of the forests consist of conifers, mainly Norway spruce ( Picea abies L.) and Scots pine ( Pinus sylvestris L.) (Alin and Sundberg 2003 ). In South America, specifically in Brazil, pine trees, mainly P. taeda and Pinus elliottii Engelm., have been planted on commercial scale for over 30 years (Ahrens 2000 ). Currently, there are about 2 million hectares of reforested pine plantations, in large continuous areas and generally in narrow genetic base stands, mainly in the South and Southeast regions (Iede et al. 2004 ). The product of these plantations is destined mostly to the timber and cellulose industries (Cardoso and Lázzari 2003 ). Insects use volatiles as their major method to find food, mates or an appropriate site to lay their eggs (El-Shafie and Faleiro 2017 ). For plants, which cannot escape threats, chemical constituents play an important role in their defense system. Their response to feeding or oviposition of herbivorous insects can be direct, by feeding deterrents, impairing digestion or producing toxins (Kessler and Baldwin 2002 ), or indirect, by the release of volatiles which attract natural enemies of the herbivorous insects (Dicke and van Loon 2000 ; Arimura and Pearse 2017 ). However, there are cases that the induced compounds may act as kairomones (Nordlund and Lewis 1976 ; Keeling Christopher et al. 2006 ; Lusebrink et al. 2016 ; McCormick et al. 2016 ), i.e. chemical compounds emitted by an organism that induce a benefic response to the receiving organism from another species, or have a synergistic effect with insect pheromones – compounds used in intraspecific communication –, as happens with Pinus ponderosa when damaged by Dendroctonus brevicomis Lec. (Bedard et al. 1969 ). Conifers can use a large array of structurally diverse mono-, sesqui- and diterpenoids as a chemical defense against herbivores (Lewinsohn et al. 1991 ; Phillips and Croteau 1999 ; Trapp and Croteau 2001 ; Martin et al. 2002 , 2003 ). Induction of volatiles terpenoids may result in a quantitative or qualitative difference in the chemical profile produced by a plant (Fitzgerald 2003 ). The herbivore-induced change in the plant volatiles can be specific for the plant and herbivore species, the plant age and the herbivore stage (Takabayashi et al. 1994 ; De Moraes et al. 1998 ). Defense responses of induced terpenoid have been intensively studied using spruce ( Picea spp.) as model plant. Anatomical defenses like traumatic resin duct formation or polyphenolic parenchyma cell activation are induced by wounding or by attacking insects and pathogens (Tomlin et al. 1998 ; Nagy et al. 2000 ; Franceschi et al. 2002 ; McKay et al. 2003 ). Besides, induction of terpenoids have been observed in spruce after mechanical wounding or treatment with methyl jasmonate, a plant hormone known to upregulate plant defenses (Nault and Alfaro 2001 ; Martin et al. 2002 ; Faldt et al. 2003 ). These terpenoids can act as repellents or toxins against those herbivores, defending conifers directly, or mimic juvenile hormones (Lewinsohn et al. 1991 ; Langenheim 2003 ; Mumm et al. 2004 ). Likewise, conifer volatiles can also attract predatory and parasitic insects, consequently defending conifers indirectly against herbivores (Nadir and Raffa 2001 ; Mumm et al. 2003 ; Sullivan and Berisford 2004 ; Hilker et al. 2005 ). In previously studies it was described that P. castaneus adults were attracted to the volatiles emitted by P. taeda (Marques et al. 2012 ) and previously stressed plants showed a higher infestation (Iede et al. 2004 ). In the present study, we conducted detailed GC–MS analyses of volatiles emitted by twigs of P. taeda , both healthy and attacked. We analyzed how the attack by P. castaneus males and females affects the volatile pattern emitted by P. taeda twigs. We also carried out laboratory and field assays to evaluate the attraction of P. castaneus to healthy and stressed host plants. 2. Materials And Methods 2.1. Field evaluation of the attraction of P. castaneus by P. taeda 2.1.1. Characterization of the experimental area The attractiveness experiments were performed in two plantations with density of 1,666 1 year old plants/ha, located in Três Barras (TB), Santa Catarina, Brazil (26°07'41"S, 50°19'30"W, 802m altitude) and Cambará do Sul (CS), Rio Grande do Sul, Brazil (29°02'52"S, 50°08'41"W, 1031m altitude). In each area, the experiment was repeated on three plots, 200m apart from each other. For the experiment’s purpose, plants found in CS were considered stressed due to poor planting sites in flooded areas with poor drainage, whereas plants found in TB were considered healthy. 2.1.2. Evaluation of the attractiveness of P. castaneus to P. taeda log traps To evaluate the attractiveness of P. castaneus to P. taeda , two variables were analyzed: the effect of stressed (CS) x non-stressed (TB) environments, and the effect of seasonal variations through a 12-month experiment. In each plot of each area, log traps composed of 20 freshly cut 1m long and 8–10 cm diameter P. taeda logs were installed every month between July 2012 and June 2013 for adults of P. castaneus to lay eggs in. After 30 days, the logs were removed from the field, new log traps were installed in the same plots, and the removed ones were transported to the laboratory. For each batch of 20 logs, 17 were stored for 1 year in screened cages in a room at 20 o C, humidity on 70 ± 10% and photoperiod of 12h. The logs were monitored weekly to assess the emergence of viable adults. Any emerged adult was removed from cages. The average number of adults emerged in each area per month was obtained by averaging the total number of adults emerged in the three plots in each area using BioEstat 5.0 (Ayres et al. 2007 ). To access the effect of seasonality and location in the attractiveness of the insects, a factorial analysis of variance (ANOVA) was performed using Statistica software (version 8.0; StatSoft Inc, Tulsa, OK, USA). The homogeneity of data was evaluated through a Levene’s test, and a Duncan post-hoc test was performed to evaluate which means were statistically different at 5%, both using Statistica 8.0 software. 2.2. Chemical analysis of the attraction of P. castaneus by P. taeda 2.2.1. Insects For chemical assays, the insect colony was started with specimens collected from log traps installed in Três Barras plantation (26°07'41"S, 50°19'30"W, 802m altitude). To obtain the insects for the bioassays, P. taeda freshly cut logs were placed in the field. Log traps were installed from July 2012 to June 2013. Each log trap was composed of 20 freshly cut 1m long and 8–10 cm diameter pine logs for adults of P. castaneus to lay eggs in. After 30 days, the logs were removed from the field and placed in screened cages measuring 30 cm height by 30 cm diameter until adult emergence. After their emergence, the adults were separated by sex and each group were kept for 5–10 days in an incubator at 22 ± 2°C, 70 ± 10% relative humidity and 12L:12D photoperiod. Females and males were selected for the trials and placed alone in plastic screened cages with ventilation at the sides under the same conditions as described above. Adults were fed a natural diet consisting of pieces of fresh P. taeda branches. The cages were cleaned, and the food supply replaced at regular intervals of five days. 2.2.2. Plants All trees of P. taeda used in this work were 1–2 years old and were obtained from greenhouses near Curitiba, Paraná, Brazil. 2.2.3. Y-tube bioassays Bioassays were conducted with a vertical glass Y tube (2.5 cm ID, bottom arm 20 cm, top arms 20 cm) with ground glass female joints at the end of each arm, and matching male joints terminating in hose nipples. In the system, the speed of humidified, charcoal filtered air was adjusted to 2.0 L/min. All connections were made of Tygon tubing (0.6 cm ID). After each trial, all materials were washed with neutral soap, dipped in 70% ethanol, and placed in the oven at 50 o C for 30 min to avoid any residual volatile compounds. Test insects were introduced individually into the bottom of the Y tube and, after 5 min of acclimation, allowed 20 min to respond. When the insect moved through the arm containing the stimulus (male or female-attacked P. taeda branches), the result was considered positive while the result was negative when it moved through the control arm (healthy P. taeda branches). However, when the insect did not move towards any of the arms within 20 min, it was considered non-responsive. The location of stimulus and control were rotated after each test. The insects used in the tests were given no food 12 h before the bioassays. The branches of P. taeda used as stimuli were collected one day before conducting the bioassays, placed in the thermal box, and transported to the laboratory where they were kept in the refrigerator at 10 ± 2°C until tests. Bioassays were conducted in a temperature- (23 ± 2 o C) and humidity-controlled room (70 ± 10% relative humidity). Each stimulus was tested with 30 sexually mature virgin adult insects of each sex. The data obtained with insects that reached the corresponding odor sources in the Y tube assay were compared by the χ 2 test using BioEstat 5.0 (Ayres et al. 2007 ). Results showing p ≤ 0.05 were considered statistically significant. 2.3. Tentative Identification of healthy and attack-induced compounds produced by P. taeda 2.3.1. Healthy trees Four branches of healthy trees of P. taeda (200g) were aerated in a 1 L glass chamber. Aerations were carried out under controlled conditions at 23 ± 2°C, relative humidity of 70 ± 10% and a photoperiod of 12L:12D. Volatiles were trapped on a 0.4 cm long bed of Super Q resin (Alltech, Deerfield, Illinois, USA) held in place by glass wool plugs in a glass tube (4 mm ID). Volatiles were collected for two days (flow 1.0 L/min), then eluted with hexane (3 x 0.5 mL). Extracts were concentrated as required (c.a. 100 µL) under Argon. All procedures were carried out in triplicate. 2.3.2. P. castaneus attacked trees Saplings of P. taeda (1–2 years old) were placed in plastic screened cages with ventilation at the sides, at 22 ± 2°C, 70 ± 10% relative humidity and 12L:12D photoperiod, with 10 males or 10 females of P. castaneus . After 72 hours of attack, branches of these trees were cut and aerated the same way healthy trees were. 2.3.3. Chemical analysis Extracts and n -alkane standards were analyzed by coupled GC/MS with a Shimadzu CGMS-QP2010 Plus system equipped with a quadrupole detector using a Rtx-5MS (Crossbond 5% diphenyl / 95% dimethyl polysiloxane) low bleeding column (30 m x 0.25 mm x 0.25 µm), using Helium as carrier gas, at flow of 1.02 mL/min. Oven temperature was initially held at 60°C for 1 min and increased at a rate of 3°C/min to 250°C. The mass spectrometer operated in electron impact mode (70 eV) with a mass range set from 40 to 350 m/z. The interface and source temperatures were set at 250°C. Kovats retention index was calculated for the compounds and their mass spectra were compared with the literature (Adams 2007 ). The C 8 H 18 -C 18 H 38 and C 20 H 42 n -alkanes standards were purchased from Aldrich (Deisenhofen, Germany). Compounds detected in at least 2 of 3 samples of the attack-induced pine branches were considered. 3. Results 3.1. Number of P. castaneus that emerged from log traps of P. taeda The average number of adults of P. castaneus that emerged from the log traps differed between the two study sites. In CS, higher average number of insects (196.778 ± 27.552) were collected when compared to TB, where in average 110.556 ± 22.775 insects were collected (Table 1 ). Table 1 Mean number (± SE) of adults of Pissodes castaneus that emerged from the logs trap installed in periods (months) compared within in a Pinus taeda forest in Três Barras (TB), Santa Catarina, Brazil, and Cambará do Sul (CS), RS, Brazil, July/2012 up to June/2013. Period (month) Site CS TB Average nº of insects (n = 3) Significantly distinct months within area (Duncan) Average nº of insects (n = 3) Significantly distinct months within area (Duncan) Jul.12 283 ± 40.427 * ABC 46.333 ± 31.424 * ABC Ago.12 149.667 ± 15.431 DEF 61.333 ± 12.548 DE Set.12 116.333 ± 38.942 AGHI 82.333 ± 35.366 F Out.12 230 ± 79.775 J 240.333 ± 20.497 ADFGHI Nov.12 308.667 ± 17.023 DGKLM 198 ± 68.000 BJKL Dez.12 185 ± 65.957 N 118 ± 40.633 Jan.13 288 ± 33.307 HOP 212 ± 30.238 CEMNO Fev.13 233 ± 83.720 Q 180.333 ± 22.821 PQ Mar.13 304 ± 55.426 * EIRS 106.333 ± 66.520 * Abr.13 0 ± 0 BFJKNOQRT 44 ± 7.234 GJM Mai.13 100 ± 37.005 CLPS 27.333 ± 14.769 HKNP Jun.13 163.667 ± 41.378 * MT 10.333 ± 4.702 * ILOQ Mean 196.778 ± 27.552 110.556 ± 22.775 * at “Average nº of insects” rows indicate mean values that were statistically different at 5% significance level among different areas within the same month. Similar letters at “Duncan” rows indicates that the mean values were statistically different at 5% significance level among different months within the same area Although ANOVA results revealed a correlation between seasonality treatment and mean number of insects obtained (F = 6.618, p < 0.001; Table 2 ), no such correlation was observed when both sites and seasonality were analyzed together (F = 1.763, p = 0.0878; Table 2 ). This can be explained by the differences found in the average number of insects collected per month for each area (Table 1 ). For CS, the lowest number of insects was found during the months of April (0 ± 0) and May (100 ± 37.005), whereas the highest number was during the months of November (308.667 ± 17.023) and March (304 ± 55.426). On the other hand, for TB the lowest values were those of June (10.333 ± 4.702) and May (27.333 ± 14.769), whereas the highest ones were those of October (240.333 ± 20.497) and January (212 ± 30.238). Table 2 ANOVA results for the two treatments (period and site), as well as for the two factors combined. Degr. of freedom F p Period (Month) 11 6.618 < 0.001 Site 1 24.381 < 0.001 Month*Site 11 1.763 0.088 Since more insects were collected from the traps coming from a plantation in which the trees were visually more stressed, caused by abiotic factor such as floods, we decided to evaluate in laboratory if the stress caused by P. castaneus males and females attack could also result in a greater insect attraction. 3.2. Y-tube bioassays In Y-tube bioassays, test insects were separated by sex and responded to two different stimuli: healthy and P. castaneus attacked branches of P. taeda (Table 3 ). It was observed that males and females were more attracted to attacked branches than to healthy ones. Table 3 Responses of individual male and female P. castaneus adults to treatments in Y-tube olfactometer Bioassay Sex Responding Stimuli Positive Negative χ² P 1 Female Male attacked P. taeda 22 8 6.533 0.0176 2 Female Female attacked P. taeda 22 8 6.533 0.0176 3 Male Male attacked P. Taeda 23 7 8.533 0.0062 4 Male Female attacked P. taeda 23 7 8.533 0.0062 3.3. Tentative Identification of P. castaneus Attack-induced compounds The volatiles produced by branches of P. castaneus attacked trees were identified by GC-MS and the results are shown in Table 4 . When comparing volatiles produced by non-attacked and male and female P. castaneus attacked plants, qualitative and relative amount differences were detected. Table 4 Volatile compounds produced by healthy P. taeda and by female- and male P. castaneus -attacked plants Relative mean area (%) Terpene K.I. † Healthy P. taeda Attacked by females Attacked by males 1 α-pinene 939 22.73 ± 1.5 63.76 ± 15.8 68.93 ± 8.1 2 camphene 954 -- 0.77 ± 0.2 0.73 ± 0.1 3 β-pinene 979 10.37 ± 1.6 11.59 ± 1.6 12.67 ± 2.2 4 myrcene 991 16.14 ± 3.0 13.72 ± 12.0 1.77 ± 1.8 5 limonene 1030 3.21 ± 0.3 -- 1.10 ± 0.3 6 β-phellandrene 1032 17.08 ± 5.3 -- -- 7 ( Z )-β-ocimene 1040 -- 4.16 ± 3.0 -- 8 Unknown 1 1121 -- 1.37 ± 0.4 1.67 ± 0.4 9 methyl octanoate 1125 5.26 ± 0.5 -- -- 10 ( E )-caryophyllene 1419 1.23 ± 0.3 -- -- 11 prezizaene 1448 -- 1.34 ± 0.7 1.21 ± 0.7 12 α-humulene 1455 0.69 ± 0.1 -- -- 13 Unknown 2 1480 -- 0,24 ± 0.1 -- 14 germacrene D 1485 15.91 ± 3.8 -- -- 15 ( E )-muurola-4(14),5-diene 1491 -- 2.59 ± 1.5 5.51 ± 2.9 16 α-muurolene 1501 1.11 ± 0.3 -- -- 17 zonarene 1526 -- 0.19 ± 0.1 0.19 ± 0.1 † Kovats Index The chromatograms (Fig. 1 ) showed significant differences in the compounds produced by healthy (A) and female- (B) and male- (C) P. castaneus attacked trees. Compounds as β-phellandrene (6), methyl octanoate (9), E -caryophyllene (10), α-humulene (12), germacrene D (14) and α-muurolene are produced only by healthy trees and their production is suppressed when the plant is attacked by P. castaneus . Several compounds such as camphene (2), Z-β-ocimene (7), prezizaene (11), ( E )-muurola-4(14),5-diene (15) and zonarene (17) were produced only by attacked plants. The production of α-pinene (1), β-pinene (3), myrcene (4) and limonene (5), differed in relative percentage in the extracts from healthy and attacked plants; α-pinene (1) and β-pinene (3) were present in higher amounts in attacked-plants extracts than in healthy plants; myrcene (4) and limonene (5) were present in lower percentage in plants attacked by P. castaneus . 4. Discussion 4.1. Attractiveness of P. castaneus in a plantation of P. taeda The results obtained show that the log traps are effective to collect P. castaneus in P. taeda plantations, since they are good sites for oviposition. It was expected, once it was demonstrated in a previous work, that males and females of P. castaneus , were attracted by branches of the host plant, P. taeda , in Y-tube bioassays (Marques et al. 2011 ). The number of insects collected varied according to the plantation site. The number of insects collected in CS was higher than in TB, probably due to a stress caused by environmental conditions, such as planting sites in flooded areas with poor drainage. This apparently led to a higher infestation in CS, so it can be proposed that P. castaneus feed preferentially on damaged or stressed trees, which could favor the dissemination of the insect. The number of insects collected on each area also varied according to the time the trap was installed (seasonality treatment), which can be related with P. castaneus population variation, that is associated with abiotic factors of each region. For the areas studied, such variation was not simultaneously observed for both areas. Therefore, although for each area the parameter “month” has had a significative variation, those did not coincide among the evaluated areas. 4.2. Laboratory bioassays 4.2.1. Y-tube bioassays In Y-tube bioassays, test insects walked rather than flew when they responded to stimuli. The attractiveness of males and females to volatiles emitted by the healthy versus attacked host plant was determined. The bioassays showed that females and males were significantly more attracted by attacked host plant when compared to the healthy host plant, as shown in Table 3 . 4.2.2. Tentative identification of Attack-induced compounds Volatiles of P. castaneus attacked P. taeda have significant differences from the ones produced by the healthy trees. The relative amount of α-pinene is about three times higher and the production of β-phellandrene and germacrene D is suppressed (Fig. 2 ). On the other hand, attacked trees produce compounds not produced by the healthy ones, such as prezizaene, ( E )-muurola-4(14),5-diene and ( Z )-β-ocimene, the latter one interestingly produced only by female-attacked P. taeda (Fig. 3 ). In a search for compounds that contribute to the perception of the host in Pissodes castaneus (= P. notatus ), Bichão and co-workers (2003) identified olfactory receptor neurons that respond to α-pinene, β-pinene, limonene, β-phellandrene, ( E )-caryophyllene, camphene and other compounds. So, any change in the amount of those compounds will have influence in the perception of the host by the insect. In a recent published work, Skrzecz and co-workers ( 2019 ) showed that feeding P. castaneus were baited in olfactory bioassays and field traps using α-pinene and ethanol as attractant. Some of the volatiles produced only by the wounded trees, such as camphene, ( Z )-β-Ocimene and ( E )-muurola-4(14),5-diene were previously detected in unhealthy Pinus sylvestris trees, either by pollution (Judžentienė et al. 2006 ; Kupcinskiene et al. 2008 ) or by insect feeding (Mumm et al. 2003 ), but also in P. sylvestris healthy plants. However, this is the first report that detect prezizaene and zonarene being produced in the genus Pinus . Volatiles of many species of Pinus induced when attacked by different insects (Barnola et al. 1994 ; Sadof and Grant 1997 ; Mumm et al. 2003 , 2004 ; Miller et al. 2005 ) including other Pissodes (Tilles et al. 1986 ; Nordlander 1991 ) were studied. In most cases, there was a decrease of α-pinene and an increase of limonene production, differently from what was observed in this study. ( S )-(-)-limonene is a known repellent and α-pinene is an attractant to some weevils (Nordlander 1990 ) and many others species of herbivores (Sadof and Grant 1997 ). ( S )-(-)-limonene is also known to be more toxic to several Curculionidae compared to other monoterpenes, typically α-pinene, β-pinene, 3-carene and myrcene (Smith 1965 ; Werner 1995 ; Chiu et al. 2017 ). Likewise, another compound known to be a repellent to many insects, ( E )-caryophyllene (Bedini et al. 2015 ; Bougherra et al. 2015 ; Alquézar et al. 2017 ), had its production interrupted when the tree was attacked by P. castaneus . So, as compounds that act as repellent or are toxic to insects (( S )-(-)-limonene and ( E )-caryophyllene) have its production decreased and attractants (α-pinene) production increase, this can explain why the P. castaneus prefer to previously wounded or stressed trees, either by abiotic factors or other insects species. 5. Conclusions Our results indicate that it is possible to discriminate, using GC-MS analysis, between healthy plants of P. taeda and P. castaneus attacked ones based on the terpenes emitted. The preference of P. castaneus adults to attacked P. taeda makes it likely that this pest locates the host plants using the specific volatiles that it emits and that the presence for stressed trees can contribute to the attraction of P. castaneus to the site. Declarations ACKNOWLEDGEMENTS The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-INCT “Controle Biorracional de Insetos Praga”) and Fundação Araucária for financial support. Funding This work was supported by Fundação Araucária and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Gustavo Frensch, Scheila R. M. Zaleski, Francisco A. Marques, Beatriz H. L. N. S. Maia and Sonia M. N. Lazzari contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gustavo Frensch, Scheila R. M. Zaleski and Marina Krasniak. Statistical analysis was perfomed by Gustavo Frensch and Liliane G. Dantas. The first draft of the manuscript was written by Gustavo Frensch and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Adams RP (2007) Identification of Essential Oil Components by Gas Chromatography/Mass Espectrometry. 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Nature 393:570–573 Dicke M, van Loon JJA (2000) Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context. Entomol Exp Appl 97:237–249 El-Shafie HAF, Faleiro JR (2017) Semiochemicals and Their Potential Use in Pest Management. In: Shields VDC (ed) Biological Control of Pest and Vector Insects. InTech, Rijeka. Ch. 01 Faldt J, Martin D, Miller B et al (2003) Traumatic resin defense in Norway spruce (Picea abies): Methyl jasmonate-induced terpen synthase gene expression, and cDNA cloning and functional characterization of (+)- 3-carene synthase. Plant Mol Biol 51:119–133 Fitzgerald TD (2003) Role of Trail Pheromone in Foraging and Processionary Behavior of Pine Processionary Caterpillars Thaumetopoea pityocampa. J Chem Ecol 29:513–532 Franceschi VR, Krekling T, Christiansen E (2002) Application of methyl jasmonate on Picea abies (Pinaceae) stems induces defense-related responses in phloem and xylem. Am J Bot 89:578–586 GREZ OR, FONTECILLA LF, NUNEZ RA et al (2000) Manual de plagas cuarentenárias potencialmente daninas para o Chile com especial énfasis em plantaciones de pino y eucalipto. Chile Hilker M, Stein C, Schröder R et al (2005) Insect egg deposition induces defence responses in Pinus sylvestris: characterisation of the elicitor. J Exp Biol 208:1849–1854. doi: 10.1242/jeb.01578 Iede ET, RF W, Penteado SRC do et al (2004) Ocorrência de Pissodes castaneus (De Geer) (Coleoptera: Curculionidae) em Pínus, na Região Sul do Brasil. Comun Técnico Embrapa Floresta 114:1–6 Judžentienė A, Slizyte J, Stikliene A et al (2006) Characteristics of essential oil composition in the needles of young Scots pine (Pinus sylvestris L.) stands growing along an aerial ammonia gradient. Chemija 17:67–73 Keeling Christopher I, Bohlmann J, Keeling CI, Bohlmann J (2006) Genes, enzymes and chemicals of terpenoid diversity In the constitutive and induced defence of conifers against insects and pathogens. New Phytol 170:657–675. doi: 10.1111/j.1469-8137.2006.01716.x Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53:299–328 Kupcinskiene E, Stikliene A, Judzentiene A (2008) The essential oil qualitative and quantitative composition in the needles of Pinus sylvestris L. growing along industrial transects. Environ Pollut 155:481–491 Langenheim JH (2003) Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany. Timber Press, Portland Lewinsohn E, Gijzen M, Croteau R (1991) Defense mechanisms of conifers: Differences in constitutive and wound-induced monoterpene biosynthesis among species. Plant Physiol 96:44–49 Lusebrink I, Erbilgin N, Evenden ML (2016) The Effect of Water Limitation on Volatile Emission, Tree Defense Response, and Brood Success of Dendroctonus ponderosae in Two Pine Hosts, Lodgepole, and Jack Pine. Front Ecol Evol 4:2 Marques FA, Frensch G, Zaleski SRMSRM et al (2012) Differentiation of five pine species cultivated in Brazil based on chemometric analysis of their volatiles identified by gas chromatography-mass spectrometry. J Braz Chem Soc 23. doi: 10.1590/s0103-50532012005000042 Marques FA, Zaleski SRM, Lazzari SMN et al (2011) Identification of (1R, 2S)-Grandisal and (1R, 2S)-Grandisol in. J Braz Chem Soc 22:1050–1055 Martin D, Tholl D, Gershenzon J, Bohlmann J (2002) Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems. Plant Physiol 129:1003–1018 Martin DM, Gershenzon J, Bohlmann J (2003) Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce (Picea abies). Plant Physiol 132:1586–1599 McCormick AC, Reinecke A, Gershenzon J, Unsicker SB (2016) Feeding Experience Affects the Behavioral Response of Polyphagous Gypsy Moth Caterpillars to Herbivore-induced Poplar Volatiles. J Chem Ecol 42:382–393. doi: 10.1007/s10886-016-0698-7 McKay SAB, Hunter WL, Godard K-A et al (2003) Insect attack and wounding induce traumatic resin duct development and gene expression of (-)-pinene synthase in Sitka Spruce. Plant Physiol 133:368–378 Miller B, Madilao L, Ralph S, Bohlmann J (2005) Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and putative octadecanoid pathway transcripts in sitka spruce. Plant Physiol 137:369–382 Mumm R, Schrank KAI, Wegener R et al (2003) Chemical Analysis of Volatiles Emitted by Pinus sylvestris After Induction by Insect Oviposition. J Chem Ecol 29:1235–1252 Mumm R, Tiemann T, Schulz S, Hilker M (2004) Analysis of volatiles from black pine (Pinus nigra): significance of wounding and egg deposition by a herbivorous sawfly. Phytochemistry 65:3221–3230 Nadir E, Raffa KF (2001) Modulation of Predator Attraction to Pheromones of Two Prey Species by Stereochemistry of Plant Volatiles. Oecologia 127:444–453 Nagy NE, Franceschi VR, Solheim H et al (2000) Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): Anatomy and cytochemical traits. Am J Bot 87:302–313 Nault JR, Alfaro RI (2001) Changes in cortical and wood terpenes in sitka spruce in response to wounding. Can J For Res 31:1561–1568 Nordlander G (1991) Host finding in the pine weevil Hylobius abietis - Effects of conifer volatiles and added limonene. Entomol Exp Appl 59:229–237 Nordlander G (1990) Limonene inhibits attraction to -pinene in the pine weevils Hylobius abietis and H. pinastri. J Chem Ecol 16:1307–1320 Nordlund DA, Lewis WJ (1976) Terminology of chemical releasing stimuli in intraspecific and interspecific interactions. J Chem Ecol 2:211–220. doi: 10.1007/BF00987744 Phillips MA, Croteau R (1999) Resin-based defenses in conifers. Trends Plants Sci 4:184–190 Sadof CS, Grant GG (1997) Monoterpene compostion of Pinus sylvestris varieties resistant and susceptible to Dioryctria zimmermani. J Chem Ecol 23:1917–1927. doi: 10.1023/B:JOEC.0000006479.39087.60 Skrzecz I, Wolski R, Sowinska A et al (2019) Evaluation of attractants and traps for monitoring small banded pine weevil Pissodes castaneus. J Appl Entomol 0. doi: 10.1111/jen.12610 Smith RH (1965) Effect of Monoterpene Vapors on the Western Pine Beetle. J Econ Entomol 58:509–510 Sullivan BT, Berisford CW (2004) Semiochemicals from Fungal Associates of Bark Beetles May Mediate Host Location Behavior of Parasitoids. J Chem Ecol 30:703–717. doi: 10.1023/B:JOEC.0000028426.37482.17 Takabayashi J, Dicke M, Posthumus MA (1994) Volatile herbivore-induced terpenoids in plant-mite interactions: variation caused by biotic and abiotic factors. J Chem Ecol 20:1329–1354 Tilles DA, Nordlander G, Nordenhem H et al (1986) Increased release of host volatiles from feeding scars – a major cause of field aggregation in the pine weevil Hylobius abietis (Coleoptera, Curculionidae). Environ Entomol 15:1050–1054 Tomlin ES, Alfaro RI, Borden JH, He FL (1998) Histological response of resistant and susceptible white spruce to simulated white pine weevil damage. Tree Physiol 18:21–28 Trapp S, Croteau R (2001) Defensive resin biosynthesis in conifers. Ann Rev Plant Phys Plant Mol Biol 52:689–724 Werner RA (1995) Toxicity and Repellency of 4–Allylanisole and Monoterpenes from White Spruce and Tamarack to the Spruce Beetle and Eastern Larch Beetle (Coleoptera: Scolytidae). Environ Entomol 24:372–379 Supplementary Files supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2023 Read the published version in Chemoecology → Version 1 posted Editorial decision: Major revisions 30 Sep, 2022 Reviewers agreed at journal 10 Jun, 2022 Reviewers invited by journal 02 Jun, 2022 Editor assigned by journal 01 Jun, 2022 First submitted to journal 26 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1696409","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":110619346,"identity":"1c4c3eb2-8364-47e0-8633-7f0e7e364502","order_by":0,"name":"Gustavo Frensch","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2774-4934","institution":"Universidade Federal do Vale do Sao Francisco","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Frensch","suffix":""},{"id":110619347,"identity":"d47d45fc-1180-4c77-84d8-2f122ba413ec","order_by":1,"name":"Scheila R. M. Zaleski","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Scheila","middleName":"R. M.","lastName":"Zaleski","suffix":""},{"id":110619348,"identity":"e1fbb5f1-5ea5-4646-aaf2-2fd5290786f3","order_by":2,"name":"Renan R. Schorr","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Renan","middleName":"R.","lastName":"Schorr","suffix":""},{"id":110619349,"identity":"a9b53f21-dad5-4732-a37f-c86ee7c1b5de","order_by":3,"name":"Liliane G. Dantas","email":"","orcid":"","institution":"Universidade Federal do Vale do Sao Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liliane","middleName":"G.","lastName":"Dantas","suffix":""},{"id":110619350,"identity":"f685cfd7-899b-453a-888b-bdad2d091a5c","order_by":4,"name":"Marina Krasniak","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Krasniak","suffix":""},{"id":110619351,"identity":"eda9ec78-76e6-4f4a-b468-b5276b6db6f9","order_by":5,"name":"Sonia M. N. Lazzari","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sonia","middleName":"M. N.","lastName":"Lazzari","suffix":""},{"id":110619352,"identity":"92164e5b-c12f-4b20-ba58-3d0e7aff19ba","order_by":6,"name":"Beatriz H. L. N. S. Maia","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"H. L. N. S.","lastName":"Maia","suffix":""},{"id":110619353,"identity":"a10a8956-8129-40c4-b4f4-e666cd8d136d","order_by":7,"name":"Francisco A. Marques","email":"","orcid":"","institution":"Universidade Federal do Parana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"A.","lastName":"Marques","suffix":""}],"badges":[],"createdAt":"2022-05-26 12:45:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1696409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1696409/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00049-023-00383-1","type":"published","date":"2023-06-10T21:06:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":22416102,"identity":"c52dbb2d-73d8-4530-9842-1684dd6cbf7c","added_by":"auto","created_at":"2022-06-08 15:41:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44226,"visible":true,"origin":"","legend":"\u003cp\u003eTotal ion chromatogram of the aeration of (A) healthy, (B) female- and (C) male-attacked \u003cem\u003eP. taeda. \u003c/em\u003eAll identifications are tentative and made by comparation with Adams (2007). Compounds: 1) α-pinene; 2) camphene; 3) β-pinene; 4) myrcene; 5) limonene; 6) β-phellandrene; 7) (\u003cem\u003eZ\u003c/em\u003e)-β-ocimene; 8) Unknown 1; 9) methyl octanoate; 10) (\u003cem\u003eE\u003c/em\u003e)-caryophyllene; 11) prezizaene; 12) α-humulene; 13) Unknown 2; 14) germacrene D; 15) (\u003cem\u003eE\u003c/em\u003e)-muurola-4(14),5-diene; 16) α-muurolene; 17) zonarene.\t\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1696409/v1/2775e6212f588c515327656f.jpg"},{"id":22416101,"identity":"84a5bc1e-96b8-4393-bdd2-e30a6e850286","added_by":"auto","created_at":"2022-06-08 15:41:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29284,"visible":true,"origin":"","legend":"\u003cp\u003eRelative amount of volatiles produced by healthy \u003cem\u003eP. taeda \u003c/em\u003etrees and by \u003cem\u003eP. castaneus\u003c/em\u003e female- and male-attacked plants. All identifications are tentative and made by comparation with Adams (2007).\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1696409/v1/b90e8d8e68ad4dc661f1fc97.jpg"},{"id":22416103,"identity":"9d966e07-171d-4cdc-b6f2-3212704f76ff","added_by":"auto","created_at":"2022-06-08 15:41:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29302,"visible":true,"origin":"","legend":"\u003cp\u003eRelative amount of volatiles produced only by female- and male-attacked \u003cem\u003eP. taeda. \u003c/em\u003eAll identifications are tentative and made by comparation with Adams (2007).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1696409/v1/fcf9c5e9694e9db978be3aac.jpg"},{"id":44730543,"identity":"86c6a133-ce9c-428c-8964-ab4c549c07a1","added_by":"auto","created_at":"2023-10-16 21:31:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":686889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1696409/v1/37829080-d43f-4822-ba32-357097e06b3c.pdf"},{"id":22416104,"identity":"e460a028-aa48-40ad-9f78-a7b3e0e1aac1","added_by":"auto","created_at":"2022-06-08 15:41:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":476729,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-1696409/v1/0eb09a3dbd3a95b1638af832.docx"}],"financialInterests":"","formattedTitle":"Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: Laboratory and Field Evaluation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe banded pine weevil, \u003cem\u003ePissodes castaneus\u003c/em\u003e (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of \u003cem\u003ePinus\u003c/em\u003e (Pinaceae) in South America (Iede et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). It was detected in Brazil in 2001 in the county of S\u0026atilde;o Jos\u0026eacute; dos Ausentes, Rio Grande do Sul, on \u003cem\u003ePinus taeda\u003c/em\u003e L and later it was detected in the states of Santa Catarina and Paran\u0026aacute; (Iede et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In 2002, 7.6% of the trees of a plantation of \u003cem\u003eP. taeda\u003c/em\u003e grown in Cambar\u0026aacute; do Sul, Rio Grande do Sul, were attacked by \u003cem\u003eP. castaneus\u003c/em\u003e while in S\u0026atilde;o Joaquim, Santa Catarina, 16.5% of the trees were attacked (Iede et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis insect represents a threat to Brazilian forest production, as it has the potential to cause economic losses, such as those that occurred in Uruguay where there was a mortality rate higher than 10% (Grez et al. 2000). More than 50% of trees in a stand can be attacked in one year (Cadahia et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConifers are long lived gymnosperms, which includes many species that successfully inhabit large areas of our planet. As raw material for many products (wood, paper, plastic, fuel, and many chemicals), their economic impact on our society is of great importance. In European countries such as Sweden, 83% of the forests consist of conifers, mainly Norway spruce (\u003cem\u003ePicea abies\u003c/em\u003e L.) and Scots pine (\u003cem\u003ePinus sylvestris\u003c/em\u003e L.) (Alin and Sundberg \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In South America, specifically in Brazil, pine trees, mainly \u003cem\u003eP. taeda\u003c/em\u003e and \u003cem\u003ePinus elliottii\u003c/em\u003e Engelm., have been planted on commercial scale for over 30 years (Ahrens \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Currently, there are about 2\u0026nbsp;million hectares of reforested pine plantations, in large continuous areas and generally in narrow genetic base stands, mainly in the South and Southeast regions (Iede et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The product of these plantations is destined mostly to the timber and cellulose industries (Cardoso and L\u0026aacute;zzari \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsects use volatiles as their major method to find food, mates or an appropriate site to lay their eggs (El-Shafie and Faleiro \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For plants, which cannot escape threats, chemical constituents play an important role in their defense system. Their response to feeding or oviposition of herbivorous insects can be direct, by feeding deterrents, impairing digestion or producing toxins (Kessler and Baldwin \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), or indirect, by the release of volatiles which attract natural enemies of the herbivorous insects (Dicke and van Loon \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Arimura and Pearse \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, there are cases that the induced compounds may act as kairomones (Nordlund and Lewis \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Keeling Christopher et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lusebrink et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McCormick et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), i.e. chemical compounds emitted by an organism that induce a benefic response to the receiving organism from another species, or have a synergistic effect with insect pheromones \u0026ndash; compounds used in intraspecific communication \u0026ndash;, as happens with \u003cem\u003ePinus ponderosa\u003c/em\u003e when damaged by \u003cem\u003eDendroctonus brevicomis\u003c/em\u003e Lec. (Bedard et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1969\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConifers can use a large array of structurally diverse mono-, sesqui- and diterpenoids as a chemical defense against herbivores (Lewinsohn et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Phillips and Croteau \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Trapp and Croteau \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Induction of volatiles terpenoids may result in a quantitative or qualitative difference in the chemical profile produced by a plant (Fitzgerald \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The herbivore-induced change in the plant volatiles can be specific for the plant and herbivore species, the plant age and the herbivore stage (Takabayashi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; De Moraes et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDefense responses of induced terpenoid have been intensively studied using spruce (\u003cem\u003ePicea\u003c/em\u003e spp.) as model plant. Anatomical defenses like traumatic resin duct formation or polyphenolic parenchyma cell activation are induced by wounding or by attacking insects and pathogens (Tomlin et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Nagy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Franceschi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; McKay et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Besides, induction of terpenoids have been observed in spruce after mechanical wounding or treatment with methyl jasmonate, a plant hormone known to upregulate plant defenses (Nault and Alfaro \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Faldt et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese terpenoids can act as repellents or toxins against those herbivores, defending conifers directly, or mimic juvenile hormones (Lewinsohn et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Langenheim \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mumm et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Likewise, conifer volatiles can also attract predatory and parasitic insects, consequently defending conifers indirectly against herbivores (Nadir and Raffa \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Mumm et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sullivan and Berisford \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Hilker et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn previously studies it was described that \u003cem\u003eP. castaneus\u003c/em\u003e adults were attracted to the volatiles emitted by \u003cem\u003eP. taeda\u003c/em\u003e (Marques et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and previously stressed plants showed a higher infestation (Iede et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the present study, we conducted detailed GC\u0026ndash;MS analyses of volatiles emitted by twigs of \u003cem\u003eP. taeda\u003c/em\u003e, both healthy and attacked. We analyzed how the attack by \u003cem\u003eP. castaneus\u003c/em\u003e males and females affects the volatile pattern emitted by \u003cem\u003eP. taeda\u003c/em\u003e twigs. We also carried out laboratory and field assays to evaluate the attraction of \u003cem\u003eP. castaneus\u003c/em\u003e to healthy and stressed host plants.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Field evaluation of the attraction of \u003cem\u003eP. castaneus\u003c/em\u003e by \u003cem\u003eP. taeda\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Characterization of the experimental area\u003c/h2\u003e \u003cp\u003eThe attractiveness experiments were performed in two plantations with density of 1,666 1 year old plants/ha, located in Tr\u0026ecirc;s Barras (TB), Santa Catarina, Brazil (26\u0026deg;07'41\"S, 50\u0026deg;19'30\"W, 802m altitude) and Cambar\u0026aacute; do Sul (CS), Rio Grande do Sul, Brazil (29\u0026deg;02'52\"S, 50\u0026deg;08'41\"W, 1031m altitude). In each area, the experiment was repeated on three plots, 200m apart from each other. For the experiment\u0026rsquo;s purpose, plants found in CS were considered stressed due to poor planting sites in flooded areas with poor drainage, whereas plants found in TB were considered healthy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Evaluation of the attractiveness of \u003cem\u003eP. castaneus\u003c/em\u003e to \u003cem\u003eP. taeda\u003c/em\u003e log traps\u003c/h2\u003e \u003cp\u003eTo evaluate the attractiveness of \u003cem\u003eP. castaneus\u003c/em\u003e to \u003cem\u003eP. taeda\u003c/em\u003e, two variables were analyzed: the effect of stressed (CS) x non-stressed (TB) environments, and the effect of seasonal variations through a 12-month experiment.\u003c/p\u003e \u003cp\u003eIn each plot of each area, log traps composed of 20 freshly cut 1m long and 8\u0026ndash;10 cm diameter \u003cem\u003eP. taeda\u003c/em\u003e logs were installed every month between July 2012 and June 2013 for adults of \u003cem\u003eP. castaneus\u003c/em\u003e to lay eggs in. After 30 days, the logs were removed from the field, new log traps were installed in the same plots, and the removed ones were transported to the laboratory. For each batch of 20 logs, 17 were stored for 1 year in screened cages in a room at 20\u003csup\u003eo\u003c/sup\u003eC, humidity on 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% and photoperiod of 12h. The logs were monitored weekly to assess the emergence of viable adults. Any emerged adult was removed from cages.\u003c/p\u003e \u003cp\u003eThe average number of adults emerged in each area per month was obtained by averaging the total number of adults emerged in the three plots in each area using BioEstat 5.0 (Ayres et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To access the effect of seasonality and location in the attractiveness of the insects, a factorial analysis of variance (ANOVA) was performed using Statistica software (version 8.0; StatSoft Inc, Tulsa, OK, USA). The homogeneity of data was evaluated through a Levene\u0026rsquo;s test, and a Duncan post-hoc test was performed to evaluate which means were statistically different at 5%, both using Statistica 8.0 software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chemical analysis of the attraction of \u003cem\u003eP. castaneus\u003c/em\u003e by \u003cem\u003eP. taeda\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Insects\u003c/h2\u003e \u003cp\u003eFor chemical assays, the insect colony was started with specimens collected from log traps installed in Tr\u0026ecirc;s Barras plantation (26\u0026deg;07'41\"S, 50\u0026deg;19'30\"W, 802m altitude). To obtain the insects for the bioassays, \u003cem\u003eP. taeda\u003c/em\u003e freshly cut logs were placed in the field. Log traps were installed from July 2012 to June 2013. Each log trap was composed of 20 freshly cut 1m long and 8\u0026ndash;10 cm diameter pine logs for adults of \u003cem\u003eP. castaneus\u003c/em\u003e to lay eggs in. After 30 days, the logs were removed from the field and placed in screened cages measuring 30 cm height by 30 cm diameter until adult emergence. After their emergence, the adults were separated by sex and each group were kept for 5\u0026ndash;10 days in an incubator at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity and 12L:12D photoperiod. Females and males were selected for the trials and placed alone in plastic screened cages with ventilation at the sides under the same conditions as described above. Adults were fed a natural diet consisting of pieces of fresh \u003cem\u003eP. taeda\u003c/em\u003e branches. The cages were cleaned, and the food supply replaced at regular intervals of five days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Plants\u003c/h2\u003e \u003cp\u003eAll trees of \u003cem\u003eP. taeda\u003c/em\u003e used in this work were 1\u0026ndash;2 years old and were obtained from greenhouses near Curitiba, Paran\u0026aacute;, Brazil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Y-tube bioassays\u003c/h2\u003e \u003cp\u003eBioassays were conducted with a vertical glass Y tube (2.5 cm ID, bottom arm 20 cm, top arms 20 cm) with ground glass female joints at the end of each arm, and matching male joints terminating in hose nipples. In the system, the speed of humidified, charcoal filtered air was adjusted to 2.0 L/min. All connections were made of Tygon tubing (0.6 cm ID). After each trial, all materials were washed with neutral soap, dipped in 70% ethanol, and placed in the oven at 50 \u003csup\u003eo\u003c/sup\u003eC for 30 min to avoid any residual volatile compounds. Test insects were introduced individually into the bottom of the Y tube and, after 5 min of acclimation, allowed 20 min to respond. When the insect moved through the arm containing the stimulus (male or female-attacked \u003cem\u003eP. taeda\u003c/em\u003e branches), the result was considered positive while the result was negative when it moved through the control arm (healthy \u003cem\u003eP. taeda\u003c/em\u003e branches). However, when the insect did not move towards any of the arms within 20 min, it was considered non-responsive. The location of stimulus and control were rotated after each test. The insects used in the tests were given no food 12 h before the bioassays. The branches of \u003cem\u003eP. taeda\u003c/em\u003e used as stimuli were collected one day before conducting the bioassays, placed in the thermal box, and transported to the laboratory where they were kept in the refrigerator at 10\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C until tests. Bioassays were conducted in a temperature- (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC) and humidity-controlled room (70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity). Each stimulus was tested with 30 sexually mature virgin adult insects of each sex.\u003c/p\u003e \u003cp\u003eThe data obtained with insects that reached the corresponding odor sources in the Y tube assay were compared by the \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e test using BioEstat 5.0 (Ayres et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Results showing \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Tentative Identification of healthy and attack-induced compounds produced by \u003cem\u003eP. taeda\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Healthy trees\u003c/h2\u003e \u003cp\u003eFour branches of healthy trees of \u003cem\u003eP. taeda\u003c/em\u003e (200g) were aerated in a 1 L glass chamber. Aerations were carried out under controlled conditions at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% and a photoperiod of 12L:12D. Volatiles were trapped on a 0.4 cm long bed of Super Q resin (Alltech, Deerfield, Illinois, USA) held in place by glass wool plugs in a glass tube (4 mm ID). Volatiles were collected for two days (flow 1.0 L/min), then eluted with hexane (3 x 0.5 mL). Extracts were concentrated as required (c.a. 100 \u0026micro;L) under Argon. All procedures were carried out in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. \u003cem\u003eP. castaneus\u003c/em\u003e attacked trees\u003c/h2\u003e \u003cp\u003eSaplings of \u003cem\u003eP. taeda\u003c/em\u003e (1\u0026ndash;2 years old) were placed in plastic screened cages with ventilation at the sides, at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity and 12L:12D photoperiod, with 10 males or 10 females of \u003cem\u003eP. castaneus\u003c/em\u003e. After 72 hours of attack, branches of these trees were cut and aerated the same way healthy trees were.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Chemical analysis\u003c/h2\u003e \u003cp\u003eExtracts and \u003cem\u003en\u003c/em\u003e-alkane standards were analyzed by coupled GC/MS with a Shimadzu CGMS-QP2010 Plus system equipped with a quadrupole detector using a Rtx-5MS (Crossbond 5% diphenyl / 95% dimethyl polysiloxane) low bleeding column (30 m x 0.25 mm x 0.25 \u0026micro;m), using Helium as carrier gas, at flow of 1.02 mL/min. Oven temperature was initially held at 60\u0026deg;C for 1 min and increased at a rate of 3\u0026deg;C/min to 250\u0026deg;C. The mass spectrometer operated in electron impact mode (70 eV) with a mass range set from 40 to 350 m/z. The interface and source temperatures were set at 250\u0026deg;C. Kovats retention index was calculated for the compounds and their mass spectra were compared with the literature (Adams \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003e-C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003e and C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e42\u003c/sub\u003e \u003cem\u003en\u003c/em\u003e-alkanes standards were purchased from Aldrich (Deisenhofen, Germany). Compounds detected in at least 2 of 3 samples of the attack-induced pine branches were considered.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.1. Number of \u003cem\u003eP. castaneus\u003c/em\u003e that emerged from log traps of \u003cem\u003eP. taeda\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe average number of adults of \u003cem\u003eP. castaneus\u003c/em\u003e that emerged from the log traps differed between the two study sites. In CS, higher average number of insects (196.778\u0026thinsp;\u0026plusmn;\u0026thinsp;27.552) were collected when compared to TB, where in average 110.556\u0026thinsp;\u0026plusmn;\u0026thinsp;22.775 insects were collected (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean number (\u0026plusmn;\u0026thinsp;SE) of adults of Pissodes castaneus that emerged from the logs trap installed in periods (months) compared within in a Pinus taeda forest in Tr\u0026ecirc;s Barras (TB), Santa Catarina, Brazil, and Cambar\u0026aacute; do Sul (CS), RS, Brazil, July/2012 up to June/2013.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ePeriod (month)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCS\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTB\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage n\u0026ordm; of insects (n\u0026thinsp;=\u0026thinsp;3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignificantly distinct months within area (Duncan)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage n\u0026ordm; of insects (n\u0026thinsp;=\u0026thinsp;3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignificantly distinct months within area (Duncan)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eJul.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e283\u0026thinsp;\u0026plusmn;\u0026thinsp;40.427 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.333\u0026thinsp;\u0026plusmn;\u0026thinsp;31.424 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgo.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e149.667\u0026thinsp;\u0026plusmn;\u0026thinsp;15.431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDEF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.333\u0026thinsp;\u0026plusmn;\u0026thinsp;12.548\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSet.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e116.333\u0026thinsp;\u0026plusmn;\u0026thinsp;38.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGHI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.333\u0026thinsp;\u0026plusmn;\u0026thinsp;35.366\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOut.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e230\u0026thinsp;\u0026plusmn;\u0026thinsp;79.775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e240.333\u0026thinsp;\u0026plusmn;\u0026thinsp;20.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADFGHI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNov.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e308.667\u0026thinsp;\u0026plusmn;\u0026thinsp;17.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDGKLM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e198\u0026thinsp;\u0026plusmn;\u0026thinsp;68.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBJKL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDez.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e185\u0026thinsp;\u0026plusmn;\u0026thinsp;65.957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e118\u0026thinsp;\u0026plusmn;\u0026thinsp;40.633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eJan.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e288\u0026thinsp;\u0026plusmn;\u0026thinsp;33.307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e212\u0026thinsp;\u0026plusmn;\u0026thinsp;30.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCEMNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFev.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e233\u0026thinsp;\u0026plusmn;\u0026thinsp;83.720\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180.333\u0026thinsp;\u0026plusmn;\u0026thinsp;22.821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMar.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e304\u0026thinsp;\u0026plusmn;\u0026thinsp;55.426 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEIRS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.333\u0026thinsp;\u0026plusmn;\u0026thinsp;66.520 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbr.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBFJKNOQRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44\u0026thinsp;\u0026plusmn;\u0026thinsp;7.234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGJM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMai.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;37.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCLPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.333\u0026thinsp;\u0026plusmn;\u0026thinsp;14.769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHKNP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eJun.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e163.667\u0026thinsp;\u0026plusmn;\u0026thinsp;41.378 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.333\u0026thinsp;\u0026plusmn;\u0026thinsp;4.702 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eILOQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e196.778\u0026thinsp;\u0026plusmn;\u0026thinsp;27.552\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e110.556\u0026thinsp;\u0026plusmn;\u0026thinsp;22.775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* at \u0026ldquo;Average n\u0026ordm; of insects\u0026rdquo; rows indicate mean values that were statistically different at 5% significance level among different areas within the same month. Similar letters at \u0026ldquo;Duncan\u0026rdquo; rows indicates that the mean values were statistically different at 5% significance level among different months within the same area\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eAlthough ANOVA results revealed a correlation between seasonality treatment and mean number of insects obtained (F\u0026thinsp;=\u0026thinsp;6.618, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), no such correlation was observed when both sites and seasonality were analyzed together (F\u0026thinsp;=\u0026thinsp;1.763, p\u0026thinsp;=\u0026thinsp;0.0878; Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This can be explained by the differences found in the average number of insects collected per month for each area (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). For CS, the lowest number of insects was found during the months of April (0\u0026thinsp;\u0026plusmn;\u0026thinsp;0) and May (100\u0026thinsp;\u0026plusmn;\u0026thinsp;37.005), whereas the highest number was during the months of November (308.667\u0026thinsp;\u0026plusmn;\u0026thinsp;17.023) and March (304\u0026thinsp;\u0026plusmn;\u0026thinsp;55.426). On the other hand, for TB the lowest values were those of June (10.333\u0026thinsp;\u0026plusmn;\u0026thinsp;4.702) and May (27.333\u0026thinsp;\u0026plusmn;\u0026thinsp;14.769), whereas the highest ones were those of October (240.333\u0026thinsp;\u0026plusmn;\u0026thinsp;20.497) and January (212\u0026thinsp;\u0026plusmn;\u0026thinsp;30.238).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOVA results for the two treatments (period and site), as well as for the two factors combined.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDegr. of freedom\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeriod (Month)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.381\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth*Site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.088\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eSince more insects were collected from the traps coming from a plantation in which the trees were visually more stressed, caused by abiotic factor such as floods, we decided to evaluate in laboratory if the stress caused by \u003cem\u003eP. castaneus\u003c/em\u003e males and females attack could also result in a greater insect attraction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.2. Y-tube bioassays\u003c/h2\u003e\n \u003cp\u003eIn Y-tube bioassays, test insects were separated by sex and responded to two different stimuli: healthy and \u003cem\u003eP. castaneus\u003c/em\u003e attacked branches of \u003cem\u003eP. taeda\u003c/em\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). It was observed that males and females were more attracted to attacked branches than to healthy ones.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResponses of individual male and female \u003cem\u003eP. castaneus\u003c/em\u003e adults to treatments in Y-tube olfactometer\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBioassay\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex Responding\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStimuli\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;\u0026sup2;\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale attacked P. taeda\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.533\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0176\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale attacked P. taeda\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.533\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0176\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale attacked P. Taeda\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.533\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0062\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale attacked P. taeda\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.533\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0062\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.3. Tentative Identification of \u003cem\u003eP. castaneus\u003c/em\u003e Attack-induced compounds\u003c/h2\u003e\n \u003cp\u003eThe volatiles produced by branches of \u003cem\u003eP. castaneus\u003c/em\u003e attacked trees were identified by GC-MS and the results are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. When comparing volatiles produced by non-attacked and male and female \u003cem\u003eP. castaneus\u003c/em\u003e attacked plants, qualitative and relative amount differences were detected.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab4\" style=\"width: 529px;\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eVolatile compounds produced by healthy \u003cem\u003eP. taeda\u003c/em\u003e and by female- and male \u003cem\u003eP. castaneus\u003c/em\u003e-attacked plants\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"6\" style=\"width: 486px;\"\u003e\n \u003cp\u003eRelative mean area (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTerpene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK.I.\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026dagger;\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eP. taeda\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eAttacked by females\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eAttacked by males\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-pinene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e22.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e63.76\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e68.93\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecamphene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-pinene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e979\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e10.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e12.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emyrcene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e16.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e13.72\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elimonene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;-phellandrene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e17.08\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cspan class=\"BoldItalic\"\u003eZ\u003c/span\u003e\u003cstrong\u003e)-\u0026beta;-ocimene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnknown 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emethyl octanoate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cspan class=\"BoldItalic\"\u003eE\u003c/span\u003e\u003cstrong\u003e)-caryophyllene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eprezizaene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-humulene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnknown 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e0,24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003egermacrene D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e15.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cspan class=\"BoldItalic\"\u003eE\u003c/span\u003e\u003cstrong\u003e)-muurola-4(14),5-diene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-muurolene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 15.6406px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 166.359px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ezonarene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 28px;\"\u003e\n \u003cp\u003e1526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 63px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 112px;\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 486px;\"\u003e\u003csup\u003e\u003cstrong\u003e\u0026dagger;\u003c/strong\u003e\u003c/sup\u003e Kovats Index\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eThe chromatograms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) showed significant differences in the compounds produced by healthy (A) and female- (B) and male- (C) \u003cem\u003eP. castaneus\u003c/em\u003e attacked trees. Compounds as \u0026beta;-phellandrene (6), methyl octanoate (9), \u003cem\u003eE\u003c/em\u003e-caryophyllene (10), \u0026alpha;-humulene (12), germacrene D (14) and \u0026alpha;-muurolene are produced only by healthy trees and their production is suppressed when the plant is attacked by \u003cem\u003eP. castaneus\u003c/em\u003e. Several compounds such as camphene (2), Z-\u0026beta;-ocimene (7), prezizaene (11), (\u003cem\u003eE\u003c/em\u003e)-muurola-4(14),5-diene (15) and zonarene (17) were produced only by attacked plants. The production of \u0026alpha;-pinene (1), \u0026beta;-pinene (3), myrcene (4) and limonene (5), differed in relative percentage in the extracts from healthy and attacked plants; \u0026alpha;-pinene (1) and \u0026beta;-pinene (3) were present in higher amounts in attacked-plants extracts than in healthy plants; myrcene (4) and limonene (5) were present in lower percentage in plants attacked by \u003cem\u003eP. castaneus\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e4.1. Attractiveness of \u003cem\u003eP. castaneus\u003c/em\u003e in a plantation of \u003cem\u003eP. taeda\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe results obtained show that the log traps are effective to collect \u003cem\u003eP. castaneus\u003c/em\u003e in \u003cem\u003eP. taeda\u003c/em\u003e plantations, since they are good sites for oviposition. It was expected, once it was demonstrated in a previous work, that males and females of \u003cem\u003eP. castaneus\u003c/em\u003e, were attracted by branches of the host plant, \u003cem\u003eP. taeda\u003c/em\u003e, in Y-tube bioassays (Marques et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). The number of insects collected varied according to the plantation site. The number of insects collected in CS was higher than in TB, probably due to a stress caused by environmental conditions, such as planting sites in flooded areas with poor drainage. This apparently led to a higher infestation in CS, so it can be proposed that \u003cem\u003eP. castaneus\u003c/em\u003e feed preferentially on damaged or stressed trees, which could favor the dissemination of the insect.\u003c/p\u003e\n \u003cp\u003eThe number of insects collected on each area also varied according to the time the trap was installed (seasonality treatment), which can be related with \u003cem\u003eP. castaneus\u003c/em\u003e population variation, that is associated with abiotic factors of each region. For the areas studied, such variation was not simultaneously observed for both areas. Therefore, although for each area the parameter \u0026ldquo;month\u0026rdquo; has had a significative variation, those did not coincide among the evaluated areas.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003e4.2. Laboratory bioassays\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec21\"\u003e\n \u003ch2\u003e4.2.1. Y-tube bioassays\u003c/h2\u003e\n \u003cp\u003eIn Y-tube bioassays, test insects walked rather than flew when they responded to stimuli. The attractiveness of males and females to volatiles emitted by the healthy versus attacked host plant was determined. The bioassays showed that females and males were significantly more attracted by attacked host plant when compared to the healthy host plant, as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec22\"\u003e\n \u003ch2\u003e4.2.2. Tentative identification of Attack-induced compounds\u003c/h2\u003e\n \u003cp\u003eVolatiles of \u003cem\u003eP. castaneus\u003c/em\u003e attacked \u003cem\u003eP. taeda\u003c/em\u003e have significant differences from the ones produced by the healthy trees. The relative amount of \u0026alpha;-pinene is about three times higher and the production of \u0026beta;-phellandrene and germacrene D is suppressed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). On the other hand, attacked trees produce compounds not produced by the healthy ones, such as prezizaene, (\u003cem\u003eE\u003c/em\u003e)-muurola-4(14),5-diene and (\u003cem\u003eZ\u003c/em\u003e)-\u0026beta;-ocimene, the latter one interestingly produced only by female-attacked \u003cem\u003eP. taeda\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn a search for compounds that contribute to the perception of the host in \u003cem\u003ePissodes castaneus\u003c/em\u003e (=\u0026thinsp;\u003cem\u003eP. notatus\u003c/em\u003e), Bich\u0026atilde;o and co-workers (2003) identified olfactory receptor neurons that respond to \u0026alpha;-pinene, \u0026beta;-pinene, limonene, \u0026beta;-phellandrene, (\u003cem\u003eE\u003c/em\u003e)-caryophyllene, camphene and other compounds. So, any change in the amount of those compounds will have influence in the perception of the host by the insect. In a recent published work, Skrzecz and co-workers (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) showed that feeding \u003cem\u003eP. castaneus\u003c/em\u003e were baited in olfactory bioassays and field traps using \u0026alpha;-pinene and ethanol as attractant.\u003c/p\u003e\n \u003cp\u003eSome of the volatiles produced only by the wounded trees, such as camphene, (\u003cem\u003eZ\u003c/em\u003e)-\u0026beta;-Ocimene and (\u003cem\u003eE\u003c/em\u003e)-muurola-4(14),5-diene were previously detected in unhealthy \u003cem\u003ePinus sylvestris\u003c/em\u003e trees, either by pollution (Judžentienė et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kupcinskiene et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e) or by insect feeding (Mumm et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e), but also in \u003cem\u003eP. sylvestris\u003c/em\u003e healthy plants. However, this is the first report that detect prezizaene and zonarene being produced in the genus \u003cem\u003ePinus\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eVolatiles of many species of \u003cem\u003ePinus\u003c/em\u003e induced when attacked by different insects (Barnola et al. \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sadof and Grant \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e; Mumm et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Miller et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) including other \u003cem\u003ePissodes\u003c/em\u003e (Tilles et al. \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e; Nordlander \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e) were studied. In most cases, there was a decrease of \u0026alpha;-pinene and an increase of limonene production, differently from what was observed in this study. (\u003cem\u003eS\u003c/em\u003e)-(-)-limonene is a known repellent and \u0026alpha;-pinene is an attractant to some weevils (Nordlander \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e) and many others species of herbivores (Sadof and Grant \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e). (\u003cem\u003eS\u003c/em\u003e)-(-)-limonene is also known to be more toxic to several Curculionidae compared to other monoterpenes, typically \u0026alpha;-pinene, \u0026beta;-pinene, 3-carene and myrcene (Smith \u003cspan class=\"CitationRef\"\u003e1965\u003c/span\u003e; Werner \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e; Chiu et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Likewise, another compound known to be a repellent to many insects, (\u003cem\u003eE\u003c/em\u003e)-caryophyllene (Bedini et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bougherra et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Alqu\u0026eacute;zar et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), had its production interrupted when the tree was attacked by \u003cem\u003eP. castaneus\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eSo, as compounds that act as repellent or are toxic to insects ((\u003cem\u003eS\u003c/em\u003e)-(-)-limonene and (\u003cem\u003eE\u003c/em\u003e)-caryophyllene) have its production decreased and attractants (\u0026alpha;-pinene) production increase, this can explain why the \u003cem\u003eP. castaneus\u003c/em\u003e prefer to previously wounded or stressed trees, either by abiotic factors or other insects species.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur results indicate that it is possible to discriminate, using GC-MS analysis, between healthy plants of \u003cem\u003eP. taeda\u003c/em\u003e and \u003cem\u003eP. castaneus\u003c/em\u003e attacked ones based on the terpenes emitted. The preference of \u003cem\u003eP. castaneus\u003c/em\u003e adults to attacked \u003cem\u003eP. taeda\u003c/em\u003e makes it likely that this pest locates the host plants using the specific volatiles that it emits and that the presence for stressed trees can contribute to the attraction of \u003cem\u003eP. castaneus\u003c/em\u003e to the site.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq-INCT \u0026ldquo;Controle Biorracional de Insetos Praga\u0026rdquo;) and Funda\u0026ccedil;\u0026atilde;o Arauc\u0026aacute;ria for financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Funda\u0026ccedil;\u0026atilde;o Arauc\u0026aacute;ria and Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGustavo Frensch, Scheila R. M. Zaleski, Francisco A. Marques, Beatriz H. L. N. S. Maia and Sonia M. N. Lazzari contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gustavo Frensch, Scheila R. M. Zaleski and Marina Krasniak. Statistical analysis was perfomed by Gustavo Frensch and Liliane G. Dantas. The first draft of the manuscript was written by Gustavo Frensch and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAdams RP (2007) Identification of Essential Oil Components by Gas Chromatography/Mass Espectrometry. Allured Publishing Corporation, Illinois\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAhrens S (2000) Manejo e Silvicultura de Planta\u0026ccedil;\u0026otilde;es de Pinus na Pequena Propriedade Rural. 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Environ Entomol 24:372\u0026ndash;379\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemoecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chem","sideBox":"Learn more about [Chemoecology](https://www.springer.com/journal/49)","snPcode":"49","submissionUrl":"https://submission.nature.com/new-submission/49/3","title":"Chemoecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"banded pine weevil, induced volatiles, infochemicals, loblolly pine","lastPublishedDoi":"10.21203/rs.3.rs-1696409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1696409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConiferous trees of the genus \u003cem\u003ePinus\u003c/em\u003e (Pinaceae) are under continuous threats by numerous herbivorous insect species and pathogens attacking nearly all parts and tissues of the plants. To defend themselves, pine trees produce large amounts of oleoresin that is accumulated in a highly developed network of specialized resin ducts, which are distributed in the wood, bark, and needles. Such defense reactions in pines can be induced by the attack of herbivores. The banded pine weevil, \u003cem\u003ePissodes castaneus\u003c/em\u003e (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of \u003cem\u003ePinus\u003c/em\u003e in Brazil, where it has been an invasive species since 2001. The female lays its eggs under the tree bark of trees and the larvae feed in the phloem of the trunk and branches, interrupting the sap circulation and eventually causing its death. In the present study, we conducted detailed GC\u0026ndash;MS analyses of volatiles emitted by twigs of \u003cem\u003ePinus taeda L\u003c/em\u003e. We analyzed how the attack by \u003cem\u003eP. castaneus\u003c/em\u003e males and females affects the volatile pattern emitted by the twigs. When comparing volatiles produced by healthy plants and by female- and male-attacked \u003cem\u003eP. taeda\u003c/em\u003e, qualitative and quantitative differences were detected, as the decreased production of limonene, germacrene D and (\u003cem\u003eE\u003c/em\u003e)-caryophyllene and the increase of α-pinene. Laboratory bioassays showed that plants attacked by male and female \u003cem\u003eP. castaneus\u003c/em\u003e were more attractive to the insects. Understanding about what compounds may attract or repel the insects may help in the development of more effective traps, as well as preventing stress to avoid infestation.\u003c/p\u003e","manuscriptTitle":"Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: Laboratory and Field Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-08 15:41:45","doi":"10.21203/rs.3.rs-1696409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2022-09-30T06:39:13+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-06-10T12:50:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-02T09:09:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-01T20:41:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemoecology","date":"2022-05-26T08:43:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemoecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chem","sideBox":"Learn more about [Chemoecology](https://www.springer.com/journal/49)","snPcode":"49","submissionUrl":"https://submission.nature.com/new-submission/49/3","title":"Chemoecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d9038aec-a67e-4cf4-a05a-2ae83ca47d76","owner":[],"postedDate":"June 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:15:28+00:00","versionOfRecord":{"articleIdentity":"rs-1696409","link":"https://doi.org/10.1007/s00049-023-00383-1","journal":{"identity":"chemoecology","isVorOnly":false,"title":"Chemoecology"},"publishedOn":"2023-06-10 21:06:48","publishedOnDateReadable":"June 10th, 2023"},"versionCreatedAt":"2022-06-08 15:41:45","video":"","vorDoi":"10.1007/s00049-023-00383-1","vorDoiUrl":"https://doi.org/10.1007/s00049-023-00383-1","workflowStages":[]},"version":"v1","identity":"rs-1696409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1696409","identity":"rs-1696409","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}