Electroantennographic and Behavioral Response of Female Moroccan locusts [Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae)] to Male-Specific Aldehydes

Simple Summary

The Moroccan locust Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae) is a destructive pest of grasses and crops that has traditionally been managed with synthetic chemical pesticides. However, current regulation policies on pesticides highlight the need to develop environmentally friendly control strategies. In this context, the identification of chemical compounds capable of manipulating insect behavior (semiochemicals) emerges as a promising approach. In this study we addressed the behavioral activity of male-specific aldehydes, namely tetradecanal, pentadecanal and hexadecanal, on virgin and mated females D. maroccanus under laboratory conditions. Adult males release these compounds in an age-dependent pattern, whereas they are absent from the volatile profile of fifth-instar male nymphs. Females of both mating statuses perceive these aldehydes, although differences in the behavioral response depending on the mating status are observed in double-choice olfactometric assays. While virgin females only show a preference for hexadecanal, mated individuals are significantly attracted to tetradecanal and pentadecanal. Overall, our findings provide a preliminary basis for identifying putative pheromones in the Moroccan locust, although comprehensive research is still needed to determine the role of these aldehydes in the species’ chemical communication.

Abstract

The Moroccan locust Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae) is recognized as a serious pest of pastures and crops in many Mediterranean countries. So far, the identification of chemical compounds involved in intraspecific communication has been elusive, with only one male-specific compound, namely (2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-enal (2E,7R,11R-phytal), proposed as candidate sex pheromone component. Previous works reported that males also release the sex-specific long-chain aldehydes tetradecanal and hexadecanal, although no evidence of their role in chemical signaling has been presented so far. Here we additionally report another male-specific compound, viz. pentadecanal, and also report the electroantennographic and behavioral responses of virgin and mated females to these three aldehydes. We first demonstrated that their release is age-dependent, with emission overall peaking at 1–2 weeks after fledging. Solid-phase microextraction analyses from male hind legs suggest that these aldehydes are released from this body part. Overall, the antennae of virgin and mated females exhibited a dose-dependent response to increasing concentrations of each aldehyde, except for the response of virgin females to hexadecanal. Significant differences were observed between virgin and mated females at all doses of hexadecanal, while the mating status did not influence the response to tetradecanal and pentadecanal. Furthermore, the behavioral response of females varied according to their mating status. Specifically, mated females showed a significant preference for tetradecanal and pentadecanal, whereas a slight but non-significant attraction to hexadecanal was observed on virgin females. These findings provide new insights into the sexual communication of the species, although further research is needed to decipher their ecological function, especially on mated females.

1. Introduction

The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae) is a polyphagous pest of crops and pastures. This species shows a fragmented distribution, spanning about 10,000 km from the Atlantic islands to Central Asia, including North Africa, Mediterranean islands, Europe, the Middle East and Southwest Asia [1]. It is considered as one of the most economically significant locust species worldwide [2,3]. Population outbreaks of this species have historically constituted a major agricultural threat. Similar to other acridid species, such as the desert locust Schistocerca gregaria (Forskål, 1775) and the migratory locust Locusta migratoria (Linnaeus, 1758), favorable environmental and climatic conditions can trigger phase polyphenism, leading to the formation of hopper bands and large swarms capable of devastating entire landscapes [4]. Although these outbreaks are generally sporadic, they can cause severe damage when they occur. Recent records indicate small-scale but significant swarming events in Spain [5], France, Hungary, Italy [1], Uzbekistan and Tajikistan [6]. Furthermore, climate change is expected to intensify its distribution area and population dynamics, particularly under scenarios of consecutive drought years combined with temperatures higher than average. Preventive locust management is therefore essential to mitigate population upsurges before they reach outbreak levels. Current management strategies for these outbreaks remain dependent on chemical pesticides as diflubenzuron [7] and lambda-cyhalothrin, fipronil, chlorpyrifos or deltamethrin [8], among others, which entail substantial economic costs and environmental risk. In contrast, the implementation of environmentally sustainable approaches remains limited. Biological control includes the use of entomopathogenic fungi, such as Beauveria bassiana [9] and Metarhizium acridum [9,10], as well as innovative approaches consisting of plant-based granules impregnated with avermectins [11].

Advancing knowledge on intraspecific chemical communication and the mechanisms underlying sexual attraction could lead to the development of new semiochemically based tools for managing this pest. Previous studies on the chemical ecology of the Moroccan locust have identified the male-specific candidate sex pheromone 3,7,11,15-tetramethylhexadec-2-enal (hereafter referred to as phytal), which is released as both Z- and E-isomers [12]. Among the four possible diastereomers of the molecule, the (R,R) diastereomer has been proven to be the most behaviorally active on females [13]. In a previous laboratory study conducted on virgin and solitarious individuals of both sexes (aged 1–2 weeks), only females positively responded to the (R,R) diastereomer, while it resulted inactive for males [13]. Additional male-specific aldehydes, including tetradecanal (14:Ald) and hexadecanal (16:Ald), have also been reported from headspace collections of solitarious males, although their behavioral activity and the emission profile across the lifespan of males remain undefined [12]. As the volatile profile released by an insect species is reported to vary in an age-dependent way [12,14,15,16,17] we wondered whether the emission of these three aldehydes might show temporal variations as males age. Similarly, we also hypothesized that, if active, females may respond to these compounds in different ways depending on their reproductive status [18,19,20].

To shed light on these questions, the present study aims to (i) determine the release pattern of male-specific aldehydes according to male age, and (ii) evaluate the electroantennographic and behavioral response of virgin and mated females to these compounds.

2. Materials and Methods

2.1. Insects

Feral nymphs were collected during May and June 2025 from various locations with reported D. maroccanus outbreaks in the provinces of Aragon and Extremadura (Spain; see Table S1 for further information about collection sites). Immediately after collection, insects were taken to CITA (Centro de Investigación y Tecnología Agroalimentaria, Zaragoza, Spain), where they were sorted by sex and shipped to the Institute of Advanced Chemistry of Catalunya (IQAC-CSIC, Barcelona, Spain).

Upon arrival, nymphs were kept separated by sex in Bugdorm rearing cages (30 × 30 × 30 cm, Entomopraxis, Barcelona, Spain) under controlled conditions (25 ± 5 °C, 50 ± 10% RH and 16:8 L:D). After fledging, a group of males and females was kept isolated to ensure virgin status, while another group was placed together in cages to allow mating. Subsequently, those females found while mating were selected for further electroantennographic and behavioral assays.

All locusts were provided with fresh alfalfa (Medicago sativa L.) and common reed (Phragmites australis Cav.). Food was replaced every day, and insects were also sprayed with water to provide humidity. White light bulbs (60 W) were placed close to the cages to ensure heating and illumination.

2.2. Chemicals

14:Ald (>96%), pentadecanal (15:Ald) (>97%) and 16:Ald (>97%) were purchased from TCI Chemicals (Tokyo Chemical Industry, Tokyo, Japan). Dodecyl acetate, was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). n-Hexane (GC grade, ≥98.50%, Supelco, Merck Sigma-Aldrich, Madrid, Spain) was used as the solvent.

2.3. Volatile Collections

The volatiles of fifth-instar male nymphs and adult virgin males were collected through dynamic headspace collections (DHS) and headspace solid phase microextraction (HS-SPME), and further analyzed by GC-MS. These methodologies were chosen to first quantify the release of male-specific aldehydes according to developmental stage and age (i.e., DHS), and later to determine by SPME whether the hind legs are the releasing source of these compounds, as it occurs with (R,R)-phytal [12]. This technique allows the extraction of the analytes present in the headspace of a sample, without using any solvent, and therefore it was used to directly collect the volatile compounds released from excised male hind legs-. The volatile profiles of adult males of different ages (<1 week, 1–2 weeks, 3–4 weeks and >5 weeks) were analyzed by DHS, as previously described by Fürstenau et al. [12]. Fifth-instar male nymphs were also considered for the analysis because it is the last developmental stage prior to molting to adults, and some volatile compounds may be shared across stages [21]. Either adults (n = 7 males) or nymphs (n = 7) were placed in an Erlenmeyer glass flask (1 L, 21 cm tall × 12 cm Ø), and kept for 8 h at room temperature. The Erlenmeyer was sealed with a two-inlet glass cap, so that a constant charcoal-filtered air stream (500 mL/min) passed through one of the inlets, while the second inlet held a Super Q glass tube (30 g, 80/100 mesh size, Ars Inc. Gainesville, FL, USA). After the sampling period, the Super Q glass tube was eluted with 200 μL of hexane. One μL of a 100 ng/μL solution of dodecyl acetate was added as internal standard to DHS samples for further quantification of male-specific aldehydes. A total of three samples (excepting >5 weeks males, with n = 4 samples) were collected for each developmental stage and age condition. For each HS-SPME collection (n = 3), six hind legs from virgin males aged 1–2 weeks were placed in a glass vial (8 cm tall × 25 cm Ø) and exposed to a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated-fiber (65 µm, fused silica, 24-gauge needle; Supelco, Merck-Sigma Aldrich) for 8 h at room conditions and illuminated with a 60 W bulb.

All collection samples were analyzed using a Thermo Finnigan Trace 2000 gas chromatograph (Thermo Fisher Scientific, Barcelona, Spain) coupled to a Trace MS quadrupole mass spectrometer (Thermo Fisher Scientific) operating in electron impact mode (70 eV). The mass range was defined at 40–600 amu (1.0 scan/s). Both DHS and HS-SPME samples were injected in splitless mode into a non-polar TR-5MS column (30 m × 0.25 mm I.D. × 0.25 μm; Thermo Fisher Scientific) and with helium as the carrier gas. For DHS samples, oven temperature was initially set at 40 °C and maintained for 5 min, then raised to 280 °C at 10 °C/min, with a hold time of 10 min. For HS-SPME samples, initial temperature was programmed at 50 °C for 1 min, followed by an increase of 5 °C/min to 150 °C, and then further increased to 310 °C at 10 °C/min, with a hold time of 10 min. The injection port for both sample types was set at 250 °C. Male-specific compounds were identified by comparison of their mass spectra with those of synthetic standards.

2.4. Electroantennographic Response

The antennal olfactory response of D. maroccanus to increasing quantities (1, 10 and 100 µg) of male-specific aldehydes was determined for virgin (14:Ald, n = 15; 15:Ald, n = 13; 16:Ald, n = 8) and mated females (14:Ald, n = 13; 15:Ald, n = 14; 16:Ald, n = 15). All individuals tested were 2–3 weeks days old. For sample preparation, one antenna was cut off with the aid of a microscalpel, and the distal 2–3 segments were excised. Both ends were placed on a microelectrode holder (Syntech, Kirchzarten, Germany) with a drop of conductive gel (Ultrasonic gel, TELIC Group, Barcelona, Spain). The holder was connected to an EAG Combi-Probe (10×, Syntech) in turn connected to a MP-15 micromanipulator (Syntech).

To test the compounds, an aliquot of 10 μL of the corresponding hexane dilutions (0.1, 1 or 10 μg/μL) was applied to a round paper filter (2.5 cm diameter, Whatman^®, Merck-Sigma Aldrich, Madrid, Spain) to obtain the quantities of 1, 10, or 100 μg. The paper was placed into a 150 mm Pasteur glass pipette, and two consecutive 100 ms puffs (ca. 300 mL/min) were applied on the antenna for each tested compound and amount. The antenna was stimulated with two puffs of hexane—10 µL loaded on the filter paper—just before and after each pair of testing stimuli, in order to determine the baseline depolarization. A recovery interval of 60 s was allowed between each puff. A connecting glass tube (7 cm long × 5 mm Ø) with two lateral openings was placed 1 cm above the sample to hold the Pasteur pipette and to allow a continuous humified pure air flow (ca. 650 mL/min) upon the sample to avoid desiccation and thereby prolonging antennal lifespan. An Agilent ADM 1000 flowmeter (Agilent Technologies, Santa Clara, CA, USA) was used to determine both the continuous airflow upon the antenna and the flow of the chemical stimulus. EAG-Pro version 2.0 software (Syntech) was used to obtain the EAG recordings and to process the data.

2.5. Behavioral Assays

The walking response of 2–3-week-old virgin (n = 30–45) and mated females (n = 30–63) to 10 and 100 µg of 14:Ald, 15:Ald, and 16:Ald was assessed in a glass double-choice Y-shaped olfactometer (main arm 24.5 cm × 50 mm I.D., secondary arms 9 cm × 50 mm I.D., and 90° angle between arms). The olfactometer was placed horizontally, and a charcoal-purified airflow (ca. 700 mL/min, measured with an Agilent ADM 1000 flowmeter), passed through each secondary arm. One of the secondary arms contained the aldehyde to be evaluated, while the opposite arm contained hexane as the solvent control.

The chemical stimuli were released from round Whatman filter papers (2.5 diameter) loaded with 10 μL of either the corresponding hexane dilution (1 μg/μL or 10 μg/μL) of each aldehyde or only hexane. Every five insects, the filter papers were replaced, and the position of the olfactometer arms was switched to avoid any directional bias. A white light bulb (100 W) was placed 70 cm upon the olfactometer to provide a homogenous illumination of 300 lux approximately, and the whole system was surrounded with black cardboard to avoid the interference of visual stimuli. Prior to the beginning of the assay, females were individually placed in 50 mL Falcon tubes and acclimated at room temperature for 30 min. In each trial, a single female was placed at the base of the main arm and it was given 5 min to choose between either the control or the chemical stimulus. Walking response was regarded as positive when a female walked into a secondary arm at least 2 cm. Those females that did not make a choice after 5 min were considered as non-responders and therefore excluded from the statistical analysis. All assays were conducted from 10:00 a.m. to 6:00 p.m. at 25 ± 5 °C and 50 ± 10% RH, and for each compound and stimulus a minimum of 10 insects were tested daily, thereby ensuring at least three replicates per trial.

2.6. Statistical Analysis

Data from the electroantennographic recordings were log transformed when necessary to avoid violating the assumption of homogeneity of variances. Net antennal responses to each aldehyde were subjected to a two-way analysis of variance (ANOVA), with mating status and dose set as fixed factors, followed by Tukey HSD test for multiple comparisons. Differences in the release rate for each aldehyde according to male age were subjected to one-way ANOVA followed with Bonferroni post hoc tests for multiple pairwise comparisons between groups [22]. When necessary, data were subjected to log transformation (x + 1) to meet the assumption of homogeneity of variances. Walking response of females was analyzed with a chi-square goodness of fit test, at a significance level of 0.05. Only those treatments with at least 15 responding females were subjected to the statistical test. All analyses were conducted with JASP statistical software version 0.95.

3. Results

3.1. Headspace Collection and Quantification

Dynamic headspace collections from virgin adult males corroborated the release of 14:Ald and 16:Ald, along with the detection of another male-specific aldehyde, namely pentadecanal (15:Ald) (Figure 1a). Further HS-SPME analyses of excised hind legs from male adults revealed the emission of these three aldehydes from this body part (Figure 1b). Additional collections on virgin females (1–2 weeks) also verified the absence of 15:Ald (Figure 2). These three compounds were also absent from volatile collections from fifth-instar male nymphs (Figure 3). According to the quantification from DHS samples, the release of these compounds in adult males was age-dependent, as shown in Figure 3. In the case of 14:Ald (F_3,9 = 4.631, df = 3, p = 0.032), significant differences in the amount of compound released were only detected between males aged <1 week (3.5 ± 6.0 ng/male/h) and 1–2 weeks (50.0 ± 26.0 ng/male/h) (Figure 3).

Figure 1.

Illustrative chromatographic profiles from (a) DHS collections from adult D. maroccanus virgin males (1–2 weeks) and (b) HS-SPME from excised hind legs of adult males (1–2 weeks). Numbered peaks correspond to male-specific compounds: (1) tetradecanal, (2) pentadecanal, (3) hexadecanal, (4) (2Z,7R,11R)-phytal, (5) (2E,7R,11R)-phytal. (IS): internal standard (dodecyl acetate).

Figure 2.

Illustrative chromatographic profiles from DHS collections of adult D. maroccanus (a) males and (b) females. Numbers correspond to male-specific aldehydes: (1) tetradecanal (2) pentadecanal, and (3) hexadecanal. (IS): internal standard (dodecyl acetate).

Figure 3.

Mean release rate (ng ± SEM) per individual per hour of male-specific compounds tetradecanal (14:Ald), pentadecanal (15:Ald), and hexadecanal (16:Ald) released by adult males at different ages. Different letters within a single compound indicate statistical differences (one-way ANOVA followed by Bonferroni post hoc tests).

Significant differences were also detected in the emission of 15:Ald (F_3,9 = 44.12, df = 3, p < 0.001) and 16:Ald (F_3,9 = 7.476, df = 3, p = 0.008) (Figure 3). In the case of 15:Ald, males aged 1–2 weeks (15.2 ± 9.9 ng/male/h) and 3–4 weeks (12.7 ± 5.6 ng/male/h) released significantly higher amounts of compound than males of <1 (2.1 ± 0.7 ng/male/h) and >5 weeks old (Figure 3). Indeed, the emission of 15:Ald from males aged less than one week (2.1 ± 0.66 ng/male/h) differed from that of the oldest males (>5 weeks old), in which the emission of the compound ceased (Figure 3). Concerning 16:Ald, the emission rate peaked in males aged 1–2 weeks (298.2 ± 58.3 ng/male/h) (Figure 3). This maximum release rate significantly differed from that of young (<1 week old: 26.0 ± 22.3 ng/male/h) and males aged more than 5 weeks (81.9 ± 36.0 ng/male/h), although it was similar to the emission from males aged 3–4 weeks (225.1 ± 130.1 ng/male/h) (Figure 3).

3.2. Electroantennographic Response to Male-Specific Aldehydes

For all aldehydes tested, the dose factor was found significant in the EAG response, while mating status was reported as significant in response to 15:Ald and 16:Ald (Table 1). Conversely, the interaction between mating status and dose had no significant effect in either aldehyde (Table 1).

Table 1.

Two-way ANOVA results of the effect of mating status, dose and their interaction on the EAG response of D. maroccanus females to tetradecanal (14:Ald), pentadecanal (15:Ald) and hexadecanal (16:Ald).

Compound	Source of Variation	Mean Square	F	df	p
14:Ald	Mating status	0.019	0.238	1	0.628
	Dose	2.085	26.132	1	<0.001
	Mating × Dose	0.059	0.737	2	0.484
15:Ald	Mating status	0.374	5.718	1	0.022
	Dose	1.227	18.740	1	<0.001
	Mating × Dose	0.006	0.090	2	0.766
16:Ald	Mating status	5.759	53,669	1	<0.01
	Dose	0.758	7.065	1	<0.001
	Mating × Dose	0.120	1.115	2	0.339

With the exception of mated females in response to 16:Ald, all EAG profiles followed a dose–response pattern (Figure 4). The response to 14:Ald and 15:Ald at each quantity did not differ between mating statuses (Figure 4a,b), except at 10 µg of 15:Ald, where none of the antennae from virgin females responded to this stimulus (Figure 4b). In the case of 16:Ald, antennae from virgin females significantly responded to all stimuli in a higher magnitude than mated ones (Figure 4c).

Figure 4.

Mean EAG response (mV ± SEM) of virgin and mated D. maroccanus females in response to increasing stimuli (1, 10 and 100 µg) of (a) 14:Ald (virgin females, n = 15; mated females, n = 13), (b) 15:Ald (virgin females, n = 13; mated females, n = 14) and (c) 16:Ald (virgin females, n = 8; mated females, n = 15). Letters indicate significant differences in the response magnitude among doses and reproductive statuses, (two-way ANOVA followed by Tukey HSD test).

3.3. Behavioral Response to Male-Specific Aldehydes

Overall, 50% of the total of D. maroccanus females made a choice for either the test aldehyde or the solvent control (233 out of 462 females). The lowest mobility percentages were observed when virgin and mated females were exposed to 10 µg of 15:Ald and 16:Ald, respectively, with only 27–30% of individuals making a choice.

Different behavioral responses in response to the stimuli were detected depending on the mating status. While mated females showed a significant preference for 10 µg of 14:Ald and 15:Ald (14:Ald, χ² = 4.235, df = 1, p = 0.040; 15:Ald, χ² = 9.529, df = 1, p < 0.01), neither compound appeared attractive for virgin females (Figure 5a,b). Similarly, no significant attraction was observed in response to 100 µg of 14:Ald and 15:Ald, irrespective of the mating status (Figure 5a,b).

Figure 5.

Percentage of attraction in a Y-tube olfactometer of virgin (n = 30–43) and mated (n = 30–63) D. maroccanus females to 10 and 100 µg of (a) 14:Ald, (b) 15:Ald, and (c) 16:Ald. Significant differences in the attraction are denoted by asterisks (chi-square goodness of fit test; ns = not significant, ‡: data not included in the analysis due to the low number of individuals making a choice). The numbers beside each bar indicate the number of females tested and those that made a choice for either arm of the olfactometer (in parentheses).

With regard to 16:Ald, none of the quantities tested elicited a positive chemotactic response on virgin and mated females, although 100 µg of compound evoked a slight preference on virgin females (χ² = 3.267, df = 1, p = 0.071) (Figure 5c).

4. Discussion

Seeking alternative and environmentally sustainable control measures has become the Holy Grail for locust management, in order to reduce the widespread reliance on insecticide use. Within this context, pheromone isolation and identification appear promising, due to their species-specific activity and proven efficacy in pest management. Recent findings on the identification of the aggregation pheromone of L. migratoria, viz. 4-vinylanisole [23], have opened an optimistic scenario for future management strategies, not only focused on monitoring and trapping the species [24], but also inhibiting the biosynthesis of 4-vinylanisole and subsequent suppression of swarm formation [25]. While S. gregaria and L. migratoria have received considerable attention in terms of deciphering their chemical communication [21,23,26,27,28] knowledge about the Moroccan locust remains limited, being restricted to the identification of the male-specific compound (R,R)-phytal as sex pheromone candidate [12,13].

Here, we present new laboratory insights into the intraspecific chemical communication of this species, addressing the electrophysiological and behavioral activity of male-specific compounds. In addition to the two aldehydes (14:Ald and 16:Ald) previously identified in the volatile blend of adult males [12], our study reveals the presence of 15:Ald as an additional male-specific compound. The release of these three aldehydes followed the same trend as that reported for (R,R)-phytal [12], being absent in fifth-instar male nymphs and peaking in adult males at 1–2 weeks after fledging. Despite the observed biological activity, the role of these sex-specific aldehydes within the chemical signaling system of the species remains unclear. The overlapping emission pattern with (R,R)-phytal, together with the observed intersexual attraction, may suggest a putative role of these compounds as pheromone components. While (R,R)-phytal is mainly attractive for virgin females [13], our work reveals that 14:Ald and 15:Ald are found attractive to mated females in double-choice trials. Conversely, virgin females do not show any positive chemotactic response to either aldehyde, except the slight although not significant response observed for 16:Ald. In locusts, differences in the behavioral response to conspecific odors are not only related to phase state, but also to mating status. In L. migratoria, odors from virgin gregarious females are attractive to virgin gregarious males [28]. Conversely, this attraction is not observed in mated gregarious males and solitary virgin and mated males [28]. The mating-status-dependent response to 14:Ald and 15:Ald may suggest that the attraction evoked on mated females is linked either to mating partner location or to alternative mating strategy traits. Female locusts are polyandrous, frequently mating with multiple males [29], and the last male to mate fertilizes the eggs [30]. This phenomenon, known as last-male sperm precedence, is a post-copulatory outcome within the cryptic female choice, a widespread strategy in animals in which females control and bias which sperm fertilizes their eggs through various mechanisms, thereby benefitting the offspring by favoring males with particular traits [31]. In this regard, male-specific chemical cues are suggested to influence cryptic female choice in insects [32,33]. In addition to female-adopted strategies, males’ traits can be sensed by females during pre-copulatory or copulatory courtship and affect cryptic female choice. Concerning locusts, male-released pheromone bouquets in Schistocerca americana (Drury) and Schistocerca piceifrons (Walker) are regarded as mate assessment pheromones, as females preferred the sperm of those males releasing high pheromone quantities [34,35]. Even though the number of responding individuals in the behavioral tests was relatively low, probably due to the absence of other pheromone components females exhibited a significant preference for both individual compounds and the complete pheromone bouquet, while males were only arrested by the complete pheromone bouquet [35]. In light of these previous studies, 14:Ald and 15:Ald released by virgin D. maroccanus males may play an undetermined effect on mated females related to cryptic female choice and/or mate assessment and acceptance. Nonetheless, our hypothesis is based exclusively on behavioral evidence in response to these compounds, and further comprehensive research on courtship and mating behavior of the species would be essential to address this question. We are also aware that the biological activity of these aldehydes has only been assessed individually in females. Moreover the response of females was generally weak, with a mean mobility percentage of approximately 50% and even below 30% in two of the experiments—mated females responding to 10 µg of 16:Ald and virgin females to 10 µg of 15:Ald—, which may suggest the absence of additional key compounds necessary to evoke a robust behavioral response. Therefore, and in spite of the positive chemotactic response observed to 14:Ald and 15: Ald, additional laboratory trials are needed to determine the biological implications of these aldehydes and the potential activity of the binary mixture and their synergism with (R,R)-phytal in both sexes, at both adult and nymphal stages.

Both 14:Ald and 15:Ald have been reported as key components in the chemical communication of insect species belonging to different Orders, such as Mantodea [36], Hymenoptera [37], and Hemiptera [38]. In addition, both compounds are found in minor quantities in the marking pheromones from mandibular glands produced by males of various beewolf species (Hymenoptera: Sphecidae) [37]. So far, most of the biologically active chemical cues from nymphal and adult stages of Locusta and Schistocerca species are aromatic derivatives, such as phenol, guaiacol, phenylacetonitrile, 4-vinylanisole, anisole, veratrole and benzaldehyde [23,39,40]. In S. americana, nonanal and two monounsaturated alcohols—(Z)-3-nonen-1-ol and (Z)-2-octen-1-ol—are regarded as the male-produced sex pheromone [34], whereas S. piceifrons males release a mixture comprising an aromatic derivative (i.e., 2-phenylethanol), (Z)-3-nonen-1-ol and (Z)-2-octen-1-ol [34]. The role of these compounds is diverse, including aggregation [23,26,40], oviposition [41], maturation [34,42,43], cryptic female mate choice [34,35], and courtship inhibition [44]. To the best of our knowledge, this is the first time in which 14:Ald and 15:Ald are reported as attractive in a locust species.

Headspace collection analyses through HS-SPME corroborate the emission of all aldehydes from male hind legs, as also occurs with (R,R)-phytal in D. maroccanus [12] and pheromone compounds in other locust species [23,34,44]. For instance, more than 56% of 4-vinylanisole is released from the hind legs of L. migratoria [23]. Similarly, mature gregarious males of S. gregaria release phenylacetonitrile from hind legs and especially from forewings [44]. A detailed anatomical and histological study of the hind legs of D. maroccanus may aid in the identification of the production and release sites of these compounds, such as the epidermal glands in the hind legs releasing phenylacetonitrile in S. gregaria [44] and pheromone components in S. americana [34]. In the absence of further tests, the release of these three aldehydes from different body parts of D. maroccanus should not be discarded.

Overall, our findings provide a very preliminary basis for delving into the putative role of male-specific compounds in the intersexual communication of the Moroccan locust. It is worth noting that all related findings so far have been obtained from solitarious phase individuals [12,13]. Indeed, reports on pheromone cues from solitarious individuals are scarce in locusts, in marked contrast with the vast research devoted to identifying key chemical cues from the gregarious phase [23,26,34]. Recently, Cui and coworkers [45] identified dibutyl phthalate from solitarious L. migratoria females, and laboratory tests on males support the role of this compound as a sex pheromone. Key volatiles involved in the gregarious phase are also reported to attract solitarious individuals [23,46]. In any case, the sporadic D. maroccanus outbreaks in Spanish mainland—the last severe swarming event occurred in 2022 [5]—have significantly limited further progress in understanding the chemical ecology of the species and potential inter-phase chemical interactions. Accordingly, future studies will be aimed to explore the implications of the male-specific chemical burden in the sexual communication of the solitarious phase of the species, including the evaluation of trapping efficacy under natural conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects17020176/s1, Table S1: Location and coordinates of Dociostaurus maroccanus collection sites in Spain (2025).

Author Contributions

Conceptualization, C.Q. and S.L.; methodology, E.T., B.F., M.M.C.-A., C.Q. and S.L.; formal analysis, E.T., B.F., C.C.-M. and I.R.; investigation, E.T., B.F., C.C.-M., I.R. and M.M.C.-A.; data curation, C.Q. and S.L.; writing—original draft preparation, S.L.; writing—review and editing, E.T., B.F., C.Q. and S.L. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Ministry of Science, Innovation and Universities (Spain) and the State Research Agency (Spain) (PID2022-138040OB-I00).