Gender identity and sexual experience affect mating behaviour and chemical profile in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

Alphitobius diaperinus (Coleoptera: Tenebrionidae), the lesser mealworm, is one of the most significant pests of the poultry industry worldwide. These insects cause structural damage in poultry houses and transmit several diseases, impacting chickens’ productivity and rearing costs. Although semiochemicals may offer alternative insect pest management strategies, basic information regarding pheromone identity and their role on the behavioural ecology according to their circadian pattern of sexual behaviour of A. diaperinus is essentially lacking. This study is aimed to analyse the relation of gender identity and sexual experience of adults of A. diaperinus on their mating behaviour and whether this response is related to their CHC profiles secreted. The following steps were taken to achieve the study’s goal. First, the circadian pattern of their sexual activity was observed in newly emerged pairs for at least twenty-one days (virgin adults) and experienced adults collected from the field to identify a difference based on their sexual experience and achieve the optimal mating season to develop the following assays. Subsequently, Y-tube olfactometer bioassays were conducted to evaluate their odour bouquet attraction based on gender and sexual experience. Additionally, mating behaviour bioassays were conducted to evaluate the two factor effects. Finally, cuticular analysis was performed using gas chromatography-mass spectrometry to evaluate possible chemical differences based on the two factors. With statistical and multivariate analysis, we found that behavioural, mating and chemical responses are different based on their sexual experience. The mating sequences were described into precopulatory, copulatory and postcopulatory phases. This finding gives us a deeper understanding of the sexual communication during mating. In summary, our findings provide new insights into the mating system and chemical ecology of A. diaperinus. The results presented here may serve as a base for further studies to develop strategies for managing this pest.

In this regard, sexual communication in insects is based on visual, olfactory, tactile and even auditory stimuli (62,65,66). From those mentioned, olfactory stimuli based on chemicals are by far the most critical signals (64,(67)(68)(69). Chemical signalling in sexual behaviour systems involves highly volatile compounds that promote attractiveness over long distances and compounds that can influence close-range orientation; these chemicals are perceived by direct antennal contact with the insect cuticles (58,60,67,68). Insect cuticles are made by a lipid wax layer, in which cuticular hydrocarbons (CHC) are a significant contributor (49,70,71). Besides their function of limiting water loss and reducing external damage by toxins and pathogens, CHCs also play a critical role in chemical communication for species, nest or mate recognition, and signalling reproductive status (49,61,62,64,70,(72)(73)(74). In beetles, volatiles associated with intra-specific long attraction is usually associated with aggregation pheromones; in contrast, low volatile and non-volatile compounds are associated with contact sex pheromones (58,72,75,76). CHC compounds are shared by males and female adults in some beetle species (61,70,76), which explain the observed homosexual behaviour upon antennal contact, e.g., in some Scarabaeidae, Silphidae and Tenebrionidae beetles (68,70,(77)(78)(79).
Since homosexual behaviour is known in A. diaperinus (38,40,41), it is expected that both genders share part of their CHC profiles. It has been shown that aggregation pheromones are produced by virgin males of A. diaperinus, which attracts males and females (5). However, it is unknown whether this attraction can be affected by gender identity or sexual experience.
Furthermore, we expect gender identity and sexual experience in a circadian context can impact semiochemical and pheromone activity. Given that insect CHC profiles are associated with gender identity and sexual experience (61,70,73,80,81), we were interested in finding out whether or not these two factors affect mating behaviour responses and, if so, could those differential behaviours be related to differences associated with their chemical profiles? To answer these questions, we first identified the circadian sexual behaviour of A. diaperinus adults. We then tested the attractiveness and behavioural mating responses of males and females with different sexual experiences to different CHC profiles of other A. diaperinus adults.

Studied species
Adults of A. diaperinus were obtained from a commercial poultry production located in the surroundings of Lima (12°09'27.8" S 76°53'46.2" W). All four live stages of A. diaperinus were reared in our laboratory in aquarium glass boxes (30 × 25 × 20 cm). In one of our glass boxes, hundreds of male and female adults were kept and let free to reproduce; new emerging larvae were then removed and transferred to a second glass box. To facilitate the handling of the larvae, they were kept in groups within Petri dishes. This second box was monitored twice a week to feed them and to find pupae. Each new pupa was sexed (following (82); i.e., females "F" and males "M"), and then isolated in a Petri dish to avoid they mate in order to obtain non-mated adults. In case of gender recognition could not be carried out at pupa stage (e.g., first collected adults), gender recognition was carried out by pressing their protracted ventral abdomen to see the genitalia under a stereomicroscope. Larvae and adults of A.
diaperinus were fed using commercial wheat flour "Blanca flor" (Alicorp, Lima, Peru) and tap water, which was provided by using a humidifying towel paper within the boxes. Glass boxes were kept in an environmentally controlled climate chamber (Memmert HPP750, Memmert GmbH, Schwabach, Germany) at a constant temperature of 30 °C and humidity of 50 %, with a photoperiod of 12:12 h (light : dark) (photophase 07:00 to 19:00 h and scotophase 19:00 to 07:00 h; GMT-5). Adults being 21-d-old or older were used to guarantee sexual maturity. Non-mated adults were considered as sexually inexperienced (referred to as 'virgin' [v]), whereas adults exposed to high densities of males and females were catalogued as experienced ('exp').

Circadian pattern of sexual activity
We recorded the mating behaviour of adult pairs (i.e., one female and one male) for 15 min each hour along the day for 5 days. The observations were performed over a Petri dish (60 × 15 mm), and a total of 10 pairs were utilized each day, totalizing 50 adult pairs. When a male exposed his genitalia while mounting a female, it was classified as a matting attempt. Males were not allowed to accomplish mating to continue observations along the day. The proportion of mating attempts was then calculated at each hour. During periods of darkness, observations were performed under an incandescent bulb with red light. One hour prior and between observations, experienced adults were individually kept in Petri dishes to increase sexual desire. After each observation, Petri dishes were washed with methanol.

CHC attractiveness based on gender and sexual experience
A three-arm olfactometer (i.e., Y-tube, 8 mm thickness, 10 cm apparatus trunk followed by 5 cm length arm) was used to test the attractiveness of CHC compounds of males and females.
Each Y-tube arm was connected to one of the five different treatments, which consisted of a glass chamber bearing one of the following options: (i) an empty control (C), (ii) 20 experienced females (Fexp), (iii) 20 virgin females (Fv), (iv) 20 experienced males (Mexp), or (v) 20 virgin males (Mv) (see Fig 1a). The odour released by each treatment was then driven to the olfactometer by an air compressor that provided an airflow at a constant rate of 0.3 L/min through activated charcoal filters. A virgin male or female was placed within the trunk of the olfactometer. Then, it was observed which arm the insect chose within a time of up to 5 min. The beetle odour decision was registered when the beetle entered at least 1 cm into one of the arms. All tested beetles were used only once, whereas beetles within the chambers were allowed to acclimatize for 1 h before the tests to avoid them producing unattractive compounds such as alarm pheromones. The assignment of odour sources to each arm was reversed after each trial to avoid potential illumination and directional bias (59).
After each test, the olfactometer and chambers were washed with methanol. All trials were carried out at the peak of circadian sexual behaviour (S1 Fig).

Effect of gender and sexual experience on mating behaviour
Behavioural experiments with treatments based on all combinations of gender and sexual experience were prepared (i.e., Fv-Mv, Fv-Mexp, Fexp-Mv, Fexp-Mexp). Each pair (i.e., one male and one female) of adults of A. diaperinus was placed in a Petri dish (60 × 15 mm) containing a filter paper at the inner bottom and observed for a total of 10 min. The following behaviours developed during mating attempts were timed: touching (i.e., time in touching the other insect's cuticle with their antennae or their prothoracic leg), mounting (i.e., time upon a male is over and bends his abdomen exposing the genital organ until mating), and copulation (i.e., time in copula). The recorded time per response per trial was then standardised by using a percentage scale (i.e., total trial time was transformed to 100 %).
Additionally, the number of times of successful mating attempts (i.e., copulation occurred), unsuccessful mating attempts (i.e., unsuccessful copulation), and total mating attempts (i.e., total successful and unsuccessful mating attempts) were recorded. The recorded number of times per trial was also standardised using a percentage scale based on the total mating attempts. After that, filtering was performed using principal component analysis (i.e., PCA of log10transformed and Z-score-normalized data) and one-way ANOVA (p-anova<0.01) through MATLAB vR2019b. This filtering allowed us to track data quality, reduce the data dimensionality, identify potential outliers in the dataset, and identify sample clusters (84)(85)(86)(87). To final filtering of each feature was considered the following threshold: that the average area of the sample was at least three times the average area of the C16 and the blank. After data curation, to ensure the identification of compounds, selected samples were analysed by gas chromatography coupled to an APPI-Q-Exactive HF mass spectrometer (Thermo Fisher

Statistical analysis
Differences in mating activity throughout a circadian cycle and differences in the total time of male mating reactions (i.e., touching, mounting, copulation and mating time), as well as the total mating attempts (i.e., successful, unsuccessful and total mating attempts), among all tested treatments, were performed by non-parametric Kruskal-Wallis tests, followed of pairwise comparisons by Mann-Whitney-U tests with a Bonferroni correction. Choices made by A. diaperinus adults in the olfactometer bioassays were analysed by exact binomial tests (50 % chance of selecting each arm). All these statistical tests were performed using the stats package of the R software (R version 3.6.1 (88)).
A non-metric multidimensional scaling (NMDS) was performed to display the dissimilarities among groups graphically. NMDS ordination was based on Bray-Curtis similarity of the square-root transformed dataset. Furthermore, to identify statistical differences in the CHC profiles among groups, a permutational multivariate analysis of variance (PERMANOVA) was run based on Bray-Curtis similarity of a square-root transformed dataset. A total of 99999 permutations and a Holm correction was carried out, considering "gender" as a fixed factor and "sexual experience" as a nested factor within "gender". The CHC dataset was based on the relative proportions of all the identified compounds (see results). NMDS and PERMANOVA were performed using the vegan package (89), whereas Holm correction using the RVAideMemoire package (90) of R.

Circadian pattern of sexual activity
Independently of their sexual experience (i.e., virgin or experienced), the mating behaviour of A. diaperinus adults was displayed throughout the day (S1 Fig). On the one hand, virgin adults showed more mating activity between 2 h and 9 h after the light was on (i.e., photophase) and at 17 h to 18 h and 23 h, under dark conditions (i.e., scotophase). In contrast, the lowest mating activity was recorded between 13 h and 14 h (S1 Fig). On the other hand, experienced adults were more sexually active between the 3 h and 5 h on the photophase (S1
However, due to not enough Mv individuals, no experiments were carried out.

CHC attractiveness based on gender and sexual experience
The three distinct phases of mating behaviour in A. diaperinus are summarized in Fig 1c. The precopulatory phase (i.e., during the insect localization) began when both adults approached between them. After approaching, they touched their cuticles with their antennae or their prothoracic leg. When the male recognised the female, he attempts to mount her from the back, bending his abdomen and exposing the genital organ. Females also contributed to both adults have a successful attempt mating while placing herself under the male and exposing her genitalia by aperture the last sternite. In the copulatory phase, the male grasped the female's cuticle with his prothoracic and mesothoracic leg and touched her prothorax with his antenna and maxillary palp. Interestingly, copula was on average accomplished only once per trial. In the postcopulatory phase, both adults kept touching and stay that way. When a male did not have a successful mating due to his small size related to the female, both insects attempted mating again (S1 Table).
The Kruskal-Wallis test shows that the touching responses of A. diaperinus adults were not significantly different among treatments (Fig 3, Table 1; p>0.05). On the contrary, the pattern of mounting and copulation responses was quite different among treatments (Table 1; Kruskal-Wallis test; p<0.0001). Specifically, Fv-Mv and Fexp-Mv resulted in significantly longer mounting than Fv-Mexp (Fig 3; Mann-Whitney-U test with Bonferroni correction, p<0.05); whereas we did not find differences in mounting in Fexp-Mexp compared to any other treatment. However, we did find significant differences in copulation when comparing   In the case of mating attempts, Fv-Mv and Fexp-Mv resulted in significantly longer attempts compared to Fv-Mexp (Fig 4a; Mann-Whitney-U test with Bonferroni correction, p<0.05); whereas we did not find differences in mating attempts in Fexp-Mexp compared to any other treatment. From the total mating attempts, no significant differences in unsuccessful and successful mating attempts among all the four treatments were found (Fig 4b and 4c; Kruskal-Wallis test, p>0.05). However, Mv was slightly more successful in mating with Fv compared to Fexp (Fig 4c). Moreover, from the total mating attempts showed by Fv-Mexp and Fexp-Mexp, only half was successful (Fig 4b and 4c, S1 Table).

CHC profile of A. diaperinus
A total of 19 CHC compounds were obtained by GC-MS followed by a data curation (S2 Fig), which belonged to the following chemical groups alkanes, quinones, benzothiazoles, benzophenones, fatty acids and terpenes ( Table 2). All the 19 identified compounds were detected in experienced adults but different concentrations, while only 14 compounds were detected in virgin adults. Among these CHC compounds, 2-methyl-1,4-benzoquinone and 2ethyl-1,4-benzoquinone were detectable as significant components in the CHC profile for both males and females, although they were predominant in experienced adults (Table 2; S3   Fig). Interestingly, limonene, oleic acid, linoleic acid and nonacosane were identified only in experienced adults.
The (dis)similarity of CHC profile between the sexual experience groups of males and females of A. diaperinus was visually displayed by our NMDS analysis (Fig 5), which were highly congruent with a PCA also based on CHC profile (S2 Fig). Indeed, our PERMANOVA analyses did not show differences in CHC profiles based on gender identity (i.e., males vs females; Pseudo-F (1,35) = 0.757, p = 0.459), or within sexual experience groups (i.e., between virgins, between experienced adults; both p = 0.687). However, we did find statistical differences in CHC profiles according to the nested factor sexual experience (Pseudo-F (2,35)

Discussion
The circadian cycles play an essential role in describing the mating behaviour of insect life (52)(53)(54), which might be considered a key to developing effective strategies for pest control (55,57). Alphitobius diaperinus seems not to show significant differences in mating activity along the day. However, a lower mating behaviour activity was observed at the end of the photophase and beginning of scotophase 13 h, which might be related to their cryptic behaviour when hiding from a threat or external factors (i.e., environmental changes) or to particular reproductive strategies of females to reduce her attractiveness and rejection to mate (5,56,57,68). In insects, the attractiveness of intra-specific odour relies on the interaction context (e.g., protection from environmental conditions, predators, food source or mate recognition), as recorded in some Curculionidae, Dermestidae, Tenebrionidae and other nonbeetle species (37,55,59,68,80,91). In mating behaviour, the mate preference and attraction of males toward the odour of females can be based on their physiological changes and acquired experience (i.e., reproductive status and learned signal; (91)). For instance, in some Tenebrionidae species, experienced adults have contrasting mating behavioural responses compared to virgin adults (91,92). Interestingly, males and females of A. diaperinus were more attracted to the CHC profiles of both males and females although heavily dependent on the sexual experience condition, which can be related to the gregarious behaviour of this species and similarity of CHC profiles. Effectively, it has been shown that insects with similar odour profiles are less attractive to their gender counterparts compared to others with more different profiles (e.g., Gryllodes sigillatus (93)). Although it was previously pointed out that only virgin males released attractive compounds to attract virgin insects (e.g., aggregation pheromones; (5)), we have shown that CHC profiles of both males and females can be attractive to males and females, and their attractiveness relies on gender and sexual experience. Furthermore, we have shown that the insect cuticle diffusely releases attractive odour signals, in addition to the aggregation pheromones produced by exocrine glands in other beetles (94,95). In beetles, it is known the aggregation pheromones may be made by any sex (59,68,80,96). Unlike sex pheromones that act on only one sex, aggregation pheromones induce group formation of both sexes (37,43,55,68). In this regard, it may be evolutionarily advantageous for males to call females to reduce the time spent searching for a dispersed potential mate, and females have more chances to mate (80). Thus, it is clear that different combinations of odour profiles are necessary to attract males and females of A. diaperinus successfully. Thus, a single odour blend might not be enough.
Beetles during the premating phase show an antennal behaviour (59,65,80,97) related to the close-range assessment of potential mates, where male-or female produced contact pheromone is required for mating to be successful (58,61,62,67,80,98). Based on our observations, we speculate that both females or male adults of A. diaperinus release CHC compounds that allow the partner to be recognized by the opposite gender during antennation.
This behaviour to recognise mates at short distances is mediated by semiochemicals such as a contact sex pheromone (58,61,62,67,98). In the copulatory phase, male beetles and A. diaperinus continue their antennation, touch with their maxillary palps and grasp the female's cuticles with his prothoracic and mesothoracic leg (59,65,80,97) while mounting. On the leg, males possess tibial spurs and claws, which for other species facilitate grasping and control of females, preventing aggregated males from dislodging copulating males and distributing their own CHC profile overall the female's cuticle (80). Thus, mate location and sex discrimination, the first steps of mating, is maybe due to a combination of physical and chemical recognition by the antenna and legs (59).
It is usually expected that adult males tend to mate with any available female. However, we have often recorded low mating activity of Mexp with females, compared to Mv, which could be explained by the sexual conflict (99,100). To increase reproductive success, females mate with multiple males to select their best candidate (99), giving the later males more chances to succeed. Experienced males have already mated and might be more selective to mate; therefore, they would prefer to mate with Fexp, which are less likely to have new mating partners than Fv. Moreover, the sexual experience would also reduce the amount of time in Mexp mounting and copulation since they are shorter with Fexp than Fv. Unlike Mexp, Mv has a natural-sexual need to copulate with a mate. Therefore, Fv and Fexp are both attractive to Mv, although Fexp seems to be more attractive. This behaviour is also known in other insects, where virgin males mate indiscriminately with females and do not recognise their sexual experience (101,102).
The CHC play an essential role in chemical communication during the mating behaviour of insects (49,58,(60)(61)(62)67,70,(72)(73)(74). According to our results, differences in CHC profiles of A. diaperinus adults are mainly based on their sexual experience and not gender, which explains why the odour profile of both genders can be attractive. Moreover, CHC profiles can explain the results of our mating bioassays and suggest that CHC compounds are the key to mate choice and reproduction in A. diaperinus. CHC profile similarity between males and females can also explain the expected homosexual mating behaviour observed in A. diaperinus and diverse beetle species (77,103,104). Thus, the CHC profile of the sexual experience in adults serves as a crucial species-specific mating cue, but not for sex discrimination (72). Further studies are being carried out to identify the chemical basis of A. diaperinus attractiveness.