# Endocrine responses in the pheromone induction of male sexual maturation in an insect

Stéphane Debernard, Paleo Aguilar, Annick Maria, Annabelle Fuentes, Philippe Couzi, Françoise Bozzolan, Edmundo Gassias, Evan Force

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## To cite this version:

Stéphane Debernard, Paleo Aguilar, Annick Maria, Annabelle Fuentes, Philippe Couzi, et al.. Endocrine responses in the pheromone induction of male sexual maturation in an insect. Insect Biochemistry and Molecular Biology, 2025, 183, pp.104365. 10.1016/j.ibmb.2025.104365. hal-05175427

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Submitted on 22 Jul 2025

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# Endocrine responses in the pheromone induction of male sexual maturation in an insect

Stéphane Debernard<sup>a, *</sup> ◐, Paleo Aguilar<sup>b</sup>, Annick Maria<sup>a</sup>, Annabelle Fuentes<sup>a</sup>, Philippe Couzi<sup>c</sup>, Françoise Bozzolan<sup>a</sup>, Edmundo Gassias<sup>b</sup>, Evan Force<sup>a,c,**</sup> ◐

## ARTICLE INFO

### Keywords:

Agrotis ipsilon

Sex pheromone exposure

Sexual behavior

Antennal lobe

Accessory sex gland

Juvenile hormone

## ABSTRACT

In animals, sexual maturation is marked by the development of reproductive behaviors in synchronism with the acquisition of fertility, and this timing is influenced by chemosensory experiences. In naïve and immature individuals, exposure to sex pheromones may accelerate sexual development, and mechanisms underlying this pheromone induction are not fully identified. Using the moth *Agrotis ipsilon*, we showed that pre-exposure of immature males to female sex pheromones led to early increases in the performance of sex pheromone-triggered oriented flight as well as in the maturation of accessory sex glands (ASGs) producing seminal proteins. Conjointly, biosynthesis and circulating amounts of juvenile hormone (JH) raised with an upregulation of the expression of JH receptor, *Methoprene-tolerant 1* (*Met1*) and the JH-inducible transcription factor, *Krüppel homolog 1* (*Kr-h1*) in ASGs and the primary olfactory centers, the antennal lobes (ALs). In the sex pheromone pre-exposed immature males, the loss of function of *Met1* or *Kr-h1* caused a reduction in the induction of the sex pheromone behavioral responsiveness and the ASG secretory activity. Taken together, our results showed that the accelerated effects of sex pheromone pre-exposure on male sexual maturation are mediated by increased JH biosynthesis. This ultimately leads to early induction of JH signaling in ASGs for seminal protein production and in ALs for the central processing of pheromone information, which causes the display of sexual behavior in male *A. ipsilon*. Finally, this study expands our understanding of endocrine mechanisms by which animals can modulate their fitness according to past olfactory experiences.

## 1. Introduction

In animals, sexual maturation is a period of life during which the development of the reproductive behaviors, the initiation of sexual signaling as well as the acquisition of fertility occur (Roff, 1993; Stearns, 1992). The achievement of these behavioral and physiological events needs to be timely synchronized to prevent waste of reproductive effort and energy (Roff, 1993; Stearns, 1992). For instance, animals acquire the ability to respond to sexual signals only when they reach their full sexual maturity (Dey et al., 2015; Gadenne et al., 2016; Lin et al., 2016). This physiological synchrony is under the control of endocrine actors such as lipophilic hormones (i.e., steroids and sesquiterpenoid hormones) which act simultaneously on the reproductive and nervous systems through organizational and activation effects (Abrieux et al.,

2014; Bozzolan et al., 2015; Force et al., 2025; Force and Debernard, 2025; Lenz et al., 2012; Lin et al., 2016; Romeo, 2003).

The timing and duration of sexual maturation vary according to the internal state of the individual and are also heavily influenced by an array of environmental factors including temperature, photoperiod, and sexual signals (Bohlen et al., 2018; Foster et al., 1988; Mlingi et al., 2024; Norberg et al., 2001; Vandenbergh, 1989). Chief among sexual signals are pheromones which might accelerate sexual development. In rodents, exposure to urinary pheromones of dominant males was found to facilitate estrous cycling (Whitten, 1956), sexual receptivity (Wölfl et al., 2023), and maternal behavior in immature females (Larsen et al., 2008); and exposure of immature males to an adult female was shown to stimulate copulatory behavior (Batty, 1978; James and Nyby, 2002) and testicular development (Fox, 1968; Svare et al., 1978). In insects, and

<footnote><sup>a</sup> Sorbonne Université, Université Paris-Est Créteil, INRAE, CNRS, IRD, Institute of Ecology and Environmental Sciences of Paris, 75005, Paris, France</footnote>

<footnote><sup>b</sup> Institute of Biology, University of Madrid, Pozuelo de Alarcon, 28223, Madrid, Spain</footnote>

<footnote><sup>c</sup> Sorbonne Université, Université Paris-Est Créteil, INRAE, CNRS, IRD, Institute of Ecology and Environmental Sciences of Paris, 78026, Versailles, France</footnote>

<footnote>* Corresponding author. iEES Paris, Sorbonne Université, 4, Place Jussieu, 75005, Paris, France.</footnote>

<footnote>** Corresponding author. iEES Paris, Sorbonne Université, 4, Place Jussieu, 75005, Paris, France.</footnote>

<footnote>E-mail addresses: stephane.debernard@sorbonne-universite.fr (S. Debernard), evan.force@sorbonne-universite.fr (E. Force).</footnote>
more particularly in migratory locusts, exposure of immature females to male aggregation pheromone accelerated oocyte maturation and oviposition (Chen et al., 2022). Furthermore, there is evidence that the promoting effect of pheromone exposure on sexual maturation is mediated by changes in the activities of the endocrine system (Macrides et al., 1975; Maruniak and Bronson, 1976). Elucidation of the mechanisms underlying this endocrine plasticity is essential for a better understanding of how animals can optimize their fitness according to the environmental context in which they live.

In insects, sesquiterpenoid hormones, particularly juvenile hormones (JHs), are produced by a pair of cephalic endocrine glands called *corpora allata* (CAs) (Tobe and Stay, 1985). These hormones play a crucial role in larval development and metamorphosis. Beyond these functions, JHs are also well-known for coordinating sexual displays with reproductive maturity in both adult females and males. For instance, in flies like *Anastrepha obliqua* and *Anastrepha suspensa*, treatment of newly emerged males with Methoprene, a synthetic analog of JHs, accelerated sexual maturation. This resulted in early development of calling behavior and sexual signaling (Chacón-Benavente et al., 2013; Teal et al., 2000). Similarly, in the fruit fly *Drosophila melanogaster*, JH-deficient females exhibited delayed mating and impaired synthesis of sex pheromones (Bilen et al., 2013). In female moths *Pseudaleta unipuncta*, JH was found to stimulate sex pheromone synthesis, and calling behavior paralleled oocyte maturation (Cusson et al., 1990, 1994).

It is well-established that most of JH biological actions are mediated by a nuclear protein complex composed of two members of the basic helix-loop-helix/Per-ARNT-Sim family of transcription factors, namely the JH receptor Methoprene-tolerant (Met) (Jindra et al., 2015b) and its dimerizing partner, known as Taiman (Tai) (Charles et al., 2011; Jindra et al., 2015a; Li et al., 2011, 2014; Zhang et al., 2011). In the presence of JH, the Met-Tai heterodimeric complex binds to an E-box response element in the promoter regions of JH target genes, leading to their transcriptional regulation (Li et al., 2011, 2014; Zhang et al., 2011). Additionally, *Krüppel homolog 1* (*Kr-h1*), a C~2~H~2~ zinc finger-like transcription factor, was identified as an early JH response gene that functions downstream of the Met-Tai complex (Cui et al., 2014; Kayukawa et al., 2012). In various insect species, RNA interference experiments have demonstrated that Met, Tai, and Kr-h1 play crucial roles in the JH signaling pathway, regulating male and female sexual development (Aguilar et al., 2023; Gassias et al., 2021; Gujar and Palli, 2016; Holtof et al., 2021; Lin et al., 2016; Smykal et al., 2014; Song et al., 2014).

The present study aimed to investigate the potential role of JH in accelerating the effects of sex pheromone exposure on male sexual maturation. We used the moth *Agrotis ipsilon* as a model organism, known for its JH-dependent aspects of male reproductive maturation, particularly the development of both sexual behavior and accessory sex glands (ASGs) that produce seminal proteins (Aguilar et al., 2023; Gassias et al., 2021). Just after emergence, male moths are sexually immature and their ASGs have low protein content (Force et al., 2023a; Gassias et al., 2021). Additionally, these immature males are incapable of responding to female sex pheromones (Aguilar et al., 2023; Duportets et al., 1998; Force et al., 2024b; Gadenne et al., 1993). However, five days after emergence, males attain full sexual maturity, with well-developed ASGs and elevated protein synthesis (Force et al., 2023a; Gassias et al., 2021). Consequently, these mature males are highly attracted to female sex pheromones and exhibit oriented flight towards females (Aguilar et al., 2023; Duportets et al., 1998; Force et al., 2024b; Gadenne et al., 1993). This heightened behavioral responsiveness is attributed to increased sensitivity of neurons responding to the pheromone signal within the primary olfactory centers, the antennal lobes (ALs) (Anton and Gadenne, 1999; Gadenne and Anton, 2000). Furthermore, in male moths like A. ipsilon, CAs produce various types of JH acids, with JH-II acid being the most prominent form. JH-II acid is converted into JH-II by the activity of Juvenile hormone acid O-methyltransferase (JHAMT) in ASGs and subsequently released into hemolymph (Guo et al., 2021; Shirk et al., 1983). Interestingly, circulating

JH-II levels in sexually mature males were found to be higher than those in sexually immature males of *A. ipsilon*. This difference was partly attributed to an increase in JH-II biosynthesis by CAs with aging (Duportets et al., 1998). Recent studies have revealed that JH-II simultaneously acts on both ASGs and ALs through the activation of the Met-Kr-h1 pathway. This pathway promotes the synthesis of seminal proteins and central processing of the sex pheromone signal, ultimately leading to the execution of the orientation flight towards females (Aguilar et al., 2023; Force et al., 2024a; Gassias et al., 2021).

Here, we first examined in *A. ipsilon* the impact of sex pheromone pre-exposure of immature males on (i) the orientation flight towards pheromone source, (ii) the development of ASGs (length, protein synthesis), (iii) the *in vitro* JH-II acid biosynthesis and the JH-II circulating titers, and (iv) the expression levels of *Met1* and *Kr-h1* in ASGs and ALs. Finally, we investigated the behavioral responsiveness to signal pheromones and the maturation of ASGs in Met1-or Kr-h1-deficient immature males pre-exposed to sex pheromones. Taken together, our results demonstrated that accelerating effects of sex pheromone pre-exposure on male sexual maturation are mediated by increased JH biosynthesis, which promotes an early induction of JH signaling in ASGs for the production of seminal proteins, and ALs for the central processing of pheromone information causing the display of male sexual behavior in *A. ipsilon*.

## 2. Materials and methods

### 2.1. Insects

Our work was conducted on newly emerged male *Agrotis ipsilon* (Lepidoptera: Noctuidae). They were reared in our laboratory at Sorbonne Université, France. Rearing was carried out according to the protocol described by Force et al. (2023a) in order to minimize potential circadian effects in insect studies. Every morning, male adults were collected and placed in transparent boxes each containing 5 individuals (20 × 11.5 × 5 cm). The collection day was recorded as the first day of adult life. The moths had *ad libitum* access to a 12 % sugar solution.

### 2.2. Chemicals

Juvenile hormone-II (JH-II) was purchased from SciTech (Prague, Czech Republic). JH-II acid was obtained by an enzymatic procedure using pig liver carboxylesterase (Sigma-Aldrich, Saint-Quentin-Fallavier, France), in which three enzyme units converted 1 µmol of JH-II into JH-II acid in 1 h at 40 °C in a phosphate buffer (50 mmol/L, pH 7.4). The JH-II acid formed was subsequently purified by reverse-phase high-performance liquid chromatography.

We prepared a synthetic sex pheromone blend at 1 µg/µL containing three components identified in natural extracts of the pheromone glands of the female *Agrotis ipsilon* (Gemeno and Haynes, 1998; Picimbon et al., 1997): (Z)-7-dodecen-1-yl acetate (Z7-12:Ac), (Z)-9-tetradecen-1-yl acetate (Z9-14:Ac), and (Z)-11-hexadecen-1-yl acetate (Z11-16:Ac) mixed at a ratio of 4:1:4. This blend was proven to be the most attractive to males in field trapping experiments (Causse et al., 1988) and it elicits a similar behavior in a wind tunnel as natural extracts of the pheromone gland (Barrozo et al., 2010; Vitecek et al., 2013). The three components were purchased from Pherobank (Wijk bij Duurstede, Netherland) and diluted in hexane (>98 % purity, CAS 110-54-3, Carlo-Erba, Val-de-R euil, France). A dose of 100 ng of the sex pheromone blend was used for the behavioral experiments in a wind tunnel, and this dose had previously been described as behaviorally and electrophysiologically active (Deisig et al., 2014; Force et al., 2024b; Gadenne et al., 1993; Gassias et al., 2019).

### 2.3. Pre-exposure experiments

Pre-exposure treatments were performed in newly emerged males.
Within a transparent box containing 5 individuals (20 × 11.5 × 5 cm), males were subjected to 10 ng, 100 ng, or 1 µg of sex pheromone stimulation for 3 min, after which they were removed and transferred to a new rearing box. The behavioral and physiological responses of the pre-exposed males were analyzed at 2 h, 24 h, 48 h, and 72 h after pre-exposure to sex pheromones. Control groups were composed of males pre-exposed to hexane for 3 min.

## 2.4. Behavioral experiments to sex pheromones in wind tunnel

The study of behavioral response to female sex pheromones in pre-exposed male *A. ipsilon* was carried out using a Plexiglas® wind tunnel (VT Plastics, Genevilliers, France) measuring 188.5 cm (length) × 73 cm (width) × 72 cm (height). This device had 2 side hatches. Upstream of the tunnel was a filtration chamber consisting in a particle filter and an activated carbon filter. Downstream, the wind tunnel included an extraction hood, which generated an air stream in it by evacuating the air outside the building (0.3 m/s). In addition, at the downstream end of the device, a cage was positioned (28 cm high and at 36.5 cm width) to host the moth before the start of the experiment. At the other end, a glass vial containing 1 µL of odorant (deposited on a Wattman filter paper) was placed on a platform 26 cm above the tunnel floor and at 36.5 cm width; this vial was renewed every 4 moths (approximatively every 20 min). A Teflon tube connected to the vial was connected to a stimulation system composed of solenoid valves controlled by a ValveBank (Valve-BankTM 4&amp;8 II, AutoMate Scientific, Inc., Berkeley, USA). An electronic flow meter (Digital flow switch, PFM7 Series, SMC Corporation) set the flow rate of air entering the vial at 25 mL/min. The experiments were performed at a temperature and relative humidity respectively equal to 23 °C and 50-60 %. The room was into darkness and the wind tunnel was side-lit by an array of 11 infrared projectors (5W, λ = 850 nm). The tunnel floor was covered with a black cloth.

Moths were moved to the experiment room and kept in the dark at least 2 h before starting experiments to allow them to acclimatize. The experiments were carried out between 13:30 and 16:00, the period of the scotophase during which the moths are most active (Barrozo et al., 2010; Force et al., 2023b). For each test, we placed an animal in the cage within the wind tunnel and directly started the olfactory stimulation for 3 min 100 ng of female sex pheromones were tested for 2 h, 24 h, 48 h, 72 h pre-exposed males. The order of moths' passage was randomized, and each moth was only tested once. A 2-min delay was observed between each test.

The behavioral response of male *A. ipsilon* to female sex pheromones is characterized by an oriented flight (flight in zigzags against the airflow and beyond half of the tunnel). The observation of this behavior occurred with 2 wide-field infrared-sensitivity cameras (Basler Pilot Ace, piA640-210m, Tamron 4-12F/1.2 lens) placed above the wind tunnel.

## 2.5. Tissue collections

Antennal lobes (ALs), *corpora allata* (CAs), and accessory sex glands (ASGs) were collected from pre-exposed males during dissections carried out in the middle of scotophase (between 1:00 p.m. and 4:00 p.m.) when animals were most active (Barrozo et al., 2010; Force et al., 2023b), under a Leica S9D stereomicroscope. Tissue collection was carried out in Ringer's solution (6.13 % glucose, 0.24 % HEPES, 0.11 % MgCl₂, 0.07 % NaCl, 0.05 % KCl, 0.01 % CaCl₂).

## 2.6. ASG length and protein content

The length of excised ASGs was visually measured using a microscope slide grid (1 mm × 1 mm). Illustrative photographs were acquired by a Leica MC170 HD digital camera mounted on a Leica S9D stereo microscope.

Furthermore, after length measurement, ASGs were immersed in 400 µL of Tris 20 mM pH 7.4 Carl Roth buffer (Karlsruhe, Germany) and

homogenized for 20 s with a Polytron PT 10-35 GT homogenizer. After sonication, the samples were centrifuged at 10 000 RPM for 10 min (Eppendorf Centrifuge 5804 R) at 4 °C and the resulting supernatant was collected. The protein concentration was then estimated using the BiCinchoninic acid Assay (BCA), method with the Bovine Serum Albu-min concentration ranging from 1 to 40 µg protein standard range. The quantification of each sample was made from 10 µL of protein extract mixed with 10 µL of Tris buffer 20 mM pH 7.4 Carl Roth (Karlsruhe, Germany) and 1 mL of reagent. The reagent consisted of 20 µL CuSO₄ 4 % and 0.98 mL BCA solution. The samples were heated at 37 °C for 30 min and the absorbance was then read at 562 nm with a spectrophotometer (Eppendorf BioPhotometer 6131). Each data point for the protein extraction was obtained from three pairs of accessory sex glands stored in a 1.5 mL centrifuge tube.

## 2.7. Hemolymph collection and JH extraction

The collection of hemolymphs was carried out in pre-exposed male *A. ipsilon*. The top of the moth's head was cut off and the individual was then placed in a 0.25 mL Eppendorf tube with a cut base (only the head of the animal came out of the tube at the level of the cut base). This tube was then positioned in a 1.5 mL Eppendorf tube, and centrifuged at 1 rcf for 3 min. Each sample contained 50 µL of hemolymph (from several males) mixed with 100 µL of pure ethanol (Carl Roth, Germany). Then, 200 µL of pure hexane was added to the solution and thoroughly vortexed again. The mixture was centrifuged at 10 000 g for 10 min at 4 °C and the supernatant containing JH was transferred into a new vial and dried under nitrogen flow and stored at -80 °C. The dried extract was re-suspended in 50 µL of acetonitrile and vortexed for 1 min and JH-II was quantified by reverse-phase high-performance liquid chromatography (RP-HPLC).

## 2.8. In vitro biosynthesis of JH-II acid by the corpora allata

CAs were dissected and incubated for 4 h at 28 °C in a salinized vial containing 150 µL of tissue culture medium M-199 (Thermo Fisher Scientific, Illkirch, France) supplemented with Ficoll (20 mg/mL) and (E,E)-farnesic acid (200 µM) (Sigma Aldrich, Saint-Quentin Fallavier, France). Each incubation was carried out with three pairs of CAs. At the end of incubation, 500 µL of ethyl acetate was added to the sample which was vortexed for 1 min and spun for 5 min at 4 °C and 2000 g. The organic phase containing JH acid was transferred to a new vial, and then dried under nitrogen flow and stored at -80 °C. The dried extract was re-suspended in 50 µL of acetonitrile and vortexed for 1 min. JH-II acid was quantified by RP-HPLC.

## 2.9. JH-II acid and JH-II quantification

JH-II acid and JH-II titers were determined by RP-HPLC using the HPLC system (Beckman System Gold HPLC, Palo Alto, CA, USA) with model 166 ultraviolet detector (Beckmann, Palo Alto, CA, USA). The column was Kromasil® C18 reverse-phase HPLC column (4.6 × 250 mm, 5 µm particles) and was eluted using a linear gradient from 5 % to 80 % acetonitrile in 5 mmol/L HEPES adjusted to pH 6.2 over 20 min at a flow rate of 1 mL/min with UV detection monitored at 245 nm. The quantification of JH-II and JH-II acid was computed by comparison of the peak area with external quantitative standards using the Beckman Gold system software.

## 2.10. cDNA synthesis

Total RNA was extracted from pooled tissues (3 ASGs and 10 ALs per biological replicates) of several pre-exposed males with TRIzol reagent (TRI Reagent®, Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions and was quantified using UV/Visible Spectrophotometer (NanoPhotometer® NP80, IMPLEN, Munich,
Germany). DNase treatment was carried out with RNase-free recombi-
nant DNase I (Roche Diagnostics GmbH, Mannheim, Germany) for 30
min at 37 °C followed by 10 min inactivation at 70 °C. The reaction also
contained RNase OUT (Invitrogen, Carlsbad, CA, USA). After DNase
treatment, single-stranded cDNA was synthesized from 5 µg total RNA
with SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer's instructions. The reaction con-
tained a mixture of deoxynucleotide triphosphates (dNTPs), Oligo(dT)
primers, and sterile water for a final volume of 20 µL. The mixture was
heated at 42 °C for 50 min and then incubated at 70 °C for 15 min.

2.11. Quantification of *Met1* and *Kr-h1* expressions in *ALs* and *ASGs*

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out from the extraction of total RNA from ALs and ASGs, by using the LightCycler 480 Real-Time PCR Detection System (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Each 10 µL reaction included 5 µL of AB-solute Blue SYBR green (Thermo Scientific, Waltham, MA, USA), 4 µL of cDNA (12.5 ng/µL), and 1 µL of 10 µmol/L specific primer pairs for *Met1* (qMet1dir and qMet1rev) or *Kr-h1* (qKrh1dir and qKr-h1rev) (Supplementary Table S1). The PCR conditions were 50 cycles of 95 °C for 10 s, 65 °C for 15 s, 72 °C for 15 s and the length of the amplified products was 223 bp for *Met1* and 190 bp for *Kr-h1*.

Fluorescence measurements on a melting curve between 55 and 95 °C confirmed the presence of a single specific peak and the absence of primer-dimer peaks for the primer pairs used. Each run included a negative control (water) and a series of five-fold dilutions of pooled cDNA (from all conditions). The five-fold dilution series (undiluted, 1/5, 1/25, 1/125, and 1/625) were used to construct a relative standard curve to determine PCR efficiency and for further quantification. In all experiments, the primers gave amplification efficiencies of 95–100 %. Each reaction was carried out in three technical replicates with seven independent biological replicates using the technical platform of the Institute of Ecology and Environmental Sciences of Paris (Sorbonne Université, France). Expression levels were analyzed with LightCycler 480 software (Roche Diagnostics GmbH, Mannheim, Germany) and the GENORM Visual Basic application for Microsoft EXCEL (Issy-les-Moulineaux, France) as described by Vandesompele et al. (2002). Cycle threshold (Ct) values were determined for each candidate gene as well as for the reference gene encoding ribosomal protein L8 (RpL8) of Agrotis ipsilon (accession number JX975720.1). This gene had the most stable expression levels among other reference genes tested (ribosomal protein L13, ribosomal protein S3, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, and β-actin). The average Ct value of each triplicate was used to normalize the expression level of the candidate gene to the geometric mean of the RpL8 level in the Q-GENE software (Simon, 2003) using the formula:

$$
\text{NE} = (\text{E RpL8})^{\text{Ct RpL8}} / (\text{E candidate gene})^{\text{Ct candidate gene}}
$$

with NE meaning normalized expression, E meaning PCR efficiency,
and Ct corresponding to the crossover point values. Then, the mean and
standard error were calculated from the NE values for the biological
replicates. The sequences of the specific primers for *RpL8* (qRpL8dir and
qRpL8rev) are presented in *Supplementary Table S1*, the length of the
amplified products was 200 pb.

2.12. RNA interference experiments

Double-stranded RNA (dsRNA) production and injection conditions
were established by following the protocols used in A. *ipsilon* (Aguilar
et al., 2023; Force et al., 2024a) for the silencing of *Met1* or *Kr-h1*. The
dsRNAs targeting *Met1* (Met1-1-dsRNA, 480 bp; and Met1-2-dsRNA, 478
bp), *Kr-h1* (Kr-h1-1-dsRNA, 466 bp; and Kr-h1-2-dsRNA, 472 bp), and
bacterial β-galactosidase (LacZ-dsRNA, 372 bp; control) were chosen by

using the E-RNAi web application (http://e-rnai.dkfz.de/) (Arziman
et al., 2005) and produced with a MEGAscript® T7 High Yield Tran-
scription Kit (Invitrogen, Carlsbad, CA, USA) according to the manu-
facturer's instructions. A PCR was performed on 1 µL plasmid (50 ng/µL)
with specific primers for each target gene (Met1-1-T7dir/Met1-1-T7rev,
Met1-2-T7dir/Met1-2-T7rev, Kr-h1-1-T7dir/Kr-h1-1-T7rev, Kr-h1-2-
T7dir/Kr-h1-2-T7rev, LacZ-T7dir/LacZ-T7rev) (Supplementary Table
S2) under the following program: 35 cycles of 95 °C for 30 s, 60 °C for
30 s, 72 °C for 1 min. PCR products were purified with a Nucleospin®
extract II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and
quantified using UV/Visible Spectrophotometer (NanoPhotometer®
NP80, IMPLEN, Munich, Germany). The transcription reaction was then
performed overnight at 37 °C on 1 µg of PCR product in a reaction mix
containing 2 µL of each nucleotide, 2 µL of T7 RNA polymerase enzyme,
and 2 µL of T7 10X Buffer in a final volume of 20 µL. After spectrometry
quantification, the reaction mix was incubated for 15 min at 37 °C with 2
U of Turbo DNase (Invitrogen, Carlsbad, CA, USA). The dsRNA precip-
itation was performed by addition of 30 µL of water treated with
diethylpyrocarbonate (DEPC) and 20 µL LiCl (Sigma Aldrich,
Saint-Quentin Fallavier, France), storage of the mixture at -20 °C for 2
h, and then centrifugation for 30 min at 16 000 g. The pellet was washed
with 1 mL 75 % ethanol, centrifuged, and dried before elution in 11 µL
DEPC water. Samples were denatured at 95 °C for 5 min followed by a
rehybridization step of 1 h 30 min at room temperature. The dsRNA
integrity was then checked by loading on an agarose gel. Before injec-
tion, dsRNA was diluted to 0.5 µg/µL in saline solution.

Newly emerged males were anesthetized with carbon dioxide and
injected with 1 µg dsRNA in the lateral region of the intersegment
membrane between the second and third abdominal segments. The in-
jections were performed at the beginning of the scotophase by using a
Hamilton syringe with a 32-gauge needle. On pre-exposed males, ALs
and ASGs were dissected for the *Met1* and *Kr-h1* expression analysis.
Control groups were composed of non-injected, bacterial LacZ-dsRNA-
injected, and saline-injected males.

2.13. Statistical analysis

Data analysis was done using R 4.1.2 (R Core Team (2021); RStudio
2021.09.2 software). The sample sizes are reported in the legend of each
figure, and the data represent the biological replicates. The alpha risk
was 0.05.

For behavioral experiments, statistical differences between groups of
pre-exposed males were evaluated using an R × C test of independence
by means of a G-test and also by applying the Williams correction (Sokal
and Rohlf, 1995).

For other dataset, the assumption of normal data distribution was
tested by the Shapiro-Wilk test. When this was satisfied, an ANOVA was
carried out, and when it was significant, post-hoc test was conducted: a
Student's t-test (for comparison between two groups) or a Tukey's test
(for multiple comparison). Alternatively, non-normal data were
analyzed with a Kruskal-Wallis' test. If it was significant, a Wilcoxon's
test (for comparison between two groups) or a Dunn's test (for multiple
comparison) was performed.

2.14. Animal ethics

French legislation on animal welfare is based on that of the European
Union (directive 2010/63/EU) according to which no invertebrates,
other than Cephalopods, benefit from ethical protection. However, all
experiments were carried out with care, limiting stressful situations for
the animals as much as possible.
# 3. Results

## 3.1. Female sex pheromone pre-exposure promotes the male sexual behavior

To examine the impact of olfactory experience on the development of sexual behavior, newly emerged males were pre-exposed at different concentrations of the sex pheromone blend (10 ng, 100 ng, or 1 µg; knowing that 100 ng of the sex pheromone has previously been described as behaviorally and electrophysiologically active, we selected the lower and higher doses by a factor of 10), or hexane (used as control) for 3 min; and then the proportion of oriented flight to 100 ng of sex pheromones was determined at 2 h, 24 h, 48 h, and 72 h after pre-exposure. The percentage of sex pheromone-guided oriented flight of pre-exposed males to 100 ng and 1 µg of sex pheromones was significantly higher from 24 h following pre-exposure than that of control males (around 40 % for 100 ng and 1 µg sex pheromone pre-exposed males against 10 % for control males) (Fig. 1; G-test, $G \ge 11.51$, $p < 0.001$).

## 3.2. Female sex pheromone pre-exposure accelerates the ASG maturation

To investigate the impact of olfactory experience on the maturation of ASGs, newly emerged males were pre-exposed at different concentrations (10 ng, 100 ng, or 1 µg) of the sex pheromone blend, or hexane (used as control) for 3 min. The protein content and the length of ASGs were evaluated at 2 h, 24 h, 48 h, and 72 h after pre-exposure. The protein amount of ASGs for pre-exposed males to 100 ng and 1 µg of sex pheromones was significantly increased from 48 h following pre-exposure than that of control males (in average 165 µg per ASG for 100 ng and 1 µg sex pheromone pre-exposed males against 64 µg per ASG for control males) (Fig. 2A; Tukey's test, $p < 0.001$). In addition, the length of ASGs was also significantly higher from 48 h following pre-exposure than that of control males (around 4.4 cm for 100 ng and 1 µg sex pheromone pre-exposed males against 2.5 cm for control males) (Fig. 2B–D; Dunn's test, $p < 0.001$).

## 3.3. Rates of JH-II acid biosynthesis and JH-II circulating are raised in sex pheromone pre-exposed males

To understand how the olfactory experience impacts the male sexual maturation, newly emerged males were pre-exposed to 100 ng of the sex pheromone blend, or hexane (used as control) for 3 min, and the

hemolymph levels of JH-II as well as the rates of JH-II acid biosynthesis *in vitro* were assessed at 2 h, 24 h, 48 h, and 72 h after pre-exposure. While the JH-II hemolymph titer for pre-exposed males to 100 ng of sex pheromones started to be higher from 24 h following pre-exposure than that of control males (Fig. 3A; t-test, $t = 2.27$, df = 38.04, $p = 0.029$), the JH-II circulating rate was strongly raised from 48 h after pre-exposure (around 254 pg/µL for sex pheromone pre-exposed males against 152 pg/µL for control males) (Fig. 3A; t-test, $t \ge 19.05$, df = 38.00, $p < 0.001$). Then, the same trend was observed for JH-II acid biosynthesis by CAs: from 24 h, a significant rise was measured in males pre-exposed to 100 ng of sex pheromones compared to control males (in average 16 pg/CA/h for sex pheromone pre-exposed males against 7 pg/CA/h for control males) (Fig. 3B; t-test, $t \ge 5.15$, df = 16.00, $p < 0.001$).

## 3.4. *Met1* and *Kr-h1* expression levels are increased in *ALs* and *ASGs* in sex pheromone pre-exposed males

Following the study of the rates of JH-II acid biosynthesis and JH-II circulating, we focused on the influence of olfactory experience on the JH signaling pathway in ALs and ASGs. Newly emerged males were pre-exposed to 100 ng of the sex pheromone blend, or hexane (used as control) for 3 min, and the expression of *Met1* and *Kr-h1* were quantified at 2 h, 24 h, 48 h, and 72 h after pre-exposure. In both ALs and ASGs, *Met1* expression for pre-exposed males to 100 ng of sex pheromones was increased 24 h after pre-exposure compared to that of control males (Fig. 4A and B; Wilcoxon's test, $W \ge 558.50$, $p < 0.001$). Furthermore, *Met1* expression was strongly upregulated from 48 h after pre-exposure (Fig. 4A; t-test, $t \ge 16.53$, df = 48.00, $p < 0.001$; and Fig. 4B; Wilcoxon's test, $W \ge 622.00$, $p < 0.001$). As for *Met1* expression, in ALs and ASGs, the expression of *Kr-h1* was upregulated 24 h after pre-exposure compared to control males (Fig. 4C and D; t-test, $t \ge 8.40$, df = 48.00, $p < 0.001$) and was strongly increased from 48 h following pre-exposure (Fig. 4C and D; t-test, $t \ge 22.34$, df = 48.00, $p < 0.001$).

## 3.5. *Met1* and *Kr-h1* silencing repress the early display of sexual behavior in sex pheromone pre-exposed males

To confirm the role of JH in promoting effect of olfactory experience on male sexual maturation, we investigated the impact of silencing of *Met1* and *Kr-h1* on the sexual behavior of sex pheromone pre-exposed males (efficiencies of dsRNA have previously been demonstrated in Aguilar et al. (2023)). Newly emerged males received an injection of saline solution, bacterial Beta-galactosidase (LacZ)-dsRNA,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Effect of pre-exposure to sex pheromones on the behavioral response of males *Agrotis ipsilon*. Newly emerged males were exposed to different concentrations (10 ng, 100 ng, and 1 µg) of the sex pheromone blend or hexane (used as control) for 3 min, and the proportion of sex pheromone-guided oriented flight for 100 ng of sex pheromones were determined at 2 h, 24 h, 48 h and 72 h after pre-exposure (for each post-exposure time, n = 65 individuals/group). Letters represent the statistical differences after G-tests ($p < 0.05$).</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Fig. 2.** Effect of pre-exposure to sex pheromones on the maturation of the ASGs. Newly emerged males were exposed to different concentrations (10 ng, 100 ng, and 1 µg) of the sex pheromone blend or hexane (used as control) for 3 min, and the protein amount (A) and length (B) of the ASGs were evaluated at 2 h, 24 h, 48 h, and 72 h after pre-exposure. The values represent mean ± SEM; letters indicate statistical difference after ANOVA or Dunn's tests (*p* < 0.05). For each post-exposure time, the ASG protein amount was quantified from 5 pairs of glands with 6 biological replicates (n = 30 individuals for each group) and the ASG length was measured from 35 dissected glands (n = 35 individuals for each group). Morphology of the ASGs of the males at 2 h (C) and 72 h (D) after an exposure to 100 ng of sex pheromone or hexane. Scale bar = 0.25 cm.

Met1-dsRNA, or Kr-h1-dsRNA, or no injection (non-injected), and were exposed to 100 ng of the sex pheromone blend for 3 min. The proportion of sex pheromone-guided oriented flight was then determined at 72 h after pre-exposure. Met1-and Kr-h1-dsRNA injections repressed the early display of sexual behavior in pre-exposed males (around 45 % for Met1-and Kr-h1 deficient males against 75 % for control males) (Fig. 5;

G-test, *G* ≥ 5.68, *p* ≤ 0.017). To consolidate our previous RNAi results, we tested the impact of second dsRNA designed against *Met1* and *Kr-h1* (not overlapping with the first ones), which caused similar alterations in the behavioral responsiveness to sex pheromone in males 72 h after pre-exposure (Supplementary Fig. S1; G-test, *G* ≥ 6.27, *p* ≤ 0.012).
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Effect of pre-exposure to sex pheromones on the JH-II circulating and the *in vitro* JH-II acid biosynthesis. Newly emerged males were exposed to 100 ng of the sex pheromone blend or hexane (used as control) for 3 min, and the hemolymph titer of JH-II (A) as well as the biosynthetic activity of the *corpora allata* for JH-II acid (B) were evaluated at 2 h, 24 h, 48 h, and 72 h after pre-exposure. The values represent means ± SEM; letters indicate statistical difference after Student's t-tests (*p* < 0.05). For each post-exposure time, the JH-II circulating was evaluated from 3 pairs of the *corpora allata* with 6 biological replicates (n = 18 individuals for each group) and the rate of JH-II acid biosynthesis was quantified from 6 to 8 males with 6 biological replicates (n = 36 to 48 individuals for each group).</figcaption></figure>

**3.6. Met1 and Kr-h1 knockdown prevent the accelerated ASG maturation in sex pheromone pre-exposed males**

We also examined the effect of knockdown of *Met1* and *Kr-h1* on the ASG maturation in sex pheromone pre-exposed males (efficiencies of dsRNA have previously been demonstrated in Gassias et al. (2021)). Newly emerged males received an injection of saline solution, bacterial Beta-galactosidase (LacZ)-dsRNA, Met1-dsRNA, or Kr-h1-dsRNA, or no injection (non-injected). They were then exposed to 100 ng of the sex pheromone blend for 3 min. The protein content and the length of ASGs were then evaluated 72 h after pre-exposure. Met1- and Kr-h1-dsRNA injections in pre-exposed males led to a decrease in the protein amount of ASGs (in average 285 µg per ASG for Met1- and Kr-h1 deficient males against 200 µg per ASG for control males) (Fig. 6A; Dunn's test, *p* < 0.001). Moreover, Met1- and Kr-h1-dsRNA injections in pre-exposed males resulted in a drastic reduction in the length of ASGs (around 3.4 cm for Met1 and Kr-h1 deficient males against 4.7 cm for control males) (Fig. 6B and C; Dunn's test, *p* < 0.001). To confirm these RNAi results, we injected a second dsRNA designed against *Met1* and *Kr-h1*, and we observed the same effects on the protein content and the length of ASGs in males 72 h after pre-exposure (Supplementary Fig. S2; Dunn's test, *p* < 0.001).

**4. Discussion**

This present study conclusively demonstrates that a pre-exposure to female sex pheromones accelerates sexual maturation through the induction of endocrine responses in the male *Agrotis ipsilon*. In newly emerged males, sex pheromones pre-exposure promotes the display of sexual behavior following maturation of antennal lobes (ALs) (Fig. 1), as well as the development of accessory sex glands (ASGs) producing seminal proteins (Fig. 2). The potentiating effects of pre-exposure to sex pheromones result from an early activation of JH biosynthesis by the *corpora allata* (CAs) (Fig. 3) and an increased expression of JH signaling pathway actors (*Met1* and *Kr-h1*) within ASGs and ALs (Figs. 4–6). Thus, our results highlight the endocrine mechanisms by which insects can promote their fitness according to past olfactory experiences.

Reproductive synchronization is a complex process that requires the integration of external and internal signals to coordinate the timing of reproduction between individuals (Dey et al., 2015; Kobayashi et al., 2002). Within a population, changes in reproductive status between individuals can be induced by numerous factors including photoperiod, temperature, and diet (Ward and Webster, 2016). As a result, reproduction appears to be essentially asynchronous between the sexes. However, social interactions through sexual signals are crucial to ensure reproductive synchronization (French and Stribley, 1985; Ims and Steen, 1990; Jovani and Grimm, 2008): a well-known example is the Whitten effect. In rodents, sheep, or even monkeys, stimulation of females by
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Effect of pre-exposure to sex pheromones on the expression of *Met1* and *Kr-h1* in accessory sex glands (ASGs) and antennal lobes (ALs). Newly emerged males were exposed to 100 ng of the sex pheromone blend or hexane (used as control) for 3 min, and the relative expression of *Met1* and *Kr-h1* were quantified in ASGs (A & B) and in ALs (C & D) at 2 h, 24 h, 48 h, and 72 h after pre-exposure. Each RT-qPCR was run in 3 technical replicates and 7 independent biological replicates (for ALs, n = 70 individuals for each group; for ASGs, n = 21 individuals for each group). Crosses denote the mean, bars indicate the median, and boxes represent the 75 % quartiles; letters indicate statistical difference after Student t-tests or Wilcoxon's tests (*p* < 0.05).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Effect of *Met1* and *Kr-h1* silencing on the behavioral response of pre-exposed males to sex pheromones. Newly emerged males received an injection of saline solution, bacterial Beta-galactosidase (LacZ)-dsRNA, Met1-1-dsRNA, or Kr-h1-1-dsRNA, or no injection (non-injected), and were exposed to 100 ng of the sex pheromone blend for 3 min. The proportion of sex pheromone-guided oriented flight was then determined at 72 h after pre-exposure (n = 50 individuals for each group). Letters represent the statistical differences after G-tests (*p* < 0.05).</figcaption></figure>

male pheromones promote the synchronization of estrus (Cahill et al., 1974; Gattermann et al., 2002; Vandenbergh, 1967). In some insects, and more particularly in migratory locusts, exposure of immature female pheromones to the male aggregation pheromone accelerates oocyte maturation and egg laying (Chen et al., 2022). However, to date, the effects of pre-exposure to female sex pheromones on the male reproductive physiology are still far from being fully deciphered in insects.

Previous studies in the male moth *Spodoptera littoralis* show that behavioral attraction to female sex pheromones increases after 24 h pre-exposure to these conspecific odorants (Anderson et al., 2003, 2007). The behavioral effect is paralleled by an increase in the sensitivity of neurons in ALs, while the peripheral olfactory system is not affected. Nevertheless, it is unclear whether this pre-exposure effect could have an impact on other reproductive events in male insects; and the underlying physiological mechanisms are still unknown. The male moth *Agrotis ipsilon* emerges as a model to fill this scientific gap. Indeed, sexual behavior development (Aguilar et al., 2023; Durand et al., 2021) and

reproductive system maturation (Force et al., 2023a; Gassias et al., 2021) are well-described in this species, thus allowing to explore the influence of sex pheromone pre-exposure at several biological levels. First, our results demonstrate that female sex pheromone pre-exposure in newly emerged males promotes the execution of sexual behavior as early as 24 h after pre-exposure (Fig. 1). Knowing that in *A. ipsilon* males, the antennal pheromone detection is already functional from the emergence (Gadenne et al., 1993) and that the development of sex pheromone-guided oriented flight occurs within the first five days of adult life under standard conditions (Aguilar et al., 2023; Durand et al., 2021), our results highlight an accelerating effect of sex pheromone pre-exposure on the sexual behavior development, probably through an early maturation of ALs as previously observed in *S. littoralis* (Anderson et al., 2003, 2007). In the future, using an electrophysiological approach, it would be informative to examine the pheromonal response of AL neurons as function of time in newly emerged *A. ipsilon* males pre-exposed to female sex pheromones.
<figure><img src="image_12.png" /><figcaption>**Fig. 6.** Effect of *Met1* and *Kr-h1* silencing on the ASG maturation of pre-exposed males to sex pheromones. Newly emerged males received an injection of saline solution, bacterial Beta-galactosidase (LacZ)-dsRNA, *Met1-1*-dsRNA, or *Kr-h1-1*-dsRNA, or no injection (non-injected), and were exposed to 100 ng of the sex pheromone blend for 3 min. The protein amount (A) and length (B) of the ASGs were determined at 72 h after pre-exposure. The values represent mean ± SEM; letters indicate statistical difference after Dunn's tests (*p* < 0.05). For each experimental group, the ASG length was measured from 35 dissected glands (n = 35 individuals/group) and the ASG protein amount was quantified from 5 pairs of glands with 6 biological replicates (n = 30 individuals/group). (C) Morphology of the ASGs of the *Met1*-or *Kr-h1*-deficient males at 72 h after an exposure to 100 ng of sex pheromones. Scale bar = 0.5 cm.</figcaption></figure>

To gain more information about the influence of the sex pheromone pre-exposure on the reproductive success of male insects, we also investigated the reproductive system, focusing on the maturation of ASGs. In *A. ipsilon* males, the latter is marked by a growth in length coupled to an increase in the biosynthetic activity of seminal proteins, which are required for the spermatophore formation, the survival of the spermatozoa, and their transit through the female genital tract (Duportets et al., 1998; Force et al., 2023a). The ASG maturation is completed from the fifth day of adult life in standard conditions (Force et al., 2023a; Gassias et al., 2021). As for sexual behavior development, sex pheromone pre-exposure in newly emerged males leads to accelerated maturation of ASGs by promoting the protein content and the growth in length within 48 h after pre-exposure (Fig. 2). Thus, pre-exposure to female sex pheromones potentiates the male reproductive capacities by inducing the development of sexual behavior concomitantly to the ASGs maturation in *A. ipsilon*.

Furthermore, it has been suggested that endogenous molecules, such as reproductive hormones, are mediators of the effects of sexual signals on the reproductive synchrony in animals (Engel et al., 2016; Noguera and Velando, 2019). Nevertheless, the mechanisms by which the interaction between sexual signals and hormones coordinates the

reproduction of individuals remain largely unknown. In *A. ipsilon* males, knowing that JH facilitates the execution of sex pheromone-guided flight (through the AL development) in synchronism with the ASG maturation (Aguilar et al., 2023; Duportets et al., 1998; Force et al., 2024a; Gassias et al., 2021), we hypothesized that JH might mediate the promotive effects of sex pheromone pre-exposure on the male sexual maturation. Sex pheromone pre-exposure triggers an increase in circulating JH levels (Fig. 3A) in response to a raise in JH biosynthetic activity by CAs from 24 h after pre-exposure (Fig. 3B). Thus, in *A. ipsilon* males, sex pheromone pre-exposure positively affects the endocrine system by stimulating the JH biosynthesis and its release into hemolymph.

In addition, in this same species, JH biological actions on both ASGs and ALs occur through the activation of the *Met-Kr-h1* signaling pathway (Aguilar et al., 2023; Force et al., 2024a; Gassias et al., 2021). Consequently, it is possible to believe that the potentiating effects of pre-exposure to sex pheromones on male sexual maturation result from an early induction of the JH signaling pathway. Indeed, in ALs and ASGs, sex pheromone pre-exposure upregulates the expression of *Met1* and *Kr-h1* from 24 h after pre-exposure (Fig. 4). To strengthen the involvement of the JH signaling pathway in the pheromonal induction of male
sexual maturation, we performed RNAi-mediated silencing of *Met1* and *Kr-h1* in newly emerged males pre-exposed to female sex pheromones. 72 h after pre-exposure, *Met1*-or *Kr-h1*-deficient males exhibit a reduction of the sex pheromone guided-oriented flight (Fig. 5) and a delay of ASG maturation (Fig. 6). Thus, JH acts as a mediator in the interplay between sex pheromone signals and the sexual maturation in *A. ipsilon*. Moreover, the biosynthetic activity of JH by CAs is known to be regulated by stimulatory and inhibitory neuropeptides, named allatostatin and allatotropin respectively; these neuropeptides are produced by a population of neurosecretory cells located in the *pars intercerebralis* and the *pars lateralis*, with peripheral axons terminating in contact with CAs (Hartenstein, 2006; Raabe, 1982, 1989; Veelaert et al., 1998). For future experiments, we plan to quantify by an enzyme-linked immunosorbent assay the amounts of allatostatin in the brain of sex pheromone pre-exposed males compared to naïve *A. ipsilon* males. In parallel, it would be informative to establish a mapping of brain regions that are activated by the sex pheromone pre-exposure by performing immuno-histochemistry experiments on biomarkers of Fos neuronal activation (Lara Aparicio et al., 2022).

In summary, for the first time, our work sheds light on the hormonal mechanisms linking sex pheromonal signals and the induction of sexual maturation in an insect. Our findings might expand to other taxonomic groups, such as vertebrates, for which such an interplay between sexual signals and reproductive hormones is also observed (Berger, 1992; Drickamer, 1977; McClintock, 1978). Indeed, it is well-established that the stimulatory effects of sexual signals on reproductive physiology and behavior result from the activation of hypothalamic neurons synthesizing gonadotropin-releasing hormone (GnRH) which increases the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland. In turn, LH and FSH control gonadal development and the synthesis of sex steroid hormones which facilitate the expression of social and reproductive behaviors by acting on the central neural circuits (Kelliher and Wersinger, 2009; Yoon et al., 2005).

One of the current challenges is to determine the functional interplay between the olfactory network and the neuroendocrine system in the modulation of reproductive function. In the mammalian brain, it has been demonstrated that Kisspeptin neurons directly innervate and stimulate the activity of GnRH neurons. In addition, the Kisspeptin system is involved in mediating the accelerating effects of pheromonal exposure on puberty (Jouhanneau et al., 2013). Even though insects and mammals represent highly diverged groups, a recent study revealed that insect allatostatin signaling operates in a similar way to mammalian kisspeptin signaling (Pan and O'Connor, 2019), thus suggesting similar molecular mechanisms in the pheromonal modulation of developmental processes such as sexual maturation between these taxa. Future investigation is needed to decipher the pathway in conducting pheromonal information from sensory neurons to kisspeptin/allatostatin neurons.

### CRediT authorship contribution statement

**Stéphane Debernard:** Writing – review & editing, Validation, Supervision, Conceptualization.
**Paleo Aguilar:** Validation, Investigation, Data curation.
**Annick Maria:** Validation, Investigation.
**Annabelle Fuentes:** Validation, Investigation.
**Philippe Couzi:** Validation, Investigation.
**Françoise Bozzolan:** Validation, Investigation.
**Edmundo Gassias:** Validation, Investigation, Data curation.
**Evan Force:** Writing – review & editing, Writing – original draft, Validation, Supervision, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare no competing or financial interests.

### Acknowledgments

We thank our technical colleagues from laboratory for rearing the animals used in the experiments.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ibmb.2025.104365.

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