DOI: 10.5061/dryad.rxwdbrvn1
title: Data from: Size-dependent behavioral and antennal responses to doses of (+)-isopinocamphone and 1,8-cineole mixed with pheromone: a potential host selection strategy in female Ips typographus L.

description: Context:Ips typographus, a major pest of Norway spruce (Picea abies) in
 Europe, is experiencing more frequent outbreaks due to climate change.
 These outbreaks involve shifts in population dynamics and phenotypic
 traits, influencing beetle responses to olfactory cues from stressed host
 trees. Aims:The study examines the size-dependent behavioral and antennal
 responses of female I. typographus to two host selection–deciding
 volatiles with contrasting ecological roles: 1,8-cineole, which inhibits
 attraction to unsuitable trees, and (+)-isopinocamphone, a pheromone
 synergist. Size-linked morphological and olfactory adaptations may
 influence females' ability to select suitable host trees for
 reproduction. Methods:In field trap experiments conducted in 2019 and
 2022, the body size of I. typographus females caught in response to
 different doses of (+)-isopinocamphone or 1,8-cineole in combination with
 pheromone was compared. Female Ips typographus were sorted based on body
 length, the size of the antennal club was measured, and size-dependent
 antennal responses to these volatiles were analyzed using
 electroantennography. Results:Larger females were more attracted to
 (+)-isopinocamphone in combination with pheromone in the field, showed
 stronger antennal detection, and had proportionally larger antennal clubs
 than smaller females. In contrast, smaller females were less repelled by
 1,8-cineole added to pheromone, but, in contradiction, antennally detected
 it more strongly than larger females despite having smaller antennal
 clubs. Conclusion: The total body length significantly influences
 semiochemical detection in I. typographus females. (+)-isopinocamphone was
 detected more effectively by larger females, implying an advantage in the
 selection of suitable host trees. In contrast, the discrepancy between
 behavioral and antennal responses to 1,8-cineole in smaller females
 suggests involvement of not only peripheral detection but also central
 nervous processing of olfactory signals driving behavior. This adaptation
 may enable smaller females to reduce competition with large ones by
 selecting less suitable trees. These findings provide new insights into
 the ecological relationship between beetle morphology and olfactory cues,
 with implications for tree–bark beetle interactions.
description: <strong>1) Study Location and
 years</strong>: The experimental areas: Norway
 spruce forest, the natural habitat of <em>Ips
 typographus</em>, at 600 m a.s.l. within the Forest CZU property,
 Kostelec nad Černými lesy, Czech Republic. 
 2019 site: Coordinates 49°56′02″N, 14°52′21″E.
 2022 site: Coordinates 49°55′57″N, 14°55′13″E.
 <strong>2) Time Frame and field
 experiments</strong>:      
  Experiments ran from June 3 to July 28 in both 2019 and 2022.
 <strong>2.1 Trap Type</strong>:
 Cross-vane Ecotraps (Fytofarm, Slovak Republic) were
 used for the experiments. 
 <strong>2.2 Trap Placement</strong>:
 Traps were positioned 15 meters apart, 1.5 meters
 above the ground, and approximately 30 meters from the forest
 edge. <strong>2.3 Experimental
 Design (2019)</strong>: Seven traps were
 used. Six traps were baited with a
 pheromone and three different doses (low, medium, high) of either
 1,8-cineole or (+)-isopinocamphone. One control trap
 was baited with pheromone alone. 
 <strong>2.4  Experimental Design
 (2022)</strong>: Two experimental blocks were
 established: One block tested 1,8-cineole
 dose. The second block tested (+)-isopinocamphone
 dose. Each block included a pheromone control trap to
 ensure reliable comparisons. Experimental setup are
 detailed in Moliterno et al. (2023).  
 Field effects of oxygenated monoterpenes and estragole combined
 with pheromone on attraction of <em>Ips typographus</em> and
 its natural enemies. <em>Front. For. Glob.
 Change</em> 6:1292581. doi: 10.3389/ffgc.2023.1292581
 2.5 Morphological Measurements i. Insects
 <strong>Study
 Subject</strong>: <em>Ips typographus</em> (F0
 generation) emerged from naturally infested Norway spruce logs (n = 12;
 ~50 x 28 cm). **Logs: **Tree logs were collected in
 Kostelec nad Černými Lesy during June–July 2024. 
 <strong>Selection Criteria</strong>: Only female insects (~3
 days old) were selected after sex sorting for further measurements and
 experiments. <strong>Size Groups
 Considered</strong>: <strong>Large-sized
 females</strong>: Body length ≥ 4.80 mm (n = 30).
 <strong>Small-sized females</strong>: Body length ≤
 4.70 mm (n = 30). 
 ii. Dose-Responses to Pheromones, 1,8-Cineole, and
 (+)-Isopinocamphone in Laboratory Experiments 
 <strong>Subjects Tested</strong>: Only female
 insects. <strong>Size Groups</strong>:
 <strong>Large-sized females</strong>:
 Body length ≥ 4.80 mm (n = 10). 
 <strong>Small-sized females</strong>: Body length ≤ 4.70 mm (n
 = 10). 
 <strong>Exclusion</strong>: The analysis did not include
 damaged insects (broken or missing elytra). 
 <strong>2.6 Statistical
 Analysis</strong> 
 <strong>Preliminary Tests</strong>: Data
 normality and variance homogeneity were assessed using the Shapiro-Wilk
 test and Levene’s test. 
 <strong>Dependent and Independent Variables</strong>:
 <strong>Dependent Variable</strong>:
 Body length. <strong>Independent
 Variable</strong>: Doses of tested compounds. 
 <strong>Comparative Analysis</strong>:
 Generalized Linear Model (GLM).
 The model employed a Gamma distribution with a log link
 function. Post hoc comparisons
 were conducted with the Tukey test, followed by a multiplicity correction
 using the Bonferroni method. 
 <strong>Isometric vs. Allometric Relationships</strong>:
 Assessed via the Standardized Major Axis (SMA) method
 for antennal clubs of large and small females; 
 <strong>Electroantennography
 Analysis</strong>: Compared responses of large
 and small females (n = 10 each) using the exact Wilcoxon rank-sum
 test; <strong>Software
 and alpha value</strong>: Analyses were
 performed in RStudio (version 4.1.1). Significance
 level (α) was set at 0.05. 
 <strong>Additional Information</strong>: 
 For further details, please refer to the related
 article.
description: # Data from: Size-dependent behavioral and antennal responses to doses of
 (+)-isopinocamphone and 1,8-cineole mixed with pheromone: a potential host
 selection strategy in female Ips typographus L. Dataset DOI:
 [10.5061/dryad.rxwdbrvn1](10.5061/dryad.rxwdbrvn1) ## Description of the
 data and file structure Size-dependent behavioral and antennal responses
 to doses of (+)-isopinocamphone and 1,8-cineole mixed with pheromone: a
 potential host selection strategy in female *Ips typographus* L. ####
 **Description of the data and file structure** ##### **Read the
 instructions step-by-step:** ###### Trapping experiments: Plot, traps and
 tested area The trapping experiments were conducted in 2019 and 2022 at
 the Forest CZU property in Kostelec nad Černými lesy, Czech Republic. The
 experiments took place in a mature, 100-year-old Norway spruce forest, a
 natural habitat for *I. typographus*, located at 600 m above sea level. In
 2019, the experiment was conducted at coordinates (49°56′02″N,
 14°52′21″E), while the 2022 experiment took place at (49°55′57″N,
 14°55′13″E). Both experiments were conducted during the same time frame:
 June 3 to July 28 of each year. In both the 2019 and 2022 experiments,
 traps were set up approximately 30 m from the forest edge in a 2-year-old
 clearing. The average temperature was 2019 16 °C (June), 18 °C (July), and
 2022 17 °C (June), 19 °C (July). They were arranged in a row, with a
 minimum distance of 15 m between each trap, and were installed on wooden
 poles 1.5 m above the ground. In 2019, seven cross-vane Ecotraps
 (Fytofarm, Slovak Republic) were used for collecting data for this
 experiment: three traps were baited with three different doses (low,
 medium, high) of 1,8-cineole or (+)-isopinocamphone, respectively, in
 combination with the synthetic pheromone. One trap was baited with
 pheromone alone and served as a control (for bait composition). To
 minimize positional bias, the positions of the tested baits among these
 seven traps were changed seven times according to a randomization scheme.
 (see the original article for more details). ###### General information
 with description of units used in this work: measure for body size length:
 millimeter (mm) measure for antennal club in length and width: micrometer
 (μm) µg= microgram and/or ng= nanogram mg= milligram ###### Doses tested
 in field experiment: Low (L), Medium (M), High (H)= treatment doses
 of 1,8-cineole or (+)-isopinocamphone) and control (pheromone=
 2-Methyl-3-buten-2-ol and Cis-verbenol) in milligram per day (mg/day).
 Nominal doses (see the original article for more details). L: 0.1 mg/day +
 control M: 1 mg/day + control H: 10 mg/day + control Control: 10:1
 (2-Methyl-3-buten-2-ol : Cis-verbenol) mg/day ###### Doses tested in
 laboratory experiment:  Dose–response tests were conducted using an
 aggregation pheromone in a 10:1 ratio of 2-methyl-3-buten-2-ol (MB) to
 cis-verbenol (cV). 1,8-cineole doses: 0.001 µg, 0.01 µg, 0.1 µg, 1 µg, 10
 µg, 100 µg, and 1000 µg. (+)-isopinocamphone doses: 0.001 µg, 0.01 µg, 0.1
 µg, 1 µg, 10 µg, 100 µg, and 1000 µg. ##### General instructions: Step 1:
 Download the file named: Data_females_ips_typographus.7z **Step 2:**
 Extract files; Here, the files were named as: 1. Field data_bodysize_It 2.
 Sma_Regression 3. EAG_analysis_dose responses **# the end.** ### Files and
 variables #### File: Data_females_ips_typographus.7z
 **Description:** **software, code and file name** The software used was R
 version 4.3.1 / RStudio 2024.04.2. The code is possible to be accessed in
 section "Files". The file name: code files_field and laboratory
 experiment with I. typographus females.txt Brief description: the code is
 presented in txt. format. The file also is dived in step-by-step, 1. Field
 data_bodysize_It, 2. Sma_Regression and 3. EAG_analysis_dose responses, as
 explained in this readme.  Therefore, following the README description and
 file code information is needed. Below is presented a description
 regarding to the file name: *code files_field and laboratory experiment
 with I. typographus females* **data folder: 1. Field data_bodysize_It**
 The folder contains the dose-response field experiments conducted in 2019
 and 2022, with the compounds and years named as follows. Insects were
 collected and measured for body size length in millimeters.
 **cine.2019.csv** **cine.2022.csv** **ipc.2019.csv** **ipc.2022.csv**
 **where is ipc =** (+)-isopinocamphone; cine= 1,8-cineole followed by
 respective experiment year **#Step 3: below are listed files save as .csv
 extension (same legend as above)** 1.Ph = Pheromone alone (control)\
 2.Ph.H = Pheromone + oxygenated monoterpenes at high dose\ 3.Ph.M =
 Pheromone + oxygenated monoterpenes at medium dose\ 4.Ph.L = Pheromone +
 oxygenated monoterpenes at low dose \#Step 4: Follow the respective R
 script for folder for further instruction. **# the End.** **Data folder:
 2. Sma_regression** Analysis of female *I. typographus* antennal
 measurements (measured for antennal club in length and width in micrometer
 SMA= (Standardised) Major Axis Estimation. **Step 1:** Open the data from
 the folder: Sma Regression **Step 2:** open the csv file called:
 "SMA.1" The data presented here represent the log-transformed
 measurements of the antennal club's length and width, expressed in
 micrometers **SMA.1 legend: Group:** small and large represent the body
 size of females *Ips typographus* **Log.leng:** represents the measurement
 of antennal club length; **Log.wid:** represents the measurement of
 antennal club width **Step 3: Follow the respective R script for (2)
 Sma_Regression for further instruction.** **# the End.**
 **##########################################** **Data folder:**
 3.EAG_analysis_dose responses Data from small and large of females *Ips
 typographus* EAG= electroantennography analysis **Step 1:** Open the data
 from the folder: 3.EAG analysis_dose responses \#Each folder from
 "EAG analysis_dose responses" provided (+)-isopinocamphone,
 1,8-cineole or Pheromone from females of *Ips typographus* L. 1758). \#Two
 group were tested: larger than 4.80 mm (n= 10) versus smaller than 4.70
 mm. The size was selected based on field experiment results.
 **Dose-response labels each from files, (+)-isopinocamphone, 1,8-cineole
 or Pheromone:** Dose–response tests were conducted using an aggregation
 pheromone in a 10:1 ratio of 2-methyl-3-buten-2-ol (MB) to cis-verbenol
 (cV). µg= microgram ng= nanogram 1,8-cineole doses: 0.001 µg, 0.01 µg, 0.1
 µg, 1 µg, 10 µg, 100 µg, and 1000 µg. (+)-isopinocamphone doses: 0.001 µg
 (or 1ng), 0.01 µg (or 10ng), 0.1 µg (100 ng), 1 µg, 10 µg, 100 µg, and
 1000 µg (or 1 mg). \#eag.1ng\ \#eag.10ng\ \#eag.100ng\ \#eag.1ug\
 \#eag.10ug\ \#eag.100ug\ \#eag.1mg **#Step 2:** open the csv accordingly:
 (+)-isopinocamphone, 1,8-cineole or Pheromone **#Both files mentioned
 above have the same columns and labels as follows:** **#id=** number of
 beetles tested **#eag =** stimulated dose response obtained, here IPC_eag
 = (+)-isopinocamphone, Cine_eag = 1,8-cineole or Phero_eag = Pheromone.
 **#group.eag =** size category small or large. **#Step 3:** Follow the
 respective R script for EAG_analysis_dose responses for further
 instructions. **# the End.**