DOI: 10.5061/dryad.kh18932bd
title: More to legs than meets the eye: Presence and function of pheromone compounds on heliothine moth legs

description: 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.
description: # 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, 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).