Lie to me to lay with me: Females deceive males via terminal investment

Abstract

Historically, males have frequently been portrayed as the manipulative and deceptive gender, while females are often seen as adopting a coy and passive role. In this context, it is proposed that males use a terminal investment strategy, misleading females about their true poor condition, while females passively opt to mate with these deceptive males. However, we hypothesize that females in suboptimal condition may also engage in a terminal investment strategy by mimicking or enhancing their attractiveness to match that of females in better conditions. We studied this hypothesis in Tenebrio molitor, by subjecting females to three varying doses of lipopolysaccharides of Escherichia coli (LPS; 0.25, 0.5, or 1 mg ml^-1), or three doses of the pro-oxidant Paraquat (PQ; 20, 40 or 80 mM), and subsequently assessing their survival and attractiveness to males. The LPS treatments and 20 mM of PQ had no significant effect on the survival or attractiveness of the females. However, females treated with 40 or 80 mM PQ survived fewer days compared to the control group. Those injected with 40 mM were more attractive than their control counterparts, while those treated with 80 mM were less attractive. Since the identical doses of LPS, which induce terminal investment in males, had no effect on females, we suggest sexual dimorphism in terminal investment. Furthermore, similar to males, if the stressor reaches a sufficiently high level, the signal becomes honest. These findings highlight how the quantity of stressors influences support for the terminal investment strategy in both males and females. Notably, this study challenges prevailing notions regarding gender roles in sexual selection, indicating that females, not just males, conceal their poor condition to attract mating partners.

1. Introduction

Since Darwin [1], sexual selection theory has largely emphasized reinforcing traditional sex roles while overlooking the active role of females in mating outcomes [2]. Given that optimal reproduction for males and females doesn’t always align, males may evolve manipulative strategies to enhance mating success at the expense of female fitness [3–5]. However, recent findings suggest that females are not as passive as previously thought [6, 7]. Instead, they can actively employ reproductive strategies, including engaging in multiple mating [8], and males may choose mates based on female attractiveness across various taxa [9]. Engaging in multiple mating provides females with advantages such as increased genetic diversity in offspring [10, 11], reduced risk of genetic incompatibility [12], and competition among sperm from diverse males, resulting in selection for high-quality sperm [13]. Additionally, females may obtain material benefits from males, such as access to resources, protection, parental care [6], or even beneficial molecules in the ejaculate [11, 14]. Nonetheless, whether females might adopt an offensive role and deceive males about their condition to mate with them remains largely unexplored [10, 11].

Most research on sexual selection assumes males are the courting sex and females are the choosing sex, leading to less emphasis on exploring female sexual traits as condition-dependent signals [15]. However, this approach overlooks that males also use female ornaments for mate choice [9, 16]. For instance, in the pink bollworm, Pectinophora gossypiella, the pheromone blend produced by females varies depending on their condition, including factors such as size, nutritional state, and age [17]. Male moths prefer females that produce more pheromones, which also correlates with higher egg production compared to females with lower pheromone levels [17], indicating that females provide honest signals about their condition [6]. However, from the perspective of terminal investment, organisms may allocate resources towards immediate reproduction at the expense of future reproduction when survival is uncertain [18]. For example, females facing imminent death invest in traits such as egg number, litter size, oviposition rate, hatching time, parental care, or food provisioning to their offspring (Table 1). Nonetheless, the hypothesis regarding whether females in poor condition, employing a terminal investment strategy, become more attractive to males when facing mortality remains unexplored. We suggest that females, not only males, may hide their condition and manipulate the reproductive interest of the opposite sex.

Table 1. Shows that terminal investment used by females to deceive males is a novel area of research, as no studies have ever focused on females cheating to attract males.

We selected studies from Duffield (2017) and literature published between 2018 and September 2022 regarding terminal investment.

Phylum	Species	Sex	Challenge	Response	Effect	Reference
Mollusca	Biomphaleria glabrata	F	Immune	Size	Mixed (dependent on time)	Minchella & Loverde, 1981 [19]
Chordata	Larus califonicus	F	Age	F2 success; behaviour	Young < middle aged < old	Pugesek et al. 1981 [20]
Chordata	Ovis canadensis	F	Age	Behaviour	Increase with age	Festa-Bianchet, 1988 [21]
Chordata	Ficedula albicollis	F	Age	Behaviour	Young < old	Pärt et al. 1992 [22]
Arthropoda	Acheta domesticus	F	Immune	Size	Treatment > control	Adamo, 1999 [23]
Chordata	Ficedula hypoleuca	F	Immune	Time	Treatment > control; mixed	Sanz et al. 2001 [24]
Chordata	Oreamnos americanus	F	Age	Size	Increase with age	Côté & Festa-Bianchet, 2001 [25]
Chordata	Alces alces	F	Age	Size	Increase with age	Ericsson et al. 2001 [26]
Chordata	Passer domesticus	F	Immune	Time; F2 success	Treatment > control	Bonneaud et al. 2004 [27]
Arthropoda	Daphnia magna	F/M	Immune	Size	Mixed	Chadwick & Little, 2005 [28]
Arthropoda	Gryllus texensis	F	Immune	Size	Treatment > control	Shoemaker et al. 2006 [29]
Chordata	Sorrateria mollissima	F	Immune	Behaviour	Treatment > control	Hanssen, 2006 [30]
Arthropoda	Gryllus texensis	F	Age	Size	Increase with age	Shoemaker et al. 2006 [29]
Mollusca	Biomphaleria glabrata	F	Immune	Size; F2 success	Mixed (dependent on dose)	Blair & Webster, 2007 [31]
Chordata	Chrysemys picta	F	Age	Size; F2 success	Young < old	Paitz et al. 2007 [32]
Chordata	Tamiasciurus hudsonicus	F	Age	Time; F2 success	Increase with age	Descamps et al. 2007 [33]
Chordata	Delicha urbica	F/M	Immune	Time; Size; F2 success	Treatment > control	Marzal et al. 2008 [34]
Chordata	Peromyscus mentculatus	F	Immune	Size; F2 success	Treatment > control	Schwanz et al. 2008 [35]
Arthropoda	Acyrthosiphon pisum	F	Immune	Size	Treatment > control; mixed	Altincicek et al. 2008 [36]
Chordata	Peromyscus mentculatus	F	Immune	Size	Treatment > control	Schwanz, 2008 [37]
Chordata	Kobus megaceros	F	Age	Size; sex ratio	Increase with age	Bercovitch et al. 2009 [38]
Arthropoda	Nicrophorus orbicollis	F	Age	Size; behaviour	Mixed	Creighton et al. 2009 [39]
Chordata	Cervus elaphus	F	Age	Size	Increase with age	Clutton-Brock et al. 1982 [40]
Arthropoda	Nauphoeta cinera	F	Diet	Size	Mixed	Barrett et al. 2009 [41]
Arthropoda	Nicrophorus vespilloides	F	Immune	F2 success	Treatment > control	Cotter et al. 2011 [42]
Chordata	Macaca mulatta	F	Age	Behaviour	Increase with age	Hoffman, 2010 [43]
Chordata	Troglodytes aedon	F	Immune	Size; sex ratio	Treatment > control	Bowers et al. 2012 [44]
Arthropoda	Cardiocondyla obscurior	F	Age	Size	Increase with age	Heinze & Schrempf, 2012 [45]
Chordata	Troglodytes aedon	F	Immune	Size; F2 success	Treatment > control	Bowers et al. 2015 [46]
Arthropoda	Heliothis verescens	F	Immune	Size (mixed); behaviour	Mixed	Staudacher et al. 2015 [47]
Chordata	Litoria verreauxii alpina	F/M	Immune	Size	Treatment > control	Brannelly et al. 2016 [48]
Arthropoda	Cardiocondyla obscurior	F	Immune	Size	Treatment > control	Giehr et al. 2017 [49]
Nematoda	Heligmosomoides vespilloides	F	Immune	Size	Treatment > control	Guivier et al. 2017 [50]
Arthropoda	Gryllus texensis	F	Predator simulation	Size	Treatment > control	Adamo & McKee, 2017 [51]
Chordata	Taeniopygia guttata	F	Immune	Size	Treatment > control	Sköld-Chiriac et al. 2019 [52]
Arthropoda	Drosophila melanogaster	F	Diet + immune	Size	Treatment > control	Hudson et al. 2020 [53]
Phylum	Species	Sex	Challenge	Response	Effect	Reference
Mollusca	Biomphaleria glabrata	F	Immune	Size	Mixed (dependent on time)	Minchella & Loverde, 1981 [19]

The table includes the sex studied, species, challenge, response, and effect. The response was classified as “size,” encompassing modifications in egg number, litter or egg size/mass, oviposition rate, and changes in oviduct or ovary size. For “time”, we included changes in the time to the first reproductive event or time to hatching. “Sex ratio” refers to the ratio of females to males or the resource allocation to one sex over other (larger mass/size for one sex). For “F2 (second generation) success”, we included results showing changes in hatching success, survival, or size to adulthood. The “behavioural” category encompasses alterations in parental care, such as variations in lactation time and shifts in oviposition sites. We excluded 18 studies (from 1988–2018) that looked for terminal investment in females but did not find evidence for it. So, only 56.09% of studies support the terminal investment by females.

To test the hypothesis, we used the mealworm beetle Tenebrio molitor. This species is promiscuous, exhibiting no morphological differences between sexes in colour and shape, and both sexes engage in mate choice [54, 55]. Mate choice and sexual communication occur through chemical signals, as males and females produce odours simultaneously to facilitate mating interactions [56]. On the one hand, males that produce a higher quantity of the pheromone (Z)-3-dodecenyl acetate (Z3-12: Ac) [57] are preferred by females compared to those producing it in lower levels [58]. This could be explained because pheromones reveal the resistance to infections and immunocompetence of their bearers [58–63], resistance to oxidative stress [55], and their nutritional status [61]. On the other hand, females produce the pheromone 4-methyl-1-nonanol [64], which reveals to potential mates their reproductive status [65] and age [66].

It has been widely demonstrated that males use the terminal investment strategy to deceive females if they experience a higher mortality rate than healthy control males [55, 67–71]. For instance, males challenged with nylon implants were found to be more attractive to females than control males, yet they invested less in immune melanization response [69]. Furthermore, males challenged with lipopolysaccharides from the Gram-negative bacteria Escherichia coli dissolved in Ringer solution (2.5 mg ml⁻¹) were significantly preferred by females over non-challenged control males [71]. Lastly, males challenged with 40 mM of the pro-oxidant Paraquat (PQ) were found to be more attractive to females than control males. However, males exposed to 80 mM of PQ were less attractive than controls [67]. Therefore, T. molitor presents an intriguing model to explore whether females, akin to males, engage in deceptive behaviour through terminal investment. We predict that females subjected to oxidative stress with PQ or immune challenges with LPS will exhibit greater attractiveness to males and shorter lifespans compared to healthy females. While this outcome is highly probable, the dose of the stressor may determine whether it leads to a terminal investment strategy or honest signalling [67]. In this regard, there are two potential explanations why females may not engage in terminal investment: the dosage could be too low to pose a threat to their survival, or the dosage could be excessively high, surpassing the organism’s capacity to enact it. Hence, we utilized varying levels of LPS or PQ stressors to assess their impact on the likelihood of females exhibiting a terminal investment strategy.

2. Material and methods

(a) Experimental design

Tenebrio molitor was reared at 27 ± 0.5°C in complete darkness and provided with ad libitum access to a mixture of cornflour and wheat bran (1:3 ratio), supplemented with apple slices every other day [55]. Following this, pupae were individually separated into 12-well boxes (Corning) containing sterile food. Pupae were sexed [72], and adults aged 10–12 days were utilized, as this age aligns with the peak of pheromone production [73]. We selected individuals that were around 13 mm (males: $\overline{\mathbf{x}}$ 13.7 ± 0.79 mm; females: $\overline{\mathbf{x}}$ 13.6 ± 0.82 mm) and made sure to pair females and males that shared the same size for each of the behavioural trials. This would sometimes reduce the amount of available adults for experiments. Moreover, as some individuals would die before the experiment, the sample size for each treatment slightly varied but we insisted on keeping a minimum of 30 individuals per treatment. We also used individuals who came from the same stock for each treatment to reduce dependent variables.

We injected females with 0.25, 0.5, or 1 mg ml-1 of lipopolysaccharides (LPS) from Escherichia coli, diluted in a Ringer solution, as it induces terminal investment in males [69, 74]. Each insect received an injection (using Hamilton syringes of 10 μl) of 1 μl of LPS diluted in Ringer or 1 μl of Ringer alone (control). To induce oxidative stress, we orally administered the herbicide Paraquat (PQ) from Sigma-Aldrich. Each female received 1 μl of PQ diluted in water using a micropipette [55]. We established three experimental groups receiving 20 mM, 40 mM, or 80 mM of PQ diluted in distilled water. The control group received only distilled water. We selected these doses based on preliminary experiments, which demonstrated their effect on chemical communication in T. molitor [55, 67] and because the same doses induce terminal investment in males [67]. Following treatments, we recorded individual survival every 24 hours (see Fig 1A). Experiments concluded once all individuals in each group had died. Specific sample sizes are provided in the results section.

Fig 1.

A) Experimental design. Adult females of T. molitor of 12 days old were treated with PQ or LPS and 3 or 24 hours after treatment, respectively, females were tested for survival and/or mate choice. B) Behavioural arena for recording mate choice. The triad is simultaneously placed inside the arena. The males are left for 5 minutes in an enclosure located in the middle of the central box (dotted square in the central part), and then the male is released to walk around the box and enter one of the two chambers where the females are located. The side chambers containing the females have small holes to allow pheromones to pass through and communicate with the male. The insect images were generously provided by Elaine Méndez Muñis.

(b) Mate choice

To assess female choice, we utilized arenas comprising one large main chamber for the male and two adjacent small chambers for each female [55]. All chambers featured perforated walls to facilitate chemical communication while keeping the females separated from the male (Fig 1B). A habituation period of 5 minutes was allotted before recording mate choice for 10 minutes per trial [55]. Following habituation, males were released to explore, and we documented each visit and its duration for each female he encountered [55]. A visit commenced when the male’s entire body entered the female’s chamber and ceased when his entire body exited [54]. Observers were blinded to each female’s treatment during all trials to maintain data integrity. Fresh individuals were used for each trial, and the chambers were thoroughly cleaned with ethanol between trials.

In all tests, we used triads (Fig 1B) that consisted of one focal male and two females: one control (either injected with Ringer solution or that drank water) and one experimental (injected with 0.25 mg ml-1 or that drank 20 mM, 40 mM or 80mM of PQ). For the PQ treatments, mate choice trials were performed 3 h post-treatment [55], whereas the trials for individuals treated with LPS were performed 24 h post-treatment [71]. Instead of utilizing male pheromone extracts, we chose to expose males to females in order to maintain the continuous production of pheromones or cuticular hydrocarbons (Fig 1B). This method acknowledges potential differences in male and female reactions in the presence of a potential mate, thus offering a more realistic representation of natural scenarios that could result in varying levels of mate attraction [55]. Details regarding sample sizes are provided in the results section. The S1 Video shows an example of mate choice by a male evaluating one female and in the opposite chamber, a rejected male in its chamber.

All animals used in this study were obtained from a culture stock in our laboratory. We ensured to use the minimal number of animals and minimized the harm by using the least invasive methods and choosing oral administration vs injection when possible. For pain management, we placed all individuals in chilled ice to cause a mild sedation.

(c) Statistical analysis

A Cox survival test was used to compare differences in mortality between treatments. To analyse mate choice, paired t-tests were performed to compare the time females spent visiting each male according to treatment. All tests were carried out in SPSS V.22.0.

3. Results

(a) Survival

Immune challenge

Females treated with 0.25 mg ml^-1 (n = 36) did not differ in survival (X² = 0.36; df = 1; p = 0.54) compared with the control group (n = 35). A similar result was observed between 0.5 mg ml^-1 (n = 41) versus its control (n = 45) (X² = 0.73; df = 1; p = 0.39) or between 1 mg ml^-1 (n = 37) versus control (n = 33) (X² = 0.005; df = 1; p = 0.94).

Oxidative stress

Females treated with 20 mM (n = 42) and control females (n = 30) did not differ in survival (X² = 0.08, df = 1; p = 0.76). However, females challenged with 40 mM (n = 25) lived less days than their control (n = 17) (X² = 7.2, df = 1; p = 0.007) (Fig 2A) and females treated with 80 mM (n = 20) lived less days than their control (n = 40) (X² = 8.5; df = 1; p = 0.004) (Fig 2B).

Fig 2.

Survival in days of PQ 40 vs Control (A) and PQ 80 vs Control (B). Experiments were conducted independently, with each experimental group compared against its respective control.

(b) Mate choice

Immune challenge

No significant differences were shown between 0.25 mg ml^-1 versus control (N = 32) (t = -0.15; df = 31; p = 0.88) nor between 0.5 mg ml^-1 and its control group (N = 33) (t = 0.182; df = 45; p = 0.86) and for 1 mg ml^-1 and its control (X² = 0.42; df = 1; p = 0.51).

Oxidative stress

No significant differences were found between 20 mM versus control (N = 40) (t = -0.255; df = 39; p = 0.8). However, females treated with 40 mM were preferred over their control (N = 40) (62.1 ± 18.11 s; t = 2.33; df = 39; p = 0.02). Whereas the control females were preferred over those treated with 80 mM (N = 38) (134.17 ± 22.76 s; t = 2.02; df = 37; p = 0.04) (Fig 3).

Fig 3. Male choice is measured as the total duration of visits for each treatment.

The treatment applied is Paraquat and each graph corresponds to one of the doses administered: A) 40 mM, and B) 80 mM. Bars represent standard errors.

4. Discussion

Since Darwin and Bateman’s proposals [1, 75], research has predominantly focused on males’ strategies to influence females’ decisions. This dominance extends to studies on terminal investment during mate choice, where males are portrayed as deceitful and manipulative to control females’ reproduction [68–71]. Our study reveals that T. molitor females can employ the terminal investment strategy to deceive males when faced with a 40 mM dose of oxidative stress from PQ. However, when stressors reach a higher level (80 mM PQ), females may struggle to deceive males, prompting them to signal their true poor condition. Females treated with 20 mM of Paraquat survived as well as healthy (control) females, suggesting that this challenge was not sufficiently costly to impair their condition signals. This finding is consistent with our immune challenge results, where the doses females encountered did not compromise their survival, thus they did not use the terminal investment strategy.

Our findings suggest that females exhibit a higher tolerance for oxidative stress and greater resistance to immune challenges compared to males. In a previous study, males treated with 20 mM of Paraquat [55] were less attractive than control males. However, in this study, females’ attractiveness was unaffected by the same dose. This disparity may be explained because females are more resistant than males to oxidative stress [76], potentially accounting for the observed differences in the threshold. Furthermore, numerous studies conducted on male T. molitor have demonstrated that immune challenges [54–69, 77] elicit a terminal investment response, wherein treated males become more attractive to females than healthy ones. However, the lack of a terminal investment strategy observed in females in this study, despite the administration of LPS at 0.25 mg ml^-1, is noteworthy. This dose induces changes in cuticular hydrocarbons in males, subsequently increasing their attractiveness to females and eliciting a terminal investment response [71]. The underlying pattern for the relationship between immune response and the presence or absence of sexual hormones presents conflicting findings [78–81]. We propose that males may be less resistant than females to immune challenges and that the presence of Juvenile Hormone (JH) could potentially be a mechanistic link. The JH is present in females and males. However, in females (and not in males) it increases haemocyte number, thereby enhancing their immune response [82]. Consequently, the threshold for triggering terminal investment in females may be higher than in males, which is also partially supported by the lack of survival cost observed in females in this study. However, we do not know the exact mechanisms for this apparent sexual dimorphism in terminal investment and grant further research.

To the best of our knowledge, this is the first study to explore the possibility of females deceiving males to secure a mating partner in the form of terminal investment, as illustrated in Table 1. Engaging in terminal investment could offer females two potential benefits: 1) securing a final mating event before death and/or 2) engaging in reproductive events with one or multiple high-quality males. The latter scenario presents an intriguing phenomenon with significant implications. On one hand, the perceived quality of a mate can drive organisms to allocate greater resources to that reproductive event, as proposed by the differential allocation hypothesis [83]. While our experimental design does not address multiple mating, it is well-documented that females of this species frequently engage in multiple mating to enhance their fecundity [84]. Consequently, it is plausible that females employing the terminal investment strategy may attract more mates due to their heightened attractiveness. In this context, females presenting themselves as high-quality mates could potentially acquire more direct benefits from these mating events. On the other hand, females, males, or both could adjust their investment towards that mating event to optimize the outcome (i.e., offspring quality/quantity) or mitigate the impact of selecting a lower-quality mate. This concept is known as the reproductive compensation hypothesis [83]. This suggests two potential outcomes: 1) if males do not perceive that they are being deceived, their investment in the reproductive event could offset the effects of mating with a lower-quality female; or 2) if the male perceives that he is being deceived, he could strategically modify the quality or quantity of the spermatophore he transfers to the female in order to conserve resources for a future reproductive event. Alternatively, if terminal investment does not affect offspring quality or quantity, cheating males or females may solely benefit from acquiring a mating partner that they would otherwise not obtain if perceived by the opposite sex as being in poor condition. These hypotheses grant further research.

It has been proposed that the terminal investment strategy is dynamic rather than fixed, with a specific temporal window for its occurrence [67, 85, 86]. Our study aligns with findings in males of T. molitor during mate choice [67], revealing a similarly dynamic strategy in females. This behaviour may be optimal under specific conditions (i.e., at 40 mM PQ), inefficient under high stress (i.e., at 80 mM PQ), or not displayed within a threshold level (i.e. at 20 mM). These findings confirm the importance of considering a gradient in stress levels and simultaneously recording the survival rates of organisms when studying terminal investment in both sexes.

Only a few studies have examined both male and female mate choice within the same species, even though it is estimated to be a widespread phenomenon [9, 87–89]. In insects, male mate choice has been reported in 58 species belonging to 37 families and 11 orders [9], representing only a small fraction of the potential species that may exhibit male mate choice. Mate choice seems to be more widespread than previously thought [16]. Hence, terminal investment in condition-dependent signals by females could be more common than we imagine. It will be important to test whether males, as well as females, can carry out the strategy of terminal investment in such species with mutual mate choice.

Antagonistic sexual conflict theory proposes that females bear elevated costs due to males’ strategies to secure mating partners [11]. However, considering females as the offensive rather than defensive sex also prompts intriguing questions about the costs of deception for males and their potential impacts on subsequent generations. Exploring whether males possess mechanisms to detect deception or memorize partners, akin to females avoiding cheating males (Cordero-Molina et al., Under review), would be insightful. This study underscores that females engage in deceptive behaviours by concealing their condition. Acknowledging that females also possess the ability to deceive their mates for reproductive advantages raises questions about the competition between sexes in reproductive strategies. We should critically examine our sexual and anthropomorphic biases in shaping research questions, as they have influenced studies in sexual selection [90–94] and delve deeper into understanding the differential costs of reproduction and offensive strategies for both sexes. This endeavour has been long overdue since the inception of sexual selection theory [94].

Supporting information

S1 Video

(MP4)

S1 Data. Complete data female terminal investment.

(XLSX)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

JCG and LMDL received a grant from Dirección General del Asuntos del Personal Académico/ Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica/ Universidad Nacional Autónoma de México (Grant No. IN225120). https://dgapa.unam.mx/index.php/impulso-a-la-investigacion/papiit The sponsors or funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.