The mating rate of Drosophila suzukii reduction due to reproductive interference from Drosophila melanogaster


 Background Drosophila suzukii is widely distributed. Research has revealed that the presence of Drosophila melanogaster can reduce the emergence and egg laying of D. suzukii. However, the reasons for these phenomena have not yet been reported. To investigate this issue, we sought to answer three questions: Can the presence of D. melanogaster reduce the longevity of D. suzukii? Does D. melanogaster dominate in larval interspecific competition with D. suzukii? Does reproductive interference occur between these species; i.e., do individuals of one species (e.g., D. suzukii) engage in reproductive activities with individuals of the other (e.g., D. melanogaster) such that the fitness of one or both species is reduced? Results The results showed that the adult offspring number of Drosophila suzukii was significantly reduced when this species was reared with Drosophila melanogaster. The larval interspecific competition had no significant effects on Drosophila suzukii longevity or population size. Surprisingly, Drosophila melanogaster imposed reproductive interference on males of Drosophila suzukii, which led to a significant decline in the rate of successful mating of the latter species. Conclusions The presence of Drosophila melanogaster causes the population size of Drosophila suzukii to decrease through reproductive interference, and the rate of successful mating in Drosophila suzukii is significantly reduced in the presence of Drosophila melanogaster.


Background
Drosophila suzukii (Diptera: Drosophilidae), known as the spotted-wing drosophila (SWD), is originally from Asia. However, it has become a severe invasive pest in South America and Europe since 2008 [1,2], causing large economic losses in the fruit production industry [3]. Usually, Drosophila suzukii females lay eggs within ripe or ripening fruit via a sclerotized and serrated ovipositor that penetrates the fruit skin, causing physical damage to the fruit [4,5]. According to the literature, D. suzukii can lay up to 600 eggs (approximately 400 eggs on average) during their life span, and the minimum development time from egg at oviposition to adult under optimal temperature is 8 days [6]. Regarding environmental adaptability, D. suzukii has a wide range of tolerance to climatic conditions, with successful production of offspring possible at temperatures from 10 to 32 °C [7]. The optimal range for larval development is usually between 20 to 25 °C [8]. In addition, this pest has a wide host range, with more than 150 host species reported to date [9,10]. Since these factors and the fact that different hosts have different ripening times. Therefore, it is almost impossible to eliminate this pest in a short period of time. Wild hosts as well as ornamental plants can serve as refuges, allowing D. suzukii to persist through the winter, even under low temperatures [11].
The use of ovipositors can cause physical damage to intact fruit, and the larvae can accelerate the process of decay. Damaged, decaying fruit is an ideal food source for other phytophagous insects, so competition of D. suzukii with other Drosophila species, such as Drosophila melanogaster, is almost inevitable. Interspeci c competition widely exists among insects [12,13] and includes intraspeci c competition and interspeci c competition. The outcome of competition depends on the relative strengths of interspeci c competition and intraspeci c competition. Both types of competition represent struggles for survival, growth and reproduction for every individual because of limited environmental resources [14,15]. As a result of interspeci c competition, the population of one species tends to decrease while that of the other tends to increase, with strong effects on species coexistence, habitat partitioning, food resource partitioning, and species replacement [16]. The importance of interspeci c competition to community structure in plant-eating insects has been discussed by many ecologists [17,18]. Denno analysed 193 pairs of competitor species and concluded that the interspeci c competition affects the abundance and distribution of plant-eating insects [19].
According to niche theory, closely related species overlap in resource needs, so competition can be very strong when two species rst meet. The results of competitive interaction are either competitive exclusion of one of the species or niche reduction until coexistence becomes possible [20]. A previous study revealed that in the lab environment, the presence of D. melanogaster can signi cantly reduce emergence and egg laying in D. suzukii through interspeci c competition [21]. One possible explanation for these effects is the production of a pheromone called cis-vaccenyl acetate (cVA) by males of D. melanogaster that is used in courting, aggression, and aggregation signalling and has a disruptive effect on D. suzukii [21]. The number of successful matings by males of D. suzukii is reduced when they encounter this pheromone. Correspondingly, in previous work [22], we found that when adults of D. melanogaster and D. suzukii were reared together for three generations, the whole population of D. suzukii eventually died. Thus, in the present study, we attempted to explore alternative potential factors contributing to this phenomenon.
The competitive interactions of two closely related species over resources entail time and energy costs and risk of injury. The allocation of time and energy by these two y species to interspeci c competition results in decreased egg laying, and individuals might suffer physical damage, affecting longevity.
Decreases in both egg laying and adult longevity could be important factors contributing to the extinction of D. suzukii populations. Recent studies show that both D. suzukii and D. melanogaster have strong appetites and overlapping feeding niches [20] [23]. Therefore, in our experiments, we investigated the effects of interspeci c competition on the longevity and fecundity of the two ies. We studied both adult competition and larval competition between D. melanogaster and D. suzukii. Reproductive interference is a kind of interspeci c sexual interaction wherein the reproductive success of females of one species could be reduced due to interference by another species [24]. The frequencies of conspeci c courting and mating, female fecundity, and female fertility can be in uenced by reproductive interference, and time, energy, nutrients, or gametes can be wasted in heterospeci c sexual interactions, causing tness losses for the individuals involved [25]. Reproductive interference might be a factor resulting in the population reduction of D. suzukii and is a possibility examined in this study.

Results
Offspring number and longevity comparison of the two y species under the two rearing conditions The offspring number of D. suzukii was signi cantly decreased from that under independent rearing when this species was reared with D. melanogaster (Fig. 1a). Under the independent rearing condition, the offspring number of D. suzukii (117.37±24.28) was signi cantly higher than that observed in the mixed rearing condition (40.47±8.23, t 17 =3.13, P= 0.006). The same pattern was found in D. melanogaster: offspring number in the independent condition (494.50 ± 36.92) was much higher than that in the mixed rearing condition (269.53±47.05, t 18 = 3.76, P< 0.01).
Regarding longevity, the two y species were affected differently under the two conditions (Fig. 1b). The longevity of D. suzukii in the independent condition (54±2.70 days) did not signi cantly differ from that in the mixed condition (61.6±3.04 days, t 36 = -1.85, P= 0.07). However, in D. melanogaster, the longevity in the independent rearing condition (70.1±1.05 days) was signi cantly higher than that in the mixed rearing condition (79.25±3.05 days, t 38 = -2.838, P= 0.007).

Larval interspeci c competition
We calculated the numbers of pupation events and eclosion events of each of the two species in a larval interspeci c competition experiment ( Table 1). The results showed that for D. suzukii, eclosion number did not signi cantly differ between the independent and mixed rearing conditions (c 2 =3.52, df=1, P= 0.061); similarly, D. suzukii pupation number did not differ between the conditions (c 2 =3.14, df=1, P= 0.077). In D. melanogaster, neither eclosion number (c 2 = 0.515, df=1, P= 0.473) nor pupation number differed substantially between the two rearing conditions (c 2 =0.002, df=1, P= 0.905).

Discussion
Consistent with a previous study [21], our results showed that the number of offspring of D. suzukii was signi cantly reduced when this species was rearing in mixed groups with D. melanogaster (Fig. 1). Datta et al. (2008) suggested that the presence of D. melanogaster could change oviposition preference, resulting in reduced egg laying by D. suzukii. Males of D. melanogaster produce a pheromone, cisvaccenyl acetate (cVA) [26], which is natural repellent to D. suzukii females searching for oviposition sites. Our results suggested that in addition to affecting oviposition preference, reproductive interference by D. melanogaster likely contributed to the signi cant decrease in offspring number in D. suzukii.
Reproductive interference between invasive and native species has received much attention. Reproductive interference and is a kind of interspeci c sexual interaction wherein the female reproductive success of one species is reduced because of the interference by another species [24]. In our behaviour experiment, we found that the presence of females and males of D. melanogaster resulted in reproductive interference with males of D. suzukii, which selected D. melanogaster females as their mating targets in some cases (see Additional le 1 f). Usually, reproductive interference is categorized into seven types: signal jamming, heterospeci c rivalry, misdirected courting, attempted heterospeci c mating, erroneous female choice, heterospeci c mating and hybridization [27]. Three types of reproductive interference, misdirected courting, attempted heterospeci c mating and heterospeci c mating, were observed in our study.
Among the tested adult males of D. suzukii, 95% (19 of 20 groups) showed misdirected courting behaviour. Usually, misdirected courting is performed or initiated by males, which are often indiscriminate in mate choice, as they invest less energy in reproduction [28]. In our study, we found that misdirected courting was initiated by males of D. suzukii. Such males may fail to recognize conspeci c females and preferentially court heterospeci c females or even males [29,30]. Previous studies reported that heterospeci c females with larger body size than conspeci c females were attractive to males because large body size indicates high fecundity [31,32]. This nding contrasts with our observations that D. suzukii males were attracted to D. melanogaster females, which are smaller than D. suzukii females. Therefore, we deduce that D. suzukii males fail to recognize conspeci c females and that D. melanogaster females possess features that can attract D. suzukii males. D. melanogaster sex pheromones are cuticular hydrocarbons (CHC) that mediate chemical communication for both sex and species recognition [33]. These CHC may make D. melanogaster females attractive to D. suzukii males and prevent scent-mediated attraction to conspeci c females.
Consistent with previous reports [34], in our observation experiments, all of the heterospeci c mating attempts followed misdirected courting, and D. melanogaster males showed no heterospeci c mating attempts. The behaviour of heterospeci c mating has been investigated in many studies, such as Takafuji (1988), Fujimoto et al (1996), and Takafuji et al (1997) [35][36][37]. However, there is no study reported D. suzukii males underwent heterospeci c mating with D. melanogaster females. Usually, reproductive interference can be regarded as a "mistake" resulting in incomplete species recognition. Thus, time, energy, nutrients, or gametes are wasted in heterospeci c sexual interactions, causing tness losses of the individuals involved [25]. The frequencies of conspeci c courting and mating, female fecundity, and female fertility can be in uenced by reproductive interference [25].
In this study, potential factors resulting in population decline in D. suzukii due to D. melanogaster were explored. However, inconsistent with our expectations, the lifespan of D. suzukii was not reduced when this species was reared with D. melanogaster. Our results suggest that competition with D. melanogaster might not lead to physical damage to D. suzukii adults thus might not affect adult longevity. Therefore, the population decline of D. suzukii was unlikely to be caused by a reduction in lifespan in D. suzukii adults reared and competing with D. melanogaster. In addition, larval interspeci c competition might not be a factor affecting D. suzukii population decline, as rearing condition had no signi cant effect on pupation rate or eclosion rate (Table 1).

Conclusions
In this study, we found that D. suzukii population size was signi cantly decreased when individuals of this species were reared with D. melanogaster vs. reared alone, which was consistent with previous research. Potential factors underlying this phenomenon, including longevity, larval interspeci c and reproductive interference, were studied. The experimental results showed that longevity and larval interspeci c competition had no signi cant effects on population size in D. suzukii. However, D. melanogaster imposed reproductive interference on D. suzukii males, signi cantly reducing the rate of selecting conspeci c females as mating targets in these males, and apparently decreasing the rate of successful mating rate. This phenomenon warrants further research, including research into the features of D. melanogaster that cause D. suzukii males to not recognize conspeci c females. We speculate that Drosophila melanogaster may release a substance, such as a sex hormone, that distracts D. suzukii males.

Material And Methods
Fly stocks adults of the two species were independently reared in nylon cube cages (35×35×35 cm) at 25±1 °C under a light: dark photoperiod of 14:10 and a relative humidity of 60 ± 5%. Both ies species were fed a solid food diet [38], which was changed out daily.

Longevity and fecundity
For this experiment, 20 male-female pairs of newly emerged (<24 h) individuals of each of the two y species were selected. Ten pairs of each species were independently reared in food-containing tubes (one pair per tube), and the remaining pairs were reared in mixed-species groups (two pairs per tube). The longevity of each adult y was recorded daily. To investigate egg laying, one pair of D. suzukii or D. melanogaster individuals was placed into a transparent tube pre-loaded with 20 ml solid food. Additionally, treatments with the two species reared together were conducted. The next day, the adult ies in each tube were anaesthetized by CO 2 and transferred to another transparent tube with new solid food and maintained there until death. The number of adult offspring that emerged was counted daily from the rst adult eclosion at three days to the death of the adult female. Ten replicates of each y species and rearing type were established. All tubes containing ies and all replacement tubes were maintained in incubators under the same conditions of light:dark photoperiod (14:10), temperature (25±1 °C), and humidity (60±5%) and were checked daily.

Larval competition
We hypothesized that larval interspeci c competition would lead to a change in D. suzukii population size. To determine the effect of larval interspeci c competition on adult emergence, we reared the two y species under two conditions. Because interspeci c competition has a density-dependent effect on population dynamics, when the density of one species increases, the density of the other decreases [24]. Furthermore, the species with the higher initial density is likely to exclude the species with lower initial density [39,40]. Therefore, in our experiment, we maintained the same density in the two rearing conditions to control for the in uence of density on competitive ability (10 larvae per 10 g solid food in the independent condition; 10 larvae per species per 10 g solid food in the mixed condition).
Both y species were fed on yeast water for 8 h, and then two cups containing solid food were used for egg collection. Twenty larvae of each species were placed in a cup containing 10 g solid food for independent rearing after egg incubation. For the mixed rearing condition, 10 larvae from each species were placed together in a cup containing solid food. All the cups were maintained in incubators with the following condition settings: light:dark photoperiod, 14:10 h; temperature, 25±1 °C; and humidity, 60±5%.
The numbers of pupation events and adult eclosions were counted daily. Ten of twenty replicates for each species were established for determining eclosion counts, and the remainder were established for pupation counts.

Observations of courting behaviour of the two ies under independent and mixed conditions
To ensure that the ies used for behavioural observations were unmated, we placed pupae into EP tubes for emergence. All tubes were maintained in incubators with the following condition settings: light:dark photoperiod, 14:10 h; temperature, 25±1 °C; and humidity, 60±5%. Observation started 24 h after eclosion. For the independent condition, one pair of D. suzukii or D. melanogaster individuals was placed in a quartz cylinder (height: 0.5 cm, diameter: 3 cm) with a 0.5 mm quartz lid. An appropriate amount of solid food was placed in each cylinder before each observation period. For the mixed condition, one pair of each y species was placed together a cylinder for observation. We recorded a 40-min video of courting and mating behaviour with a microscope (VHX-5000, Osaka Japan, Keyence Corporation). The frequency of courting behaviour and total mating time were recorded. We conducted observations of 20 replicates of each of the independent and mixed conditions for the two y species.

Data analysis
We performed a Kolmogorov-Smirnov normality test on all collected data. The longevity, offspring number, courting time, and mating time data t normal distributions, so these data were analysed by Student test (t-test). The data that did not t a normal distribution, such as the frequencies of correct courting, misdirected courting, and heterospeci c mating attempts, were analysed by the Mann-Whitney U test. The successful mating rate, pupation rate, and emergence rate were analysed by the Chi-square test. All these tests were performed with R 3.6.1.