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# Endocrine regulation of reproductive behaviors in insects: A comprehensive review

Evan Force, Stéphane Debernard

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Evan Force, Stéphane Debernard. Endocrine regulation of reproductive behaviors in insects: A comprehensive review. Current Opinion in Insect Science, 2025, 69, pp.101360. 10.1016/j.cois.2025.101360. hal-05005823

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ELSEVIER

Review

# Endocrine regulation of reproductive behaviors in insects: a comprehensive review

Evan Force* and Stéphane Debernard#

<figure><img src="image_4.png" /></figure>

Insects use pheromones in a complex system of sexual communication for reproduction. Hormones, peptides, and biogenic amines are crucial regulators involved in reproductive behaviors. Despite knowledge gaps, this review shows how hormones and related molecules influence insect reproduction and highlights the intricate endocrine network that governs reproductive behaviors through diverse signaling pathways. In the future, it will be very interesting to explore not only endocrine regulation but also the impact of environmental changes on reproductive behaviors, deepening our understanding of insect reproductive processes and their adaptability.

## Address

Sorbonne Université, Université Paris-Est Créteil, INRAE, CNRS, IRD, Institute for Ecology and Environmental Sciences of Paris, iEES Paris, F-75005 Paris, France

Corresponding authors:

Force, Evan (evan.force@sorbonne-universite.fr),

Debernard, Stéphane (stephane.debernard@sorbonne-universite.fr)

* https://orcid.org/0000-0002-0873-352X

# https://orcid.org/0000-0002-7687-1182

**Current Opinion in Insect Science** 2025, 69:101360

This review comes from a themed issue on Neuroscience

Edited by **Marlène Goubault, Charlotte Lecureuil and David Siaussat**

Available online 7 March 2025

https://doi.org/10.1016/j.cois.2025.101360

2214-5745/© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

In many vertebrate species, hormones are known to control the display of reproductive behaviors notably by finely tuning sensory responsiveness to sexually relevant cues [1,2]. As vertebrates, insects display a large variety of reproductive behaviors (e.g. calling, courtship, mating, egg-laying) by using an efficient sexual signaling system to attract, identify, and choose appropriate sexual partners [3]. This sophisticated sexual communication system is known to be regulated by endogenous signals

including hormones [4-7] and hormone-like molecules such as peptides [8] and biogenic amines [4,9].

Although studying the endocrine control of insect reproductive processes was a major topic decades ago, this field of research has since fallen into disuse. Today, it is once again being studied in depth by focusing on the cellular and molecular mechanisms by which endocrine signals exert their influence on reproductive behaviors. Therefore, this review aims to highlight advances in our understanding of the regulatory endocrine pathways of sexual behaviors in insects and also to reveal scientific gaps that would merit further in-depth investigation.

## Endocrine actors mediate pheromone biosynthesis and calling behavior

In many insect species, pheromones play a crucial role in identifying and attracting sexual partners, ultimately leading to mating behavior [10,11]. Pheromones are often secondary products derived from fatty acids or isoprenoids and are frequently blends of pheromonal compounds. The synthesis of pheromones is carried out by exocrine glands (commonly referred to as pheromone glands) composed primarily either by a single epidermal layer located in various parts of the body [12-14] or by epidermal secretory cells (called oeno-cytes) present in the abdomen [15]. Despite intense research over the last few decades, the biosynthetic pathways of pheromones, as well as their endocrine regulation, are not yet fully understood at the cellular and molecular levels. Nevertheless, a large number of studies performed mainly in Lepidoptera have revealed that pheromonogenesis is due to a photoperiodic cue initiated by the timely release into the hemolymph of a hormone-like molecule: the pheromone biosynthesis activating neuropeptide (PBAN) [16-18], which is predominantly produced by neurosecretory cells of the suboesophageal ganglion [19] and transported to the *corpora cardiaca* before its release into the hemolymph [20]. This neuropeptide is found to act through its binding to a G protein-coupled transmembrane receptor, the pheromone biosynthesis activating neuropeptide receptor (PBAN-R), located at the surface of pheromone gland cells to active a signal transduction cascade involving an influx of extracellular Ca²⁺ in the triggering of pheromonogenesis [21]. Early observations that the production and release of sex pheromones were synchronized with female reproductive cycles suggested the existence of hormonal regulation of pheromonogenesis. It has recently been proven that the two insect major hormones, juvenile
hormone (JH) and 20-hydroxyecdysone (20E), are critical regulators of pheromone production in several insect groups [22–29]. In moths, it has been demonstrated that the JH's regulatory effects on pheromone production vary according to species. Indeed, in long-lived moths, such as the armyworm *Pseudaletia unipuncta* and the black cutworm *Agrotis ipsilon*, JH induces pheromone biosynthesis by stimulating the release of PBAN in mature females [30,31], whereas in species with shorter lifespans, such as the cotton bollworm *Heliothis armigera*, JH is required for the primary induction of the pheromone gland to PBAN stimulation in female pupae [32,33]. By contrast, JH administration in adult females leads to an inhibition of both specific binding to PBAN-R and pheromone production in other moth species [24,34]. In bark beetles, JH is also shown to activate directly the aggregation pheromone synthesis by upregulating the transcriptional activity of genes encoding the pheromone biosynthetic enzymes [23]. Similarly, a direct stimulatory action of JH on the synthesis of female sex pheromones, through the involvement of its nuclear receptor Methoprene-tolerant (Met), has been described in the fruit fly *Drosophila melanogaster* [22]. More recently, experiments of RNA interference-mediated silencing of *Met* and its nuclear partner, *Taiman (Tai)*, provide evidence in the worst desert locust *Schistocerca gregaria* that phenylacetonitrile aggregation pheromone synthesis is controlled by the JH-Met-Tai signaling pathway [35]. Thus, to date, at least three endocrine factors, PBAN, JH, and 20E, are recognized to be key regulators of pheromone biosynthesis in insect sexual communication.

Calling behavior is associated with the release of pheromones in many insects [36-39]. The initiation of calling behavior has been intensively investigated about external factors such as light and temperature [40]. By contrast, only a few studies highlight the existence of the hormonal regulation of such sexual behavior. For instance, in moths and cockroaches, the removal of the *corpora allata*, endocrine glands producing JH, prevents the expression of female calling behavior; and injections of the JH analog fenoxycarb in allatectomized females restore the onset of calling [38,41]. To date, the molecular basis of this regulatory action of JH on the calling behavior remains unknown.

## Peripheral and central processing of pheromones are influenced by endocrine system

In insect sexual communication, the pheromonal signal is recognized by olfactory receptor neurons located in the antennal sensilla; inside these, the odorant-binding proteins capture and transport odorant molecules to the olfactory receptors (ORs) [42-45]. The chemical signal is transformed into an electrical signal that is first integrated into the primary olfactory centers, the antennal lobes (ALs), and then conveyed to higher-order brain centers,

ultimately leading to the execution of sexual behaviors [46]. In some moth species, several reports have revealed an increase in the antennal detection of female sex pheromones during the adulthood of males [47,48]. In the male moth *Spodoptera littoralis*, the age-related increase of antennal sensitivity to pheromone signal is dependent on the activation of 20E signaling pathway involving the nuclear ecdysone receptor (*Ecr*), its partner ultraspiracle (*USP*), and the transcription factor *E75* in correlation with induction of some regulators of olfactory transduction such as calmodulin and diacylglycerol kinase [49]. Furthermore, in *D. melanogaster*, Lin et al. [7] show that the olfactory receptor Or47b is indispensable for the courtship behavior of sexually mature old males: the sensitivity of Or47b neurons to a stimulating pheromone, palmitoleic acid, is low in young males but high in older ones. This age-related sensitization of Or47b neurons is found to be influenced by JH, which acts directly on Or47b neurons by binding to its Met receptor [7]. More recently, it has been uncovered in *A. ipsilon* males that altered JH biosynthesis or disrupted JH signaling pathway leads to reduced expression of pheromone binding protein (PBP2) and pheromone receptor OR3 coupled with a loss of the sensitivity of OR3 neurons to (Z)-7-dodecen-1-yl acetate, the major sex pheromone compound [50].

In addition to hormonal control of the peripheral antennal sensilla system, evidence has been accumulating that the olfactory centers are also the targets of endocrine signals. For instance, in *S. gregaria*, JH decreases the sensitivity of AL neurons to phenylacetonitrile with the aging of males, thus modulating the initiation of aggregation behavior [51,52]. In contrast, the emergence of sex pheromone-triggered orientated flight in *A. ipsilon* males is in part due to the increased sensitivity of AL neurons to pheromonal signal by JH [53,54]. This hormonal action is mediated by the Met-Tai-dependent cascade involving the JH inducible transcription factor, Krüppel homolog 1 [55]. In concert with JH, 20E also contributes to the functional remodeling of the AL network by upregulating the expression of synaptic proteins such as synaptotagmins [56]. In addition to JH and 20E, the monoaminergic system, especially octopamine and dopamine, is also involved in the age-dependent increase of behavioral and neuronal responses to sex pheromone in male *A. ipsilon* [4,57,6]. Indeed, the Dop1 receptor, an ortholog of vertebrate D1-type dopamine receptors known to be coupled positively to adenyl cyclase, is required for the display of orientated flight of males. Moreover, the levels of dopamine, *Dop1* expression, and cAMP in ALs rise concomitantly with the increased responsiveness to sex pheromones [57]. It has been proposed a hypothetical scenario for Dop1-mediated dopamine modulation in *A. ipsilon* ALs: the binding of dopamine to Dop1 stimulates the catalytic activity of adenylyl cyclase, which subsequently raises cAMP to initiate the cAMP-PKA signaling pathway and promotes the excitability of neurons as well as their synaptic communication [57]. Taken
<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

Basic organization of olfactory circuits to pheromones and actors of olfactory plasticity (from refs. [58,59]). Hormone-like molecules (biogenic amines) and hormones modulate the peripheral and central nervous systems (proven effects in full arrows and supposed actions in dotted arrows), leading to changes in sensitivity to pheromones that result in variations in the display of reproductive behaviors. On the left, olfactory receptor neurons (ORNs) are housed in the antennal sensilla. In the center, ORNs project axons to the antennal lobe. All ORNs that express the same ligand-specific receptor converge to a single glomerulus where a synapse is established with projection neurons (PNs) and local interneurons (LNs). On the right, PNs send axons to higher brain centers (lateral protocerebrum). Various neurons in higher brain centers, including lateral protocerebrum output neurons (LHONs), can integrate information from multiple PN populations and generate innate reproductive behaviors.

together, all these data brought evidence that hormonal
factors and biogenic amines influence the display of re-
productive behaviors by modulating the peripheral and
central processing of pheromone information (Figure 1).

Courtship, mating, and egg-laying behaviors
are controlled by endocrine actors

At the meeting of sexual partners, some insects perform
courtship displays [60-62]. It is well documented that
neuropeptides, such as neuropeptide F (NPF), are in-
volved in the regulation of sexual behaviors related to
courtship and mating 63. Indeed, ablation of NPF-pro-
ducing neurons or inhibition of NPF results in reduced
courtship behavior in *D. melanogaster* males [63]. Fur-
thermore, in *S. gregaria*, injections of NPF into males
promote their copulatory behavior [64,65]. Concomitantly
to neuropeptides, hormones are modulators of courtship
and mating behaviors. For instance, 20E- and *EcR*-defi-
cient females perform fewer complete ovipositor extru-
sion behaviors to reject male copulation attempts [66].
These females also execute male-like courtship beha-
viors, including unilateral wing extension and song pro-
duction [66]. Moreover, in other insects, such as *S.
gregaria*, *EcR* knockdown causes a delay in the onset of
male mating behavior [67]. Furthermore, in *D. melanoga-
ster*, JH-deficient males exhibit reduced courtship displays

[68]. In burying beetle *Nicrophorus vespilloides*, JH can
temporarily prevent females from mating following the
detection of a pheromone that inhibits copulation with
the male partner, allowing them to invest in caring for
their offspring [69].

In many insects, mating induces drastic modifications in the
behavior and physiology of females. The post-mating fe-
males exhibit reduced sexual receptivity and increased egg-
laying. In *D. melanogaster*, a key inducer of this behavioral
switch is a male-derived sex peptide (SP), which is trans-
ferred into the female with the seminal fluid during copu-
lation [70,71]. SP binds to the SP receptor, a G
protein-coupled receptor, and silences SP sensory neurons
in the female reproductive tract, and the signal is then
propagated into central neurons regulating receptivity in
virgins. Injection of *Drosophila* SP into *H. armigera* females
inhibits pheromone biosynthesis and calling behavior [72].
Furthermore, *D. melanogaster* females exhibit a characteristic
sequence of behaviors when they lay their eggs with a
preference for a medium containing low sucrose amounts.
The egg-laying decision is influenced by the neuropeptide
amnesiac (AMN): females lacking AMN exhibit impaired
decision-making behavior and are not able to distinguish
between unfavorable and favorable mediums for egg-
laying [73].
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

Reproductive behaviors and their regulation by endocrine actors in insects. JH, hormone juvenile; 20E, 20-hydroxyecdysone; PBAN, pheromone biosynthesis activating neuropeptide; NPF, neuropeptide F; SP, sex peptide; AMN, amnestic neuropeptide.

## Conclusions and future perspectives

Although the relevance of endocrine actors in the regulation of insect sexual communication has long been recognized (Figure 2), we are recently beginning to decipher the underlying cellular and molecular mechanisms. Recent advances, briefly reviewed above, offer new and exciting opportunities for research into the modulatory effects of hormones, biogenic amines, and peptides on the initiation of sexual signaling (e.g. pheromone production, release, and perception) and the display of reproductive behaviors (e.g. calling, courtship, mating, egg-laying) in insects.

In the future, it would be interesting to investigate the interrelationships of endocrine actors in the control of reproductive behaviors. For instance, there is growing evidence that hormonal factors interact with biogenic amines. This functional interplay has been initially described in the context of male insect reproduction. For instance, in *Apis mellifera* drones, JH has been shown to be a critical signal in the emergence of dopamine-mediated nuptial flight by simultaneously increasing the levels of dopamine and *Dop1* receptor expression in the brain [74,75]. Moreover, it has been originally characterized in *D. melanogaster*, the DopEcR receptor that belongs to the G protein-coupled receptor superfamily and exhibits a dual affinity for 20E and dopamine [76,77]. In *A. ipsilon* males, DopEcR is present in ALs and functions as a mediator of genomic actions of 20E

and dopamine, which facilitate the central processing of pheromone signal by increasing the AL neuron sensitivity, consequently leading to the onset of pheromone-guided flight behavior [4].

Finally, a better understanding of the endocrine regulation of insect reproductive processes will be useful to explore the impact of environmental changes (e.g. global warming, pesticides, nutrient depletion, exposure to endocrine disruptors) on the endocrine system pathways and their effects on the onset and display of reproductive behaviors.

## Author contributions

EF prepared the figures and the draft manuscript; then all authors contributed to the review and approved the submitted version.

## Data Availability

No data were used for the research described in the article.

## Declaration of Competing Interest

The authors declare that they do not have any commercial or financial relationships that could be construed as a potential conflict of interest.
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