Influence of ovarian maturity, age, and mating status on the antennal responses of wild and laboratory reared Xyleborus affinis (Coleoptera: Curculionidae: Scolytinae) to ethanol

Abstract

Xyleborus affinis Eichthoff is a neotropical ambrosia beetle that, in certain regions such as the United States and Mexico, has been associated with exotic phytopathogenic fungi causing extensive tree mortality. Although studies relating its olfactory response to volatile compounds and trapping systems have been published, factors such as the insect’s physiological condition, which can affect its recognition or response to odors, have not been studied. Here, we evaluated the electroantennographic (EAG) response of wild and laboratory reared X. affinis females to 70% ethanol, a compound known to attract these insects. The experimental design was developed to consider and compare the following conditions: (i) females collected inside and outside host-galleries, (ii) sexual maturity (with mature and immature ovaries), (iii) age (0, 1, 3, and 5 d after emergence), and (iv) mating status (virgins and mated). Our results indicate that most of the wild females located outside the galleries were sexually mature but exhibited significantly lower EAG response than those inside the galleries. Regarding mating status, mated females exhibited significantly stronger antennal responses compared to virgins, whereas age did not affect antennal sensitivity. Finally, we provide images of the hitherto undescribed reproductive system of X. affinis females, a key element that enabled this investigation. The information generated offers a useful foundation for future studies aimed at understanding the physiological mechanisms and other critical factors related to sensory perception and reproductive condition. For example, factors that may influence the behavior and attraction of X. affinis females to host semiochemicals.

Graphical Abstract

Graphical Abstract.

Introduction

Ambrosia beetles (Coleoptera: Curculionidae: Scolitynae and Platypodinae) are wood boring insects that maintain symbiotic relationships with specific fungi, which are carried by females in highly specialized structures called mycetangia (Wood 1982, Vega and Biedermann 2020, Joseph et al. 2025).Once a host tree is located, females drill into the branches or tree-trunks within several plant families to build gallery systems, inoculating these tunnels with fungal spores that produce a nourishing mycelium for the nutrition of adults and larvae after germination (Saucedo-Carabez et al. 2018, Biedermann 2020).

Xyleborus affinis is an ambrosia beetle that has gained importance since it was reported as secondary vector of Harringtonia lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostamatales: Ophiostomataceae), previously known as Raffaelea lauricola (Carrillo et al. 2014, De Beer et al. 2022), a phytopathogenic fungus that causes a devastating vascular disease in Lauraceae trees (Fraedrich et al. 2008). Avocado (Persea americana Mill), a crop of high economic value within the Lauraceae family, has been documented as being negatively affected by X. affinis (Castrejón et al. 2017). Notably, X. affinis was reported to be associated with other mycopathogens such as Ceratocystis lukuohia Barnes I, Harr TC, Keith LM and Ceratocystis huliohia Barnes I, Harr TC, Keith LM that cause the Rapid ‘Ōhi‘a Death, a lethal disease for ‘Ōhi‘a trees (Metrosideros polymorpha Gaud) in Hawaii (Barnes et al. 2018, Cannon et al. 2022); and to Ceratocystis fimbriata Ellis and Halst, the causal agent of mango wilt (Souza et al. 2013, Galdino et al. 2016). Like other ambrosia beetles, X. affinis females are the colonizers and seekers of new hosts, even though it is unclear what factors trigger their host-seeking behavior (Biedermann et al. 2011). To find and select their host, females mainly use ethanol (Miller and Rabaglia 2009, Ranger et al. 2010, Cavaletto et al. 2021, Moore et al. 2026) volatile fungal compounds (pathogenic or symbiotic) reported for other ambrosia beetle species (Hulcr et al. 2011, Rassati et al. 2020), and other cues such as different terpenes (Kendra et al. 2011, 2012a, 2023).

The search for and acceptance of their hosts in insects is influenced by physiological factors such as sexual maturity, age, oviposition, or mating status (Bernays and Chapman 1994, Anton and Cortesero 2022). For example, in the coffee berry borer Hypothenemus hampei (Ferr.), it has been reported that older females are more attracted to coffee berries; however, once they oviposit, their olfactory responses to the host are modified (Mathieu et al. 2001). In contrast, studies on Bactrocera tryoni (Froggatt) have shown that increased age is associated with a significant decrease in olfactory sensitivity in both males and females (Tasnin et al. 2020). In the pepper weevil Anthonomus eugenii Cano, immature females are attracted by host and non-host volatiles, while the mature ones are only attracted by host volatiles (Addesso and McAuslane 2009). In a certain way, X. affinis information about the influence of physiological factors on its olfactory response is very limited. Therefore, the objective of this study was to evaluate whether females located outside or inside of their host, with mature or immature ovaries, either mated or unmated, and of different ages, exhibit different antennal responses to ethanol. We hypothesized that mated, sexually mature females would exhibit stronger electroantennographic (EAG) responses than virgin and immature females. We further predicted that females collected outside host galleries, representing dispersing individuals, would be predominantly mature and mated, and would exhibit greater responses to ethanol, consistent with their role in founding new colonies. Finally, we predicted that the females’ antennal responses will increase with age.

Materials and Methods

Origin of Laboratory-Reared and Wild Insects

The study included X. affinis females from laboratory colonies and wild populations. Laboratory-reared individuals were obtained from a colony kept for over 24 generations under laboratory conditions in the Molecular Entomology Laboratory at INECOL (Romero et al. 2022). The wild females were obtained following 2 procedures: (i) capturing females in flight in the vicinity of wild Bursera  simaruba (L.) Sarq. trees in Zimpizahua, Coatepec, Veracruz, Mexico (19°25′52″N, 96°56′51″W, 1,095 m.a.s.l.), during February and March 2021, following the methodologies described by Kendra et al. (2012b) and Romero et al. (2022); and (ii) from wild B. simaruba branches (20 to 30 cm diameter), showing signs of infestation by X. affinis. These branches were collected from August 2020 to March 2021 at the same location where flying females were captured and transported to the Instituto de Ecología (INECOL) in Xalapa, Veracruz, Mexico, where they were maintained at 20 ± 4 °C and 82 ± 2% RH. Branches were cut into 15-cm sections and individually placed inside emergence chambers consisting of round plastic containers (25 L; 36 cm length, 38 cm outside diameter) with a lid. Two rectangular windows (17 × 19 cm) were cut on opposite sides of each container and covered with ultrafine mesh to allow ventilation of the wood while preventing the entry of other insects and the escape of emerging beetles. Besides, 2 transparent plastic jars (300 ml) were attached to circular perforations (7 cm OD) located 11 cm from the bottom and top of the chamber. The jars were easy to remove through a screw and contained a slightly moistened paper towel (Kirkland), which helped retain beetles emerging from the logs inside the chamber.

Preparing Females for Assays

In what follows, we describe how X. affinis individuals were prepared to obtain specimens representing different conditions: (i) laboratory-reared females of progressive ages; (ii) virgin laboratory females compared with wild mated females captured in flight, used to assess mating status; and (iii) wild females collected from infested branches, obtained both outside and inside host galleries. These specimens were employed to determine antennal responses to ethanol (Fig. 1, methodological overview).

Fig. 1.

Diagram summarizing our experimental design. X. affinis females were obtained from defined sources and subjected to experimental assays, including electroantennographic recordings (EAG), ovarian maturation assessments, and determination of mating status.

Laboratory-Reared Females: Progressive Ovarian Maturation and Virgins

Because the age and mating status of wild insects are usually unknown, we used laboratory females to specifically assess the effects of age and virgin status on EAG responses. Under laboratory conditions these females can survive for at least 1 month (Rojano et al. 2018). Prior to experimentation, we conducted a preliminary observational assay in which females were monitored daily from 0 to 10 d post emergence. These observations revealed that sexual maturity was reached by day 5, with no further changes thereafter. Based on this, we selected 0-, 1-, 3-, and 5-d-old females to capture the early transition to maturity. For this, laboratory females were reared in 50-ml centrifuge tubes (Thermo Scientific Nunc) containing 15 ml of sterilized artificial diet (Menocal et al. 2018). One X. affinis female (1 to 10 d old) was introduced into the center of an artificial gallery (1 cm deep) created by puncturing the diet with a fine needle. The diet tubes were maintained in an environmental chamber (Thermo Scientific, 3940, United States) at 26 ± 1 °C and 60 ± 5% RH. The rearing medium was dissected after 25 d to recover pupae along with fragments of their gallery. Pupae were individually transferred to pill boxes (1.5 cm × 2 cm) lined with a moistened paper towel and filter paper. The pill boxes were kept in an environmental chamber under the same rearing conditions and checked daily until adult emergence (age = 0 d). Newly emerged, fully sclerotized females were individually transferred to 5-cm-diameter Petri dishes containing fresh artificial diet medium and maintained in the environmental chamber until they reached 1, 3, and 5 d of age. Sets of 0-, 1-, 3-, and 5-d-old females, kept virgin by isolation from males, were used to perform EAG assays and were then dissected to determine their sexual maturity. However, only the responses of virgin females at 3 and 5 d of age were considered for comparison with mated females because at these ages they already possess mature ovaries, making them reproductively competent.

Ovaries were dissected under a stereomicroscope (Leica S6E, Leica Microsystem, Wetzlar, Germany) to determine maturation status. In 1 ml of 30% ethanol, elytra, wings, and integument were removed with dissecting forceps to gently expose the digestive tract and ovaries (Ungureanu 1972). A 0.001% KOH (Sigma-Aldrich) solution was applied for 30 s to clear the tissues prior to the Confocal microscopy studies. Isolated ovaries were then transferred to a glass-bottom FluoroDish (FD35-100, World Precision Instrument, Inc.) with 5 µl of phosphate-buffered saline (PBS) and stained with one drop of NucBlue Fixed Cell ReadyProbes (DAPI) for 1 min in the dark at room temperature, followed by washing with PBS. Samples were observed with a Leica DMI6000B epifluorescence microscope equipped with a digital camera (Leica Microsystem, Wetzlar, Germany). Image analyses were performed using Leica Application Suite X (LAS X) software. Progressive ovarian maturation was characterized according to descriptions available in the literature (Grodowitz and Brewer 1987, Martínez 2002, Vega-Petlacalco et al. 2018).

Wild Mated Females Captured in Flight

The mating status was determined only for females collected in flight. The beetles were placed in Petri dishes lined with moist filter paper and transported to the laboratory. Individuals were identified as X. affinis based on morphological characters (Pérez-Silva et al. 2015, Bateman and Hulcr 2017) and verified against reference specimens identified by Dr. Atkinson TH (University of Texas Insect Collection, Austin, Texas, United States). The verified females were first subjected to EAG assays (see EAG responses of X. affinis to ethanol). Subsequently, the remaining body was individually placed in 1 ml of Carnoy’s solution (chloroform, absolute ethanol, glacial acetic acid 3:6:1) and refrigerated until ovary dissections. To assess ovarian maturation, specimens were gradually rehydrated through a series of ethanol dilutions (100%, 96%, 70%, and 30% EtOH in H₂O), each step involved soaking for 24 h at 3 °C. Ovarian dissections followed the methodology described previously, and the ovaries were photographed with a Digital Sight DS 2Mv camera mounted on a Nikon Eclipse 50i microscope and analyzed using Nis-elements (AR) image software (Nikon Corporation 1991–2006). Ovarian condition was classified as: (i) immature, characterized by the absence of mature oocytes and low follicular differentiation; and (ii) mature, defined by the presence of mature oocytes or follicular relics (see progressive maturation of the ovaries in the Results section).

The mating status of females collected in flight was also assessed by dissecting the spermathecae and verifying the presence of sperm. Since the sperm of X. affinis had not been previously described, reference images were obtained from a preliminary imaging study. Hence, sclerotized females and males from artificial rearing media were paired in 200-ml containers maintained in an environmental chamber for 12 h. After this period, spermathecae were dissected in 0.5 ml of PBS buffer. Cleaned and isolated spermathecae were transferred to a glass-bottom FluoroDish (FD35-100, World Precision Instrument, Inc.) containing 10 µl of PBS. Spermathecae were punctured with a #3 pin to expose the contents, then stained with NucBlue Fixed Cell ReadyProbes (DAPI) for 2 min in the dark at room temperature. Observations were made using the microscopy equipment previously described. Additionally, images of spermathecae from 5-d-old virgin females served as negative controls.

Finally, to confirm the mating status of wild females collected in flight, spermathecae were dissected, placed on glass microscope slides (25.4 × 76.2 mm) with 3 µl of 80% glycerol, gently fractured to expose their contents, and covered with 24 × 24 mm coverslips. Preparations were observed and imaged using a Leica TCS SP8 confocal laser scanning microscope equipped with an HC PL FLUOTAR 10×/0.30 objective (Leica Microsystem, Wetzlar, Germany). Final observations were performed with HC PL APO 63×/1.40–0.60 OIL objectives to detect spermatozoa. Females with sperm-positive spermathecae were classified as mated, and their EAG responses were subsequently compared with the laboratory reared virgin females.

Comparison of Wild Females Inside and Outside the Host

Specimens of X. affinis were obtained from 2 conditions: outside and inside the host. For the first group, beetles were collected daily from emergence jars attached to the emergence chambers where B. simaruba branches were placed. For the second group, branches were randomly selected from the chambers and carefully dissected with a gouge (Stanley). Once the galleries were exposed, fully sclerotized females were gently collected. Individuals from both groups were visually identified, and their identities verified before use in EAG assays (see EAG responses of X. affinis to ethanol). Ovarian dissections were then performed to determine sexual maturity, following the imaging and assessment procedures described in the wild mated females captured in flight section.

EAG Responses of X. affinis to Ethanol

Antennal responses to 70% ethanol were evaluated in females of different conditions thoroughly described in the preceding sections. The head of each live female was carefully removed for EAG recording following the methodology of Kendra et al. (2012c). Antennal responses were measured using a Syntech EAG system (Syntech, Kirchzarten, Germany), comprising a micromanipulator (EAG combi probe), a signal connector (IDAC-2), a stimulus controller (CS-55), and EAG software (EAGPro Version 2.0, Syntech NL 2003). One antenna of X. affinis was carefully detached and mounted in an electrode holder (Syntech) previously coated with Spectra 360 gel (Parker Laboratories, Fairfield, United States). The basal antennal segment was positioned on one side of the electrode, with the antennal club facing the injection tube. A filtered airflow of 400 ml/min was then induced through the injection tube, located 1 mm from the electrode. Once the antennal signal stabilized, 4 ml of 70% ethanol vapor was injected using a hermetic syringe (VICI Precision Sampling, United States), and the EAG response was recorded. The odor stimulus stemmed from a 250-ml glass jar containing 15 ml of 70% ethanol, introduced 2 h prior to the assay to allow headspace saturation. The jar was sealed with a cap adapted with an airtight nozzle and a silicone rubber septum, enabling volatile extraction with a syringe without opening the jar. A negative control, consisting of air from an empty glass jar prepared under the same conditions, was also tested. Each antenna was treated as a replicate, beginning with an injection of 70% ethanol followed by the negative control (air). For each replicate, the depolarization elicited by the negative control was subtracted from the corresponding ethanol EAG recording to eliminate potential mechanical effects of injection. All assays were conducted at 27 ± 1 °C and 60 ± 5% RH.

Statistical Analyses

The assumptions of normality and homoscedasticity were assessed through graphical exploration of model residuals (residuals vs. fitted, Q–Q residuals, scale–location, and residuals vs. leverage plots) and formally tested using Shapiro–Wilk and Levene’s tests to evaluate normality and homogeneity of variance, respectively. The EAG data of female location (inside and outside of the host) and ovarian maturity (mature and immature) showed a marginal deviation from normality (W = 0.967, P = 0.0469); however, homogeneity was not rejected (F = 2.5589, P = 0.0615) and exploratory plots supported normal distribution. Therefore, these data were analyzed using a 2-way analysis of variance (ANOVA) (P < 0.05). Furthermore, the association between female sexual maturity and location (inside and outside of the host) was analyzed using the chi-square test, and the strength of the association was measured using Cramer’s V. The EAG data for laboratory-reared females of controlled age (W = 0.824, P < 0.001; F = 0.6147, P = 0.0607) and for mating status (W = 0.771, P < 0.001; F = 16. 321, P < 0.0001) did not meet normality assumptions; therefore, nonparametric tests were applied: Kruskal–Wallis to compare age-controlled groups, and Wilcoxon to evaluate differences in mating status. All statistical analyses were performed in RStudio, Inc. version 2026.05.1 + 225.

Results

Reference Images

Sperm and spermatheca images (Fig. 2) were obtained and used to identify mated females. As shown in Fig. 2A and B, the spermatheca of X. affinis exhibits a distinctive C-shaped morphology with a smooth surface, consisting of a capsule connected to a spermathecal duct (Pérez-Silva et al. 2018). The sperm of X. affinis displays a characteristic head–tail structure with a thin, elongated body typical of curculionids (Fig. 2C; Burrini et al. 1988).

Fig. 2.

Confocal and epifluorescence microscopy images representative of X. affinis spermathecae and spermatozoa. (A) Front view of empty spermatheca (white arrow); the chitinous wall of spermatheca shows autofluorescence in magenta, while nuclei were stained in green with DAPI. Image acquired under the following conditions: 63× oil/1.40 NA objective, 1× digital zoom, Bar = 20 μm. (B) Filled spermatheca (white arrow) stained with the same technique of panel A. Image acquired under the following conditions: 46 optical planes with 0.4-μm Z plane separation, 63× oil/1.40 NA objective, 1× digital zoom, Bar = 20 μm. (C) Epifluorescence microscopy image of spermatozoa, showing a head (h) and a tail (t). Nuclei were stained with DAPI; imaging conditions: UV excitation filter: BP 340 to 380. HC PL APO 100× OIL UV Objective 1.44 NA, Bar = 50 μm.

Progressive Maturation of the Ovaries

According to Fig. 3, the reproductive tract of X. affinis consists of 2 ovaries, each containing 2 ovarioles (Fig. 3A). Each ovariole is composed of a terminal filament, germarium, vitellarium, lateral oviduct, and common oviduct. At the beginning (0-d-old individuals), no oocytes are present (Fig. 3A and B). Oocytes are formed in the germarium, and mature oocytes subsequently move from the vitellarium into the common oviduct (Fig. 3B and C). A single spermatheca is present, connected to the common oviduct through the spermathecal duct (Fig. 3A).

Fig. 3.

Confocal microscopy images of the female reproductive system of X. affinis at different ages. (A) In 0-d-old females, the reproductive structures are distinguishable but undifferentiated, consisting of 2 ovaries (ov), each with 2 ovarioles (ovs), terminal filaments (tf), lateral oviducts (lo), common oviduct (co), spermatheca (s), and tracheoles (trl) with no oocytes present. (B) In 1-d-old females, the onset of cell differentiation in the germarium (gm) is observed. The spermatheca is highlighted in magenta. (C) In 3-d-old females, the germarium and vitellarium (vt) are fully differentiated, with immature and mature oocytes (o) present. (D) In 5-d-old females, follicular relics (r) are evident. Images show nuclei stained with DAPI (green), chitin autofluorescence (magenta), and transmitted light (TL) acquired under the following conditions: (HC PL APO 10×/0.30 NA. Objective. Bar = 300/200 μm).

Ovariole dissections of females at different ages revealed that 0-d-old (n = 19) and 1-d-old (n = 16) females did not have mature ovarioles (Fig. 3A and B). In contrast, most of the 3-d-old females (n = 17) exhibited mature ovarioles, with only 1 individual remaining immature (Fig. 3C). By day 5, all females (n = 18) possessed fully mature ovaries (Fig. 3D).

Effect of Age

No significant differences were detected in the antennal responses of virgin laboratory females of different ages (0, 1, 3, and 5 d old; n = 19, 16, 17, and 18, respectively) exposed to a 70% ethanol stimulus (H = 0.419, df = 3, P = 0.936; Fig. 4A), indicating that age has no significant effect on antennal depolarization.

Fig. 4.

Electroantennographic (EAG) response to 70% ethanol of X. affinis females: (A) Kernel density plots showing the distribution of antennal depolarization values across different ages (days). (B) Comparison between mated and virgin females. Boxplots show antennal responses; different letters indicate significant differences between treatments (P < 0.05).

Effect of Mating Status

The comparison of EAG responses between mated females (n = 33) and virgin females (n = 35) revealed significant differences (W = 971.5, P < 0.001); with mated females showing a marked increase in antennal depolarization (Fig. 4B).

Wild Females Inside and Outside the Host

A total of 79 X. affinis females were collected from B. simaruba branches, of which 44 were located outside the host and 35 inside. The 2-way ANOVA revealed that females inside the galleries exhibited significantly larger antennal responses than females outside the gallery (F_1,74 = 17.551, P < 0.001; Fig. 5A). Regarding sexual maturity, mature females exhibited EAG responses similar to immature females (F_1,74 = 3.751, P = 0.056; Fig. 5A). The interaction between location and maturity was not significant (F_1,74 = 0.38, P = 0.538). The chi-squared test revealed a significant association between location and maturity of females (χ² = 4.724, df = 1, P = 0.029; Fig. 5B), with a Cramer’s V coefficient of 0.245, indicating a moderate association between the variables.

Fig. 5.

Electroantennographic response (EAG) (mean ± SE) of wild X. affinis females to 70% ethanol. Different letters indicate significant differences between the treatments (P < 0.05). (A) Contrasting: Females inside and outside the galleries and with mature and immature ovaries. (B) Distribution of females according to ovarian maturity and host location: frequency and percentage.

Discussion

Ethanol emitted by stressed host trees and volatiles from associated fungi are recognized as key factors in the orientation behavior of ambrosia beetles. Nevertheless, little is known about how intrinsic physiological factors of the beetles themselves may modulate their olfactory sensitivity to these signals. In this study, we focused on X. affinis females to evaluate whether age, sexual maturity, mating status, and location (inside the gallery or in flight) influence their antennal responses to chemical stimuli.

Previous studies have reported that factors such as age, sexual maturity, and mating status can influence host-seeking mechanisms in insects (White 1989, Kendra et al. 2005, Gadenne et al. 2016). In X. affinis and other ambrosia beetles, host plants provide multiple essential functions, the galleries excavated within them serve as sites for mating, nesting, offspring development, and the establishment of symbiotic fungal gardens that supply nutrients to the larvae and adults (Wood 1982). At some point, X. affinis females leave their natal galleries in search of new hosts to colonize, exposing themselves to high-risk conditions such as adverse weather and predation (Bell 1990, Menocal et al. 2022).

Consequently, the rapid identification of a suitable host is crucial for survival and reproduction. Contrary to our predictions, wild females inside galleries exhibited stronger EAG responses to ethanol, a compound typically associated with stressed or dying trees that represent ideal hosts for colonization, than females found outside (Cavaletto et al. 2021). This differential response between females inside and outside the host may reflect adaptive host search strategies, mechanisms observed in the related species Xylosandrus germanus (Blandford). In this beetle, ethanol functions as a long-range attractant, whereas volatiles produced by its associated fungi facilitate short range orientation and final host selection (Gugliuzzo et al. 2023). In addition, females outside the host are likely evaluating multiple compounds emitted by potential hosts simultaneously, which may reduce their behavioral response to any single odor, a phenomenon well documented in other ambrosia beetles, where blends of volatiles are more attractive than individual compounds (Kendra et al. 2016). In contrast, females inside the host are exposed only to the internal odor of the galleries, where ethanol is a central component of the volatile blend. When removed from that environment and re-exposed to ethanol, they responded more strongly, likely due to familiarity with this cue (Roy et al. 2025). This pattern aligns with findings in other ambrosia beetles, where ethanol serves as an indicator of substrate suitability and also acts as a distant attractant while at the same time functioning as a defense mechanism protecting both progeny and mutualistic fungi against pathogens (Gugliuzzo et al. 2023, Moore et al. 2026). In other xyleborines, evidence shows that ethanol, in synergy with acetic acid, stimulates oviposition and significantly increases progeny production (Ranger et al. 2026). Thus, the heightened ethanol responsiveness observed here likely reflects the physiological state of females already committed to gallery establishment and reproduction, contrasting with the dispersal priorities of females collected outside the host. Prior experience, such as exposure to host plant volatiles, can modulate the response to the host plant odor and its recognition (i.e., plasticity) (Anton and Rössler 2021, Anton and Cortesero 2022). It is plausible that host localization in X. affinis involves more than chemical communication. This is consistent with recent findings showing that, when visual stimuli mimicking host silhouettes are combined with semiochemicals, X. affinis females exhibit significantly greater attraction compared to olfactory treatments alone (Conover et al. 2026). These results reinforce the idea that flying females do not rely solely on olfactory cues, but actively integrate visual guides to optimize host selection, which would explain the lower antennal response recorded in this study. Similar processes have been reported in other Scolytinae such as Xyleborus glabratus Eichhoff (Mayfield and Brownie 2013), H. hampei (Souza et al. 2020) and Dendroctonus valens LeConte (Hain et al. 2011), in which visual signals contribute to host detection.

The olfactory responses of reproductively mature and immature females of X. affinis was similar, suggesting that ovarian maturity does not directly modulate olfactory selectivity in this species. This was contrary to our hypothesis since we predicted that mature females would exhibit a higher EAG response, as has been reported with other insects, such as the pepper weevil A. eugenii, in which mature females are attracted exclusively to host plants, in contrast, immature females respond to both hosts and nonhosts (Addesso and McAuslane 2009). We acknowledge that the classification of reproductive mature females in our study was not rigorously precise, given the complexity of obtaining sufficient individuals of X. affinis emerging naturally from their hosts. Consequently, specific levels of ovarian development such as complete differentiation of ovarioles, the presence of oocytes, or follicular relics were not considered in our analyses (Tyndale-Biscoe 1984, Grodowitz and Brewer 1987). Previous research highlights how such stages of female sexual maturation modulate antennal sensitivity, as demonstrated in the fruit fly Ceratitis capitata (Wiedemann), where specific olfactory sensory neurons are activated prior to oviposition to facilitate host odor identification (Jang 1995, Dweck et al. 2013). Future studies should consider distinct maturation stages to determine whether the degree of ovarian development influences olfactory sensitivity in X. affinis.

A moderate association was found between ovarian maturity and the location of females within galleries, suggesting that position (inside or outside the host) is not the sole determinant of female maturity. The proportion of mature females outside galleries was higher than that of immature females, which may be related to the habits and social structure of X. affinis. In this species, adult females often remain in their galleries performing cooperative brood care, which allows multiple generations to cohabit the same gallery before dispersal (Masoudi et al. 2025). Moreover, once fully sclerotized, females can remain in their galleries for 12 d or more prior to dispersal (Biedermann et al. 2011, Brar et al. 2013), a suitable period to achieve ovarian maturity (Sahota et al. 1970). Our findings are consistent with those of Biedermann et al. 2011, who reported that both dispersing (outside the host) and delayed dispersing (inside the host) females of X. affinis could produce eggs. In different insects, host abandonment is typically associated with the search for oviposition sites; however, our results indicate that some immature females also leave their galleries, albeit in low proportion (Fig. 5B). In these cases, the dissemination of symbiont fungi to new hosts may be prioritized with host colonization initiated later through parthenogenesis. This hypothesis is supported by the findings of Joseph et al. (2025), who found that X. affinis females incorporate their symbiotic fungus into their mycangia only 6 h after feeding on mycelium. This rapid acquisition underscores the importance of ensuring fungal dissemination prior to flight. This strategy could optimize the allocation of energy resources between wing and ovarian maturation (Robertson 1998). However, it could also function as a mechanism for cross-fertilization. In other ambrosia beetles, females have been reported to colonize hosts that already have galleries from other females and that when they dig their galleries, these tunnels may intersect, facilitating encounters with males from other colonizing females (Scabbio et al. 2026).

Analyzing the age variable revealed a trend (nonsignificant one) toward decreased antennal response in females as age increased, suggesting a potential influence of factors related to maturity or experience. This pattern is consistent with findings in Camponotus floridanus (Buckley), where older ants exhibit more stable but less intense responses compared to younger individuals (Ferguson et al. 2023). Age has also been shown to interact with other factors, such as odor type, differentially modulating the responses of Anastrepha suspensa (Loew) females (Kendra et al. 2005). It is important to note that age-controlled females in our study were obtained from laboratory colonies maintained on artificial diets, which are known to produce lower antennal responses than individuals reared in natural environments (Guillén et al. 2016, Romero et al. 2022). These considerations highlight the need for further research on the effect of beetle age, incorporating a broader range of olfactory stimuli, working with females raised under conditions that closely mimic their natural habitat, and expanding the age range studied to determine whether the observed trend becomes significant.

The effect of mating status on antennal response and behavior has been scarcely studied in the genus Xyleborus, which reproduces through arrhenotokous parthenogenesis. In this respect, our study provides a novel finding: mated females of X. affinis exhibit greater olfactory sensitivity compared to virgin females. This represents the first report of such an effect in the genus, suggesting that mating status can influence antennal responses in these beetles. Similar effects have been documented in other species, including Diaphorina citri Kuwayama (Wenninger et al. 2009), the fruit fly Bactrocera tryoni (Froggatt) (Devescovi et al. 2021), and the Gonipterus platensis Marelli (Branco et al. 2019). In many insects, the enhanced response of mated females is often associated with the need to locate oviposition sites (Vargas et al. 2018). Finally, in this study, the reproductive structures of X. affinis females were analyzed for the first time to assess their sexual maturity (mature or immature) and determine whether wild individuals were virgin or mated. We found that the reproductive tract of X. affinis is comparable to that of other Scolytinae species, such as Xyleborus ferrugineus (Fabricius) (Norris et al. 1983), Dendroctonus pseudotsugae Hopkins (Sahota et al. 1970), Trypophloeus klimeschi Eggers (Gao et al. 2021), and H. hampei (Rubio-Gómez et al. 2007), among others. The reproductive system of X. affinis comprises 2 ovaries, each with a pair of ovarioles, lateral oviducts, and a common oviduct attached to the type I spermatheca (Fig. 3). Analogous to other Coleoptera, such as Scyphophorus acupunctatus Gyllenhaal (Vega-Petlacalco et al. 2018) and Sitophilus oryzae L. (Perez-Mendoza et al. 2004), oocyte production is asynchronous. In X. affinis, oocytes at different stages of maturation were observed within the same ovariole, and even empty ovarioles were found, whereas others contained oocytes ready for oviposition. In species such as D. pseudotsugae, ovarian maturation, including elongation and complete differentiation of the ovarioles, occurs within 96 h (Sahota et al. 1970). In this study, most 3-d-old (72 h) X. affinis females already had mature ovaries, with only 1 immature female recorded at this age. Future research should expand on these findings to better understand the structural changes that occur across different female age classes.

In conclusion, fully sclerotized females collected within the galleries exhibit stronger antennal responses to ethanol than those collected outside the host. Both mature and immature females displayed similar response patterns, indicating that ovarian maturity does not directly modulate olfactory sensitivity. By contrast, mating status significantly influenced the olfactory sensitivity of X. affinis; however, no significant differences were detected with respect to female age under the conditions evaluated in this study.

Author Contributions

Patricia Romero-Arellano (Conceptualization [equal], Data curation [lead], Formal analysis [lead], Investigation [equal], Methodology [equal], Visualization [lead], Writing—original draft [equal]), Luis A. Ibarra-Juárez (Conceptualization [equal], Funding acquisition [supporting], Investigation [equal], Methodology [supporting], Resources [equal], Supervision [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Daniel Carrillo (Conceptualization [equal], Investigation [supporting], Methodology [supporting], Supervision [supporting], Writing—original draft [supporting], Writing—review & editing [supporting]), Olinda Velázquez (Methodology [supporting], Supervision [supporting], Validation [supporting], Writing—review & editing [supporting]), and Larissa Guillén (Conceptualization [equal], Formal analysis [supporting], Funding acquisition [lead], Investigation [equal], Project administration [lead], Resources [lead], Supervision [supporting], Writing—original draft [lead], Writing—review & editing [lead])

Funding

This project received funding from the Mexican National Council for Science and Technology (CONACYT, currently Secretaría de Ciencias, Humanidades, Tecnología e Innovación—SECIHTI) through the Fondo Institucional de Fomento Regional para el Desarrollo Científico y Tecnológico y de Innovación (FORDECYT) Grant 292399 (now Programa Presupuestario F003), and from the Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) currently Secretaría de Agricultura y Desarrollo Rural (SADER) through the Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA). This study was part of the PhD dissertation of PR.

Conflicts of Interest

None declared.