More to legs than meets the eye: Presence and function of pheromone compounds on heliothine moth legs
Data files
Feb 16, 2024 version files 171.88 KB
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bmega_rep1.csv
11.84 KB
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bmega_rep2.csv
11.84 KB
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bmega_rep3.csv
11.84 KB
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ecoli_rep1.csv
11.86 KB
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ecoli_rep2.csv
11.83 KB
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ecoli_rep3.csv
11.86 KB
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FirstChoice.csv
1.48 KB
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GCAnalysis.csv
10.15 KB
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Inhibition_binary.csv
362 B
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Inhibition_data.csv
1.18 KB
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qPCR.csv
80.06 KB
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README.md
7.57 KB
Abstract
Chemical communication is ubiquitous in nature and chemical signals convey species-specific messages. Despite their specificity, chemical signals may not be limited to only one function. Identifying alternative functions of chemical signals is key to understanding how chemical communication systems evolve. Here, we explored alternative functions of moth sex pheromone compounds. These chemicals are generally produced in, and emitted from, dedicated sex pheromone glands, but some have recently also been found on the insects’ legs. We identified and quantified the chemicals in leg extracts of the three heliothine moth species Chloridea (Heliothis) virescens, Chloridea (Heliothis)subflexa, and Helicoverpa armigera, compared their chemical profiles and explored the biological function of pheromone compounds on moth legs. Identical pheromone compounds were present on the legs in both sexes of all three species, with no striking interspecies or intersex differences. Surprisingly, we also found pheromone-related acetate esters in leg extracts of species that lack acetate esters in their female sex pheromone. When we assessed gene expression levels in the leg tissue, we found known and putative pheromone biosynthesis genes expressed, which suggests that moth legs may be additional sites of pheromone production. To determine possible additional roles of the pheromone compounds on legs, we explored whether these may act as oviposition-deterring signals, which does not seem to be the case. However, when we tested whether these chemicals have antimicrobial properties, we found that two pheromone compounds (16:Ald and 16:OH) reduce bacterial growth. Such an additional function of previously identified pheromone compounds likely coincides with additional selection pressures and, thus, should be considered in scenarios on the evolution of these signals.
README: More to legs than meets the eye: presence and function of pheromone compounds on heliothine moth legs
Data repository JEB-2022-00299.R1 Zweerus et al. (2023)
Author names NL Zweerus, LJ Caton, L de Jeu, AT Groot
Contact details NL Zweerus, naomizweerus@gmail.com
Brief summary
Despite their specificity, chemical signals may not be limited to only one function. Identifying alternative functions of chemical signals is key to understanding how chemical communication systems evolve. Here, we explored alternative functions of moth sex pheromone compounds.
Responsibilities
NLZ, LJC and LdJ collected and analyzed the data
Overview of files and their content
GCAnalysis
To identify the chemical profiles of the legs of all three species and sexes, we extracted the chemical compounds from moth legs of three heliothine species. We analysed the samples in a gas chromatograph (GC) and compared the chemical profiles between species and between males and females.
To identify the chemical profiles of the legs of all three species and sexes, we extracted the chemical compounds from moth legs of three heliothine species. We analysed the samples in a gas chromatograph (GC) and compared the chemical profiles between species and between males and females.
The data file contains the following measurements and information:
Species - scientific name of species used in the study
sample_id - unique id of the collected sample
GC_id - identity of sample run in the gas chromatograph
Sex - M = male, F = female
16:Ald [all chemical compounds in absolute amounts = ng per moth (i.e., 6 legs pooled for extraction)]
16:OAc
Z11-16:OAc
16:OH
Total: sum of 16:Ald, 16:OAc, Z11-16:OAc, 16:OH
C25
C27
remark: any additional information noted by the data collector
qPCR
To determine whether moth legs could be the production site of several pheromone compounds, we quantified and compared the expression of three candidate genes putatively involved in acetate-ester production along with delta-11-desaturase (d11) in the following tissues: female sex pheromone glands (FG), male hairpencils (HP), and female (FL) and male front legs (ML). To obtain a comparison of the expression of the lesser-known candidate genes and the better-studied d11, we also determined the gene expression in the thorax tissue (T) of one species (C. subflexa).
The data file contains the following measurements and information:
Sample info:
species - scientific name of helitohine moth species
sex - M = male, F = female
line - selection line (only applicable for C. virescens, others marked NA = not applicable)
tissue - FL = front leg, G = pheromone gland, HP = hairpencils, T = thorax
nbr - identifier for tissue sample
Tech rep - technical replicate number
Run name - identifier for run in the qPCR machine
sample_no - number of sample
Ct-Threshold - set at 0.3
Reference gene:
Well - location in 96-well plate
ref gene - Eif4a hvhsha for all
Ct-ref - Ct value of reference gene
Target gene:
Well - location in 96-well plate
Gene - target genes as described in the study (primers can be found in supplementary material)
Ct (dRn) - Ct value of traget gene
FirstChoice
We evaluated whether the chemicals from moth legs could function as oviposition-deterring signals. To assess if compounds from legs have an oviposition-deterring effect, we tested how many females choose a control area compared to a site treated with leg extract to lay their first egg. For the assay, we visually divided the top of each cage into 4 equal cubic areas, numbered them, and assigned a treatment to the area randomly. We observed the activity of each female and noted the areas she oviposited eggs.
The data file contains the following measurements and information:
date: date of experiment
sample: sample identifier related to the date of experiment
extract: leg extract
hexane: hexane control
blank 1 & blank 2: filter-paper controls
Whether or not a female chose the respective area to lay an egg was recorded as 1 = oviposition, 0 = no oviposition.
Antimicrobial_properties
To assess antimicrobial properties of the pheromone compounds we found on moth legs, i.e. hexadecanal (16:Ald), hexadecanyl acetate (16:OAc), (Z)-11-hexadecenyl acetate (Z11-16:OAc) and hexadecanol (16:OH), we tested these compounds for their inhibitory effects on the growth of Bacillus megaterium (gram positive bacterium) and Escherichia coli strain DH5α (gram negative bacterium).
The data files and more detailed descriptions are available in the 2 separate folders:
1) Inhibition_zone
To assess if 16:Ald, 16:OAc, Z11-16:OAc, or 16:OH, can inhibit microbial growth, we tested synthetic pheromone compounds in inhibition-zone assays against two bacteria; B. megaterium and E. coli, and also the fungi N. niger and N. nomiae. We took pictures of every plate with a smart phone camera and determined the occurrence of inhibition zones as binary result. These 2 files contain the data:
1.1 Inhibition_binary.csv
We took pictures of every plate with a smart phone camera and determined the occurrence of inhibition zones as binary result.
1 = inhibition zone occurs
0 = no inhibition zone
"n/a" = not applicable
The raw data (as pictures) for Figure 4 are provided in the supplementary materials of the publication.
1.2 Inhibition_data.csv
We quantified the area of the inhibition zones for B. megaterium and E. coli in pixels / cm with ImageJ of the 16:Ald treatment.
The data sheet contains all the inhibition zones from both E.coli and B.megaterium and the inhibition zones from the positive controls.
2) Growth
We conducted a bacterial growth assay to determine the growth rate r and the maximum density (carrying capacity, k) of the bacterial culture in the given environment, which provides a quantitative measure of any inhibitory effects of the treatment. To quantify bacterial growth, we tested a range of concentrations of 16:Ald, 16:OAc, Z11-16:OAc, 16:OH and a control (hexane) in an optical density (OD) test on B. megaterium and E. coli DH5α in 96-well plates.
These 6 files contain the data:
bmega_rep1.csv
bmega_rep2.csv
bmega_rep3.csv
ecoli_rep1.csv
ecoli_rep2.csv
ecoli_rep3.csv
The data files bmega_rep1.csv,bmega_rep2.csv and bmega_rep3.csv contain the data of three replications of the experiment in which we tested the response of the bacterium B. megaterium to 5 concentrations of 16:Ald, 16:OAc, Z11-16:OAc and 16:OH in an optical density (OD600) test. The experiment was performed in 96-well plates for each replicate. Each well was filled with a medium with the bacteria B. megaterium and a compound at a specific concentration. We then measured the bacterial growth as optical density at 600nm (OD) at regular time intervals (time in hours). The column names of the file mean the following:
Hexane1 and hexane 2 are the controls.
ald = 16:Ald
oh = 16:OH
oac = 16:OAc
z = Z11-16:OAc
"compound"5 means 22.5 ng/µL of the compound (e.g., ald5 = 22.5 ng/µL of 16:Ald)
"compound"4 means 18 ng/µL of of the compound
"compound"3 means 13.5 ng/µL of of the compound
"compound"2 means 9 ng/µL of the compound
"compound"1 4.5 ng/µL of the compound
The data files ecoli_rep1.csv, ecoli_rep2.csv and ecoli_rep3.csv contain the data of three replications of a methodologically identical experiment but tested on E. coli instead of B. magaterium. These files have the data structure and column names (see above).