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START readme
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## Authors: Qi Wang, Yufei Jia, Hans M. Smid, Berhane T. Weldegergis, Liana O. Greenberg, Maarten Jongsma, Marcel Dicke, Alexander Haverkamp

#Contact: alexander.haverkamp@wur.nl
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## Readme for data underlying the publication "Loss of olfaction reduces caterpillar performance and increases susceptibility to a natural enemy"

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## Introduction

This project aims to evaluate the significance of olfaction in a butterfly species Pieris brassicae. By knocking out the odorant receptor co-receptor (Orco), the larval brain structure and adult oviposition changes confirmed the successful knockout. The changes in knockout caterpillars in optimal host-plant seeking, performance under the attack of natural enemies and behavioral preference to plant volatiles suggested the critical importance of olfaction in caterpillars in the tri-trophic interactions.

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## Methods:

#Axon tracing
L4 caterpillars of similar head size were anesthetized on ice and immobilized in clay. The tip of each antenna was peeled, and a layer of petroleum jelly (Vaseline) was applied around the antenna. A 0.5 µL drop of 2.5 % biotin-dextran solution was added to the cut and covered with petroleum jelly, then incubated at 4 °C overnight. Brains were dissected in PBS, fixed in 4 % formaldehyde in PBS at 4 °C overnight, dehydrated in xylene and rehydrated in PBS-T, followed by three washes in PBS-T for 2 hours each. Brains were then incubated in 10 % NGS in PBS-T for 1 hour, followed by incubation in 1:200 streptavidin 488 and 1:1000 To-Pro iodide in PBS-T at 4 °C for three days. After further rinsing in PBS-T overnight, brains were mounted in DPX. 


#Electroantennography (EAG)
Filter papers loaded with 10 µL of selected chemicals, diluted in paraffin oil (10-2 v/v), were inserted into Pasteur pipettes, clean filter paper was employed as negative control. The left antennae of butterflies were excised at the basal end and the distal tip was also removed for better conductivity. Antennae were placed into glass capillaries filled with EAG Ringer solution. The first puff of each chemical was abandoned before the first round of testing. Antennae were exposed to the chemicals in random order and were given intervals of at least 20 s for recovering until the baseline stabilized.


#Oviposition
A mating pair of newly emerged butterflies were put in a cage and supplied with a four-week-old Brussels sprouts cabbage plant as oviposition substrate and sugar water as nutrients. The numbers of eggs were counted on a daily basis for seven days. The female butterflies were then collected and dissected to compare mating frequency between WT and KO females.


#Caterpillar performance on plant
A group of ten L1 caterpillars was placed on one leaf of a four-week-old cabbage plant. Caterpillars were reared for ten days, and more plants were provided when needed. The caterpillars were then collected for weighing to compare the growth between the two genotypes. 


#Caterpillar performance on plant under disarmed parasitoids
Groups of ten L1 caterpillars were placed on individual four-week-old cabbage plants as described above. The parasitoid wasps were anesthetized on a CO2 plate and the ovipositor was removed. Two disarmed parasitoid wasps were released in each rearing cage 24 hours after the caterpillars had been placed on the plants. The disarmed parasitoid wasps were replaced by newly disarmed parasitoids every day, the caterpillars were reared on the plants for ten days and then collected for weighing. 


#Caterpillar performance on plant under unmanipulated parasitoids
A group of ten L1 caterpillars was placed on a leaf of a four-week-old cabbage plant two days prior to placing two female parasitoid wasps. The parasitoid wasps were replaced every day to ensure that caterpillars were always exposed to natural enemies. The caterpillars were collected after ten days to count the number that survived; the collected caterpillars were further reared until they pupated or C. glomerata larvae emerged. 


#Larval host-plant seeking: Cabbage vs Tomato vs Paper
A single L3 caterpillar was then placed at the center of the arena. The caterpillars were provided with a choice between either (i) a cabbage leaf disc and a paper disc, (ii) a tomato leaf disc and a paper disc, or (iii) a cabbage leaf disc and a tomato leaf disc. A choice was recorded when caterpillars began feeding on the cabbage leaf disc or contacted the tomato leaf / green paper discs. 


#Larval host-plant seeking: healthy plant vs infested plant vs infested plant with wasps
A Y-tube olfactometer (diameter 35 mm) with a built-in black Y-shaped wire, allowing caterpillars to crawl along the wire was employed. Each arm was provided with a 1.1 L/min inlet airflow which had been purified by charcoal and humidified by water before entering the treatment jars. Healthy plants were untreated, infested plants were infested with 30 early L3 caterpillars for 24 hours and with caterpillars on plants while testing. Differently treated plants were placed in jars to test caterpillar behaviors. The caterpillars had been starved for approximately 5 hours before testing.


#volatile analysis
Volatiles were collected from L3 P. brassicae caterpillars, three-day-old female C. glomerata wasps, P. brassicae caterpillars and C. glomerata wasps together, caterpillar frass collected from L3 caterpillars and caterpillar saliva also collected from L3 caterpillars using glass capillaries. Fresh frass and saliva samples were stored in a freezer at -20 °C and thawed overnight at room temperature before volatile collection. Headspace collection of volatiles was performed from five treatments: 20 female wasps (Cg) in a 10 mL glass vial; 20 L3 caterpillars (Pb) in a 10 mL glass vial; 20 L3 caterpillars and 2 female wasps (Pb-Cg) in a 10 mL glass vial; 100 mg caterpillar frass (Pb-Fr) in a 1.5 mL glass vial; and 50 µL caterpillar saliva (Pb-S) in a 1.5 mL glass vial. Clean empty glass vials (10 mL and 1.5 mL) were used as a negative control. Volatiles were collected through dynamic headspace sampling using Tenax TA adsorbent material. Synthetic air at 110 mL min-1 to the samples in 10 mL vials and 55 mL min-1 to the samples in 1.5 mL vials were constantly supplied as a carrier for the volatiles, while simultaneously the volatiles were trapped by drawing air at 100 and 50 mL min-1, respectively, through a stainless-steel tube filled with 200 mg Tenax TA for 2 h.
The collected volatiles were thermally released from the Tenax TA adsorbent using an Ultra 50:50 thermal desorption unit at 250 °C for 10 min under a helium flow of 20 mL min-1, while simultaneously re-collecting the volatiles in a thermally cooled universal solvent trap: Unity at 0 °C. Once the desorption process was completed, the volatile compounds were released from the cold trap by ballistic heating at 40°C s-1 to 280 °C, which was then kept for 10 min, while all the volatiles were transferred to a ZB-5MS analytical column (30 mL × 0.25 mm ID × 1 mm F.T. with 10 m built-in guard column), placed inside the oven of a Thermo Trace GC Ultra , for separation of volatiles. The GC oven temperature was initially held at 40 °C for 2 min and was immediately raised at 6 °C min-1 to a final temperature of 280°C, where it was kept for 4 min under a constant helium flow of 1 mL min-1. For the detection of volatiles, a Thermo Trace DSQ quadrupole mass spectrometer coupled to the GC was operated in an electron impact ionization (EI) mode at 70 eV in a fullscan mode with a mass range of 35 - 400 amu at 4.70 scans s-1. The MS transfer line and ion source were set at 275 and 250 °C, respectively. 
Automated baseline correction, peak selection (S/N > 3) and alignments of all extracted mass signals of the raw data were processed following an untargeted metabolomic workflow using MetAlign software, producing detailed information on the relative abundance of mass signals representing the available metabolites. This was followed by the reconstruction of the extracted mass features into potential compounds using the MSClust software through data reduction by means of unsupervised clustering and extraction of putative metabolite mass spectra. Tentative identification of volatile metabolites was based on a comparison of the reconstructed mass spectra with those in the NIST 2008 and Wageningen Mass Spectral Database of Natural Products MS libraries, as well as experimentally obtained linear retention indices (LRIs). 


#Caterpillar behavioral preference chemicals in Y-tube olfactometer
L3 caterpillars were tested in a custom-made set-up consisting of 25 separate arenas. Single caterpillars were placed in the central well of the individual arenas before the test. Caterpillars were given a choice between a selected volatile chemical and paraffin oil. Input odors were delivered at 1.1 L min-1 for both chemicals and solvent controls, the odors were sucked out at 2.2 L min-1 at the same time from the center. The arena was cleaned by ethanol and dried by high-pressure air. The position of chemicals was exchanged after each test. 


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## Folder content:

-- Orco_raw_data.xlsx
Contains datasheets related to caterpillar glomeruli, butterfly oviposition, caterpillar development on plants, caterpillar behaviors and performance under attack of natural enemies of both WT and GO animals. 
These datasheets can be used to check results presented in the publication.

-- Orco_eagdata_male.csv
Contains one datasheet of male butterfly antennal responses to chemical compounds of both genotypes. 
It can be used to check results presented in the publication.

-- Significance_male.csv
Contains one datasheet of significant difference test results between WT and KO male butterflies for annotating significant difference tests.
It can be used to check results presented in the publication.

-- Orco_eagdata_female.csv
Contains one datasheet of female butterfly antennal responses to chemical compounds of both genotypes. 
It can be used to check results presented in the publication.

-- Significance_female.csv
Contains one datasheet of significant difference test results between WT and KO female butterflies for annotating significant difference tests.
It can be used to check results presented in the publication.

-- Orco_script.R
Contains R scripts for all figures in the publication.
It can be used to repeat the figures presented in the publication with datasheets mentioned above.

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This sections explains each datasheet in the excel "Orco_raw_data.xlsx" and csv forms related to EAG results

## DATASHEETS IN "Orco_raw_data.xlsx" :

- glomeruli: the number of glomeruli in both WT and KO larval antennal center.

- Spermatophores: the number of spermatophores dissected in both WT and KO female butterflies from the oviposition experiment.

- Oviposition: the number of eggs on each plant every day laid by WT or KO female butterflies from the oviposition experiment.

- Development Plant: WT and KO caterpillar weight gain on the plant in the period of 10 days.

- Discs_Tomato_Paper: WT and KO caterpillar host-plant seeking when tomato leaf disc and paper disc were provided.

- Discs_Tomato_Cabbage: WT and KO caterpillar host-plant seeking when tomato leaf disc and cabbage leaf disc were provided.

- Discs_Cabbage_Paper: WT and KO caterpillar host-plant seeking when cabbage leaf disc and paper disc were provided.
 
- Y-tube_Healthy_No plant: WT and KO caterpillar host-plant locating when healthy plant and no plant (empty jar) were provided.

- Y-tube_Infested_No plant: WT and KO caterpillar host-plant locating when caterpillar-infested plant and no plant (empty jar) were provided.

- Y-tube_Wasp_No wasps: WT and KO caterpillar host-plant locating when caterpillar-infested plant without parasitoids and caterpillar-infested plant with parasitoids were provided.

- Development Disarmed：WT and KO caterpillar weight gain on plants under the pressure of parasitoids those ovipositors have been removed. 

- Survival: WT and KO caterpillar survival rate on plants under the attack of mated female parasitoids.

- behavior_multi-arena: WT and KO caterpillar behavioral choice when a synthetic chemical compound and paraffin oil are provided.

- volatile analysis: The components of abundance of caterpillar- and parasitoid-associated volatile blends. 

- FigS1Hatching: WT and KO caterpillar hatching rate from the female butterfly oviposition experiment

- FigS2CaterpillarInDish: WT and KO caterpillar development when a single leaf was provided in each Petri dish.



## DATASHEETS IN "eagdata_male.csv":

- eagdata_male.csv: EAG responses of both WT and KO male butterflies to the selected chemicals. Each column represents the EAG responses of each male butterfly antennal response to all chemicals.


## DATASHEETS IN "eagdata_female.csv":

- eagdata_female.csv: EAG responses of both WT and KO female butterflies to the selected chemicals. Each column represents the EAG responses of each female butterfly antennal response to all chemicals.



## DATASHEETS IN "Significance_male.csv"

- Significance_male.csv: Significant difference tests between WT and KO male butterfly EAG responses for each chemical.


## DATASHEETS IN "Significance_female.csv":

- Significance_female.csv: Significant difference tests between WT and KO female butterfly EAG responses for each chemical.



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## SoftwareRequired: 

+ version 4.4.0
+ Rstudio 2023.12.1 

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## FileFormats:

+ .R 
+ .xlsx
+ .csv
+ .txt

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END readme
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