Lack of discrimination of sex and maturity of conspecifics in the copulation attempts of the male stalk-eyed fly, Sphyracephala detrahens (Diptera: Diopsidae)

Among insects, males have evolved the ability to discriminate potential mates to avoid vain mating attempts. In Diptera, courtship and mating behaviors of species that do not discriminate potential mates have rarely been studied. In this study, we investigated copulation attempts of the stalk-eyed fly, Sphyracephala detrahens males, which do not engage in any observable pre-copulatory courtship behavior. We found that sexually mature males attempted to copulate with mature males and immature adults of both sexes, as frequently as with mature females. Successful copulation only occurred with mature females. Immature females rejected mounting males by vibrating their bodies and immature and mature males by incurving their abdomen downward. Comparison of the cuticular hydrocarbon components of individuals of different sexes and maturity levels indicated that the cuticular hydrocarbon profiles were similar regardless of the sex and sexual maturity. Therefore, we suggest that visual or chemical information are of little use for males S. detrahens in discriminating receptive conspecific mates. Since elaborate mate discrimination behaviors lead to additional time and energy costs when discrimination is difficult, S. detrahens may increase reproductive success simply by increasing the number of copulation attempts. Digital video images related to this article are available at http://www.momo-p.com/showdetail-e.php?movieid=momo211123sd01a, http://www.momo-p.com/showdetail-e.php?movieid=momo211123sd02a, http://www.momo-p.com/showdetail-e.php?movieid=momo211123sd03a, and http://www.momo-p.com/showdetail-e.php?movieid=momo211123sd04a.


Introduction
The efficiency of mate discrimination affects lifetime sexual reproductive success because sexual reproductive opportunities are limited. Accordingly, among insects, males have generally evolved the ability to detect cues or signals that are specific to potential mates. Dipteran males use a variety of sensory cues, such as visual, chemical, acoustic, and tactile cues, to avoid vain copulation attempts toward non-potential mates, such as heterospecifics, and males and immatures of conspecifics. A typical sequence of male copulation attempts in Diptera is as follows: when a male fly finds another individual in the vicinity, he may use visual, olfactory, or acoustic signals to discriminate its species, sex, and receptivity (Ewing 1977;Wicker-Thomas 2007;Everaerts et al. 2010). Then, he approaches and touches the counterpart to obtain physical or chemical contact cues (Ewing 1977;Krupp et al. 2008;Ferveur and Cobb 2010;Everaerts et al. 2010;Kuo et al. 2012;Ingleby 2015). The receptivity of counterpart may also be assessed by the responses to his courtship behavior (Ewing 1977;Briceño and Eberhard 2002;Lasbleiz et al. 2006;Dukas and Scott 2015). In dipteran species, mating behaviors of species that utilize sensory cues for mate discrimination have been well studied; however, the mating behaviors of species that achieve copulation without the use of such signals have been rarely studied. To the best of our knowledge, no dipteran species that constantly attempt to copulate without mate discrimination have been reported. Here, we report unique mating behavior in the stalk-eyed fly, Sphyracephala detrahens (Diptera; Diopsidae) males, which do not engage in any observable 1 3 pre-copulatory courtship behaviors and do not discriminate potential mates in the conspecifics.
Sphyracephala detrahens belongs to the tribe Sphylacephalini, which is most likely ancestral in the subfamily Diopsinae (Diptera, Diopsidae, Kotrba 2004). Stalk-eyed flies of the Diopsinae are noted for lateral projection of their heads, where the compound eyes are located, in both sexes. The degree of development and the level of sexual dimorphism of the eye stalks are highly variable among diopsid species (Meier and Hilger 2000;Baker and Wilkinson 2001;Kotrba 2004;Kotrba and Balke 2006). Several studies have shown a low level of sexual dimorphism in S. detrahens, with an overlap in the range of eye span and body length in the two sexes (Ohara 1993;Wilkinson and Dodson 1997;Takeda et al. 2020).
Sphyracephala detrahens is widely distributed in East Asia, South-East Asia, and some areas of Oceania, including Papua New Guinea (Ohara 1993). No congener of S. detrahens was found on Iriomote Island; the insects used in the present study were collected from this island. In the field, adult S. detrahens flies are found alone or in groups of several of individuals, keeping some distance from each other, on rocks or vegetation in streams. Mating behavior in this species has been relatively frequently observed in such places, although the natural history of this species is not well known (Fig. 1). Takeda et al. (2020) found that mature males attempted to copulate with other mature males, suggesting that S. detrahens males were unable to distinguish between mature females and males. However, the responses of mature males to immature individuals have not yet been investigated. In this study, we investigated the mating behavior of mature S. detrahens males on immature and mature individuals of both sexes. We then compared the cuticular hydrocarbon profiles of immature and mature adults because males may be able to detect cuticular compounds when in close vicinity or when they mount on the back of their counterparts. This study aimed to clarify whether males S. detrahens recognize the sex and sexual maturity of conspecifics and whether their visual and chemical senses are involved in this recognition.

Insect collection
Sphyracephala detrahens were collected from Iriomote Island, Okinawa, Japan, in April 2019 by Kudo (2019). Flies were maintained on banana peels with yeast at 25 °C in a 14-h light:10-h dark cycle. For all experiments, to obtain virgin individuals, new adults were collected within 48 h of eclosion. These flies were kept solitary in plastic vials (15 mm in diameter, 95 mm in height), each containing cotton wool (25 mm × 25 mm) soaked with 1.7 mL water, yeast, and a small piece of banana peel. The internal reproductive organs of males S. detrahens matured sufficiently in a month, and those of females matured sufficiently in 2 weeks (Takeda et al. 2020). In the present study, we treated adults within 5 days and > 4 weeks after isolation as "sexually immature" and "sexually mature", respectively.

Behavioral assays
For the behavioral assays, a sexually mature male (focal) and a tester individual (mature female, immature female, mature male, or immature male) were introduced into plastic vials, each containing cotton wool (25 mm × 50 mm) soaked with 3 mL water and a banana peel (10 mm × 15 mm). All behavioral assays were performed during the light phase and recorded using digital video cameras (SONY HDR-CX560; Sony, Tokyo, Japan). In the behavioral assays using pairs of males (two mature males or a mature male and an immature male), focal and tester insects were distinguished by their relative body sizes. The number and duration of mounting by the focal males in 30 min were measured by inspecting the video replay. In mounting, a male approaches a focal individuals and jumps on the individual's back. For pairs that were still engaged in copulation at 30 min, the observation was continued until completion. Copulation was regarded as a 'success' when mounting by the focal male continued for > 1 min for the following reasons. Of the copulation attempts that lasted less than a minute, the longest mounting lasted only 29 s and the male failed to insert his copulatory organ. The second longest mounting lasted 18 s, and the male also failed to insert his copulatory organ. Therefore, we assumed that mating was not successful when mounting lasted for > 1 min. In total, 25 mature females, 24 mature males, 25 immature females, and 25 immature males were observed as tester insects. Focal males were used repeatedly with sufficient intervals (> 6 h) in combination with four different types of tester insects in random order  Table 1). There were 27 focal males, 24 of which also served as tester mature males.

Cuticular hydrocarbon (CHC) analyses
We used 16 mature females, 16 mature males, 17 immature females, and 17 immature males for the analyses of CHCs. Flies used in the behavioral experiments, except for those that died during the course of the tests, were used for the CHC analyses. Flies were individually anesthetized with CO 2 and immersed in 1 mL hexane containing 100 ng of triacontane (Tokyo Chemical Industry Co., Ltd., Japan) as an internal standard for 5 min in small glass vials. The extract was applied to a glass pipette filled with ca. 1 g Wakogel ® C-200 (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) overlaid with 750 mg anhydrous sodium sulfate (Nacalai Tesque, Inc., Kyoto, Japan). The solvent was evaporated under a stream of nitrogen gas, and the residue was resuspended in 100 μL of hexane. Samples (2 μL) were injected into a gas chromatograph (GC-2014; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a DB-1 capillary column (30 m × 0.25 mm I.D., 0.25 μm film; Agilent J&W Scientific, Tokyo, Japan). Nitrogen was used as the carrier gas at a flow rate of 0.93 mL/ min. The GC oven temperature was maintained at 80 °C for 2 min, increased at a rate of 10 °C/ min to 300 °C, and maintained at 300 °C for 8 min. The peak areas of the CHCs were calculated using the Solution software (Shimadzu). The amounts of CHCs were calculated by comparing their peak areas with those of the internal standard, triacontane (C 30 H 62 ; retention time: 28.22 min). We estimated the carbon chain length of CHCs by referring to the retention times of four authentic standards, heptadecane (C 17 H 34 ; retention time: 14.78 min), heneicosane (C 21 H 44 ; retention time: 18.84 min), tetracosane (C 24 H 50 ; retention time: 21.44 min), and triacontane (C 30 H 62 ; retention time: 28.22 min).

Statistical analyses
All analyses were conducted using R (version 3.6.3; R Development Core Team 2019) in RStudio (version 1.2.5033; RStudio Team 2019). For behavioral data, the number and duration of mounting were compared between the four tester insect groups that differed in sex and maturity. The number of mounts by the focal male was analyzed using a generalized linear mixed model with a Poisson distribution. The total duration of mounting by the focal male and the duration of mounting per single event that ended without copulation were analyzed using generalized linear mixed models with gamma distributions. All models included both fixed effect (tester types) and random effects (focal male ID, assay order, and focal male's mated status). These analyses were conducted using 'glmer' in the lme4 package and 'glht' in the multcomp package.
To analyze differences in CHC profiles between the four tester insect groups differing in sex and maturity, we performed a principal component analysis. We then used discriminant analysis to identify the group of variables that best discriminated between the four groups on the extracted factors with eigenvalues > 1 from the principal component analysis using 'lda' in the MASS package.

Mating behavior of S. detrahens
Males S. detrahens did not engage in any observable precopulatory courtship behaviors (Fig. 2a-e; http:// www. momo-p. com/ showd etail-e. php? movie id= momo2 11123 sd01a). A male approached a female gradually (Fig. 2a) and then jumped onto her back (Fig. 2b). He oriented himself in the same direction as the female (Fig. 2c) and attempted to insert his copulatory organ (Fig. 2d, e). Males sometimes flapped their wings to remain on the backs of the females (Fig. 2d). Table 1 shows that most of the sexually mature S. detrahens males attempted to copulate not only with sexually mature females but also with sexually mature males ( Fig. 3a-f; http:// www. momo-p. com/ showd etail-e. php? movie id= momo2 11123 sd02a), sexually immature females ( Fig. 4a-f; http:// www. momo-p. com/ showd etail-e. php? movie id= momo2 11123 sd03a), and sexually immature males ( Fig. 5a-f; http:// www. momo-p. com/ showd etail-e. php? movie id= momo2 11123 sd04a). The proportion of mature males that attempted to copulate within 30 min with mature females was 24/25, with mature males was 23/24, with immature females was 24/25, and with immature males was 25/25 (Fisher's exact test, P = 0.90; Table 1). However, the number of mounting events observed in the tester insect groups differed significantly (Table 1). Sexually mature females were the least frequently mounted, and sexually mature males were most frequently mounted by sexually mature males (Table 1).

Copulation attempts of sexually mature males
Successful copulation occurred only with sexually mature females (22/25), and in 19 of 22 successful cases, females accepted mature males in the first copulation attempt. Of the 22 mature females that accepted copulation, 11 were mounted by the same male again, and seven accepted remating within the observation period of 30 min (the maximum number of copulations was two, and the maximum copulation duration was 58.77 min). In three of the seven pairs in which remating occurred, the duration of the first copulation was longer than that of the second. Most immature females (23/25) received copulation attempts by males but never accepted copulation. The total duration of mounting by sexually mature males was significantly longer in sexually mature females than in immature adults of both sexes and sexually mature males because only mature females accepted copulation (Table 1). On the other hand, the duration of mounting per single event that ended without copulation was not significantly different among the four types of tester insects (Table 1).

CHC profiles
We detected 38 CHCs from S. detrahens adults (Supplementary table 2). Figure 6 shows typical gas chromatograms of a single fly sample of a sexually mature female (A), sexually mature male (B), sexually immature female (C), and sexually immature male (D). No strictly sex-specific nor sexual maturity-specific CHCs were identified (Supplementary  table 2). Most mature adults yielded compound 24 (retention time: 20.86) and compound 29 (retention time: 22.03), but these compounds were present in a few immature adults of both sexes (Supplementary table 2). Compound 36 (retention time: 23.40) was present in most immature adults, but not in the majority of mature adults of both sexes (Supplementary  table 2).

Quantitative comparison in cuticular hydrocarbons
We conducted principal component analysis of the 38 CHCs. The first two principal components (PC1 and PC2) explained   7% and 19.6%, respectively). In the PC score plot, no tester insect group was clearly separated from the others (Fig. 7). The first seven principal components with eigenvalues > 1, which altogether explained 85.1% of the total variance in CHCs (PC1: 36.7%, PC2: 19.6%, PC3: 11.3%, PC4: 6.0%, PC5: 4.8%, PC6: 4.1%, PC7: 2.7%), were used for subsequent discriminant analysis. The discriminant analysis of these PCs significantly separated the four tester insect groups (Wilks' lambda = 0.243, df = 21, P < 0.001). None of the immature adults were assigned to the mature female group; however, 31.3% of mature females were not correctly assigned to the mature female group, and 18.8% of mature males were misassigned to the mature female group (Table 2).

Discussion
In many insect species, males use visual, chemical, or acoustic cues to distinguish potential mates before copulation attempts. On the other hand, some male insects are unable to accurately discriminate between females and other males (Serrano et al. 1991(Serrano et al. , 2000Harari et al. 2000;Switzer et al. 2004;Bailey and French 2012;Burgevin et al. 2013;Macchiano et al. 2018;Sales et al. 2018). A few dipteran males also engage in same-sex sexual behaviors (SSBs); however, SSBs occur infrequently or only up to midway through the courtship sequence (Tauber and Tauber 1967;Parker 1968;Curcillo and Tompkins 1987;Tobin and Stoffolano 1973;Kuba and Koyama 1985;Vaias et al. 1993;Maklakov and Bonduriansky 2009;Müller et al. 2012;Benelli et al. 2014). Accordingly, the occurrence of male-male mounting  behavior is rare (Tauber and Toschi 1965;Cavender and Goeden 1982;Preston-Mafham 2006;McLachlan 2011). These findings, when viewed from the opposite perspective, suggest that mating behavior has been extensively studied in species that have evolved the ability to discriminate potential mates. In the present study, we revealed that mature males of S. detrahens do not distinguish mature females from immature females or males. Although visual or chemical cues may be involved in species recognition, these cues are not likely to be useful for potential mate discrimination by males of S. detrahens. Sexually mature S. detrahens males made copulation attempts toward nearby individuals regardless of their sex or sexual maturity. On the other hand, the apparent frequency of mounting in the 30 min observation period was significantly different; it was least frequent with mature females and most frequent with mature males. The frequency of mounting with mature females was low because successful copulation continued for an average of > 10 min. Mounting on mature males was frequent because mature males were very active and thus they encountered their counterparts more frequently. These observations suggest that mature males were unable to distinguish mature females from males until they mounted on the backs of their counterparts. Thus, non-contact cues, such as the morphologies and odors of their counterparts, do not seem to be useful for mate recognition in this species. Males of the stalk-eyed fly species Teleopsis dalmanni, who demonstrate sexual dimorphism in their eyespan, can differentiate the fecundities of females when given a choice (Cotton et al. 2015). Similarly, in S. detrahens, the presence of choices may also provide males with some information for better discrimination between potential mates.
Mature males also attempted to copulate with immature females; however, none of the immature females accepted copulation. Immature females mounted by mature males vibrated their bodies and tried to shake the males off (Fig. 4e,f;? movie id= momo2 11123 sd03a). Such body vibrations were also observed in mature females during mating. However, when mature and immature males were mounted by other males, they incurved their abdomen downward to avoid copulation attempts. When the male rejection rates of sexually monomorphic and dimorphic stalk-eyed species were compared, Teleopsis quinqueguttata (rejection rate: 17.2%), a monomorphic species, rejected copulation attempts from males more frequently than sexually dimorphic species, T. dalmanni (4.2%) and T. whitei (8.1%). In T. dalmmani and T. whitei, which exhibit low rejection rates, females tend to mate with males that possess wider eye spans and prefer to fly to mating aggregation sites with such males that are considered to be attractive (Burkhardt andde la Motte 1988, Wilkinson andReillo 1994). In T. quinqueguttata, which does not form mating aggregation in the field (Kotrba 1996), rejection of copulation attempts 1 3 by females was not affected by male body size or stalk eye length (Wilkinson et al. 1998).
We found that male and female S. detrahens copulated more than once within the short (30 min) observation period. Remating in Diopsidae has only been studied in three species of the genus Teleopsis, and all three species have been reported to copulate multiple times (Wilkinson et al. 1998). The most sexually dimorphic species T. whitei remates most frequently, in which females and males copulate every 1.7 and 1.0 h, respectively (Wilkinson et al. 1998). The present study suggests that both sexes of S. detrahens usually remate. However, additional details regarding remating behavior would be clarified by extending the observation duration and presenting new adults to mature males. We did not find any sex-or maturity-specific CHCs in this species. Only compound 36, which was present in most immature adults but not in the majority of mature adults, had a negative eigenvector for PC1; however, its absolute value was small. Discriminant analysis using the seven principal components with eigenvalues > 1 showed that although immature males, regardless of sex, were rarely incorrectly classified as mature females, mature males were more often misclassified as mature females. For the males of S. detrahens, it may be more difficult to distinguish mature females from mature males than those from immature adults of both sexes.
The CHC profiles of the four groups, which differed with sex and level of sexual maturity, were qualitatively and quantitatively indistinguishable. The variations in the number and duration of copulation attempts by mature males were not correlated with the CHC profiles of tester insect groups. Therefore, we conclude that contact cues, such as non-and low-volatile pheromones, are unlikely to be useful for mate discrimination in S. detrahens. In the field, both males and females were observed, throughout the year, on rocks or vegetation in the stream (Ohara 1997). Newly emerged adult S. detrahens are sexually mature in a month and live for several months. Therefore, mature males are expected to encounter other mature males and immature individuals of both sexes not infrequently. This study demonstrated that mature males lack the ability to discriminate between the sex and maturity of conspecifics. It may be beneficial for males to attempt copulation without discrimination of mature females when it takes time and energy to discriminate them, or when the accuracy of the discrimination is low. Male S. detrahens do not engage in any obvious (time-consuming) pre-copulatory courtship behavior; their copulation attempt is initiated by jumping on the backs of their counterparts. If a male took too long to determine the sex of a potential mate, he would be more likely to lose a female to another male. To elucidate why male S. detrahens are poor at discriminating between potential conspecific mates, it is necessary to investigate the reproductive behaviors and operational sex ratio in the field.