Sexual reproduction is the most prevalent evolutionary strategy in Eukaryotes (Otto, 2009). Through sexual reproduction, living organisms generate original combinations of traits in offspring (new generations). Diverse progeny resulting from genetic mixture and consequent phenotypic outcomes can survive under heterogeneous environmental conditions (Forsman, 2014). However, at the same time, many individuals possess traits suboptimal for conditions they are experiencing (Birkhead and Pizzari, 2002; Matthews and Matthews, 2010). Despite being the most common, sexual reproduction is a costly process in many ways and somehow ‘inefficient’ compared to other types of reproduction, and thus strong selection pressure is expected to improve its efficiency (Otto, 2009; Lehtonen et al., 2012).

Specific mating strategies and behaviours can minimise the costs and risks of sexual reproduction (Höglund and Sheldon, 1998; Parker and Birkhead, 2013; Kappeler, 2021; Kappeler et al., 2023). For example, one’s reproductive success and fitness, can be increased with increased mating events and mating partners, potentially resulting in more offspring (e.g., polygyny and polyandry; Brown et al., 1997; Parker and Birkhead, 2013). In insects, sexual promiscuity is one of the most common mating strategies (Birkhead and Pizzari, 2002; Matthews and Matthews, 2010). Yet, this mating strategy carries ‘fecundity costs’, e.g., physiological cost (loss of energy or nutrients) and, ‘survival cost’, e.g., risk of increased predation rate, physical injuries, and exposure to pathogens and parasites (Magnhagen, 1991; Arnqvist and Nilsson, 2000). These costs may conflict with resources otherwise available for somatic growth and survivorship (Schultz and Warner, 1991; Jennings and Philipp, 1992; Kotiaho, 2001; Lehtonen et al., 2012).

In order to minimise costs, and maximise benefits (fitness), male and female insects may optimise their mating behaviour (Arnqvist and Nilsson, 2000; Arnqvist et al., 2004). Mating behaviour typically involves locating and approaching a suitable partner, evaluating the quality of a potential mate, and copulation (Alexander et al., 1997). Mating behaviours are variable among insects, and some are extremely peculiar (Thornhill and Alcock, 1983; Page, 1986; Tatarnic et al., 2014). For example, males of Stylops ovinae (Strepsiptera) pierce the female’s cephalothorax cuticle with their penis during copulation to transfer sperm, an act known as traumatic insemination (Peinert et al., 2016). In odd circumstances, insects can misidentify reproductive partners, due to flawed signals and behaviours. The jewel beetle Julodimorpha bakewelli has been recorded copulating with inanimate things such as beer bottles (Gwynne and Rentz, 1983). Males of the two spotted ladybird Adalia bipunctata have been fooled into copulating with dummies bearing male and female elytra (Hemptinne et al., 1998).

An extreme mating behaviour that sometimes occurs across various animal taxa, is mating with already dead individuals, i.e., necrophilia (Bettaso et al., 2008; Ayres, 2010; Cortés Bedoy et al., 2014). In some arthropods, e.g., cannibalistic mantises or various spider species, killing partner during copulation can be a common part of their mating system (Scardamaglia et al., 2015; Ma et al., 2022). However, in many other cases mating with a dead partner seems to be unintentional. Explanations for this behaviour can be grouped in two major categories: 1) individual sexual deviation, i.e., problem of a given animal and not whole species (Tomita and Iwami, 2015; Swift and Marzluff, 2018; Pettigrew, 2019) and 2) the inability of species to perceive chemical and physical cues that would otherwise distinguish between dead and alive individuals (Goncalves and Biro, 2018). Mating with a dead partner typically reduce fitness and may be maladaptive for a given species or population since copulation does not result in a new generation (Crespi, 2000). However, an interesting example of “necrophilic” behaviour in the small Amazonian frog, Rhinella proboscidea, shows that under specific conditions this behaviour can increase male fitness (Izzo et al., 2012). Mating with a dead partner considered as individual deviation, i.e., necrophilia, has been repeatedly reported for vertebrate species (Tomita and Iwami, 2015; Swift and Marzluff, 2018; Colombo and Mori, 2019; Pintanel et al., 2021). In contrast, in insects, it is quite common that freshly killed individuals may not be recognized as dead by other conspecifics, however, it has been rarely investigated how long this situation persists (Geiselhardt et al., 2009; Buellesbach et al., 2018).

Ladybirds represent a suitable insect group for investigating mating behaviour, as many ladybird species are promiscuous, mate frequently, and mating can last for quite a long time (generally few hours; Hodek et al, 2012). Several studies indicate that ladybird mating behaviour is induced by a combination of visual stimuli (in particular movement) and olfactory cues (semiochemicals; Hemptinne et al., 1998). The ladybird Harmonia axyridis serves as a popular model species because of its invasive success worldwide (Brown et al., 1997; Roy et al., 2016) and because a significant amount of knowledge on its biology and behaviour has been accumulated through extensive studies (Hodek et al, 2012; Awad et al., 2015a; Awad et al., 2017; Knapp and Řeřicha, 2020; Řeřicha et al., 2021). During our own laboratory and field investigations focused on various aspects of ladybird biology (e.g., Knapp et al., 2019; Knapp et al., 2020; Knapp et al., 2022), we repeatedly observed attempts to mate with dead conspecifics in Harmonia axyridis during the growing season and post-overwintering period.

In this study, we investigated several factors that could affect “necrophilic” behaviour in Harmonia axyridis using two laboratory behavioural experiments. We hypothesized that 1) the probability of mating with a carcass will be higher for unmated males compared to sexually experienced ones, 2) the attractiveness of carcasses will decrease with time, and 3) the presence of conspecific chemical cues will increase the probability of mating with carcasses. In addition, we expected that time to the first mating attempt will be higher for carcasses compared to alive individuals and mating duration will be shortened in the case of mating with dead females.

Our study showed that mating with a dead conspecific is quite common phenomenon in Harmonia axyridis under laboratory conditions, however its frequency and importance under field conditions should be evaluated in a future study. In general, males are unable to distinguish between alive and freshly dead (1-day old carcass) females. Reduced mating rates were recorded for 7-day old carcasses and ca. 90% of experienced (already mated) males refused to mate with 14-day old carcasses. However, mating probability significantly increased when males were sexually inexperienced or deprived, resulting in ca. 35% of previously unmated males mating with a decaying (14-day old) carcasses. This indicates that despite the ability of Harmonia axyridis males to distinguish low-quality females (14-day old carcasses), extremely low choosiness of sexually-deprived unmated males can result in a surprisingly high occurrence of necrophilic behaviour.

Even though female choice is considered to be more widespread among insects than male choice, accumulated evidence confirmed existence of male mating choice in diverse insects spanning all major orders (Bonduriansky, 2001). Males can evaluate the quality of females, and therefore should be able to distinguish between dead and alive females (Goncalves and Biro, 2018). Ladybirds in general, and specifically Harmonia axyridis, likely have well-developed male mate choice since choosiness is typically favoured when mating is costly (e.g., involving large spermatophores and long copulation duration) and high variability in female quality exists (Bonduriansky, 2001). Harmonia axyridis males can select females based on their age (Osawa, 1994) and colour morph (Awad et al., 2015a). The ability to distinguish between alive and dead females (1-day old or 7-day old carcasses) was confirmed for another large ladybird species Coccinella septempunctata from the same tribe (Coccinellini; Omkar and Srivastava, 2002), and thus our results indicating that Harmonia axyridis males could have a limited ability to recognize dead females is surprising. In addition, mating can be costly for males in some ladybird species when energy requirements of ejaculate production are considered (Perry and Rowe, 2010; Hodek et al., 2012). Therefore, strong selection against wasted mating attempts is expected, especially when mating with a dead female cannot result in any offspring (in contrast to the frog example; Izzo et al., 2012). However, the study by Omkar and Pervez (2005) investigating other ladybird species (Propylea dissecta) showed that 7-day old female carcasses can be attractive to males and 30% of males started mating with such carcasses, which illustrates that Harmonia axyridis is not an exceptional species among ladybirds. Weak ability of Harmonia axyridis to distinguish between alive and dead conspecifics is indicated also by absence of significant differences in mating latency. However, it is possible that this variable is mainly under female control, as it has been previously shown for another ladybird species (Cryptolaemus montrouzieri) that females are able to reduce mating latency when offered with a preferred male (Xie et al., 2014).

It seems that sensorial and cognitive constraints preclude males of various insect species to identify aliveness of their sexual partner (Goncalves and Biro, 2018). In general, insects are largely dependent on chemical cues to identify and evaluate conspecifics. Pheromones and cuticular hydrocarbons have been repeatedly identified as major tool in mate selection (Vaníčková et al., 2012; Gomez-Diaz and Benton, 2013; Chung and Carroll, 2015; Fassotte et al., 2016; Menzel et al., 2019). Ladybird mating behaviour is likely induced by a combination of visual stimuli (in particular movement) and olfactory cues (semiochemicals; Hemptinne et al., 1998), but the importance of sexual semiochemicals is still unclear. Sometimes ladybirds fail even with proper species identification, resulting in repeatedly observed interspecific copulations, and this phenomenon can become more common under laboratory conditions (Majerus, 1997; Mercado, 2023). Existing evidence indicates that the role of cuticular hydrocarbons can be limited in Harmonia axyridis as males are unable to detect differences between mated and unmated females based on cuticular hydrocarbons (Legrand et al., 2019a). Sex pheromones probably play a more important role in Harmonia axyridis (Legrand et al., 2019b), however existing knowledge is surprisingly limited. Potentially limited importance of chemicals for Harmonia axyridis mating behaviour is further supported by this study in which there was no significant difference between new Petri dishes and ones with fresh traces of ladybirds. Such traces were previously shown to be detectable by adult ladybirds and can affect oviposition behaviour (Yasuda et al., 2000; Evans, 2003).

Females that are unresponsive to the mating attempts of a male may be attractive to males in species that typically exhibit female refusing behaviour (Bonduriansky, 2001). Female ladybirds often resist mating attempts by males (Perry et al., 2009), which may decrease males preference for highly active females. In contrary, sometimes female’s inactivity can protect them from intensive harassment. For example, in a dragon fly species, female use inactivity (‘fake death behaviour’) to escape from suffering from intensive harassment in places with extremely high male densities (Khelifa, 2017). However, note that this behaviour essentially makes dragonfly females invisible to males rather than unattractive. This indicates that insect males can have a serious problem to recognize that an immobile female is really dead. Fast recognition of death requires relatively specialized evolutionary novelties as illustrated by social insects that need fast identification of carcasses for their timely transport from the nest (Sun et al., 2018). The Argentine ant (Linepithema humile) produce dolichodial and iridomyrmecin from the pygidial gland and spread it on the cuticle to mask the already present corpse removal stimuli, triglycerides (Choe et al., 2009). This mechanism allow detection of nestmate death in less than one hour. In contrast, detection by traditional necromones resulting from the decomposition process, e.g., fatty acid death cues, can take a few days (Goncalves and Biro, 2018; Sun et al., 2018). Fatty acid death cues are used by a wide range of arthropods, and it is probable that even ladybirds can recognize them (Yao et al., 2009; Goncalves and Biro, 2018).

Males of some insect species can only recognize the quality of a mating partner during copulation and adjust their investment at that time, e.g., reduce copulation duration or amount of ejaculate (Parker, 1983; Gwynne, 1984; Geiselhardt et al., 2009). This may be because some semiochemical, e.g., cuticular hydrocarbons, are relatively stable and can persist in unchanged shape (amount and composition) for many days after a death (Menzel et al., 2019), giving the impression that the potential mate is still alive even days after death. In such cases, the lack of behavioural response from females to a mating attempt may be the most important cue to males as to the status of their potential mate. Despite of a methodological limitation of our study (just 15-minute-long trials), we observed a significant decrease in mating duration with older carcasses for unmated males (no-choice experiment) and specifically for males used in the choice experiment. This suggests that the presence of alternative sexual partners can increase male choosiness when facing low-quality females (7-day old carcasses). At the same time, mating experience increased choosiness of ladybird males, which is in line with the expected costs of mating in Harmonia axyridis. The increased fitness (paternity) that results from repeated matings is outbalanced by physiological costs and an increased risk of contracting sexually transmitted diseases or parasites (Fiedler and Nedvěd, 2019). On the other hand, decreased male choosiness ensures high fitness and can be advantageous under conditions of many unmated females. Such conditions occur in late winter and early spring as Harmonia axyridis overwinter in huge aggregations where many females have empty spermathecas (Awad et al., 2015b). It is the time when we repeatedly observed necrophilic behaviour in Harmonia axyridis in nature (MK and MŘ personal observation - unquantified and unpublished data).

In conclusion, we observed surprisingly high occurrence of mating with dead females in the invasive ladybird Harmonia axyridis under laboratory conditions, even when males were provided with older (7-day or 14-day old) carcasses. Unfortunately, our experiment was not designed to distinguish whether males only initiated mating or completed a mating attempt (transmitted their ejaculate) with dead females, which would confirm an extremely high physiological costs of such behaviour. Nonetheless, mating with already dead conspecifics can exposes ladybird males to an increased risk of disease and parasite transmission. Understanding the mechanisms that would otherwise prevent such behaviour in Harmonia axyridis deserves the attention of researchers and a future research.

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

The manuscript presents research on animals that do not require ethical approval for their study.

MR and MK developed the main idea and designed laboratory experiments; MR and SM-M performed laboratory experiements; MR and FH analysed data; MR and SM-M wrote draft of the manuscript; all authors contributed to final edits of the manuscript. All authors contributed to the article and approved the submitted version.