Asymmetric reproductive interference of male Drosophila suzukii caused by both male and female Drosophila melanogaster

Reproductive interference, leading to negative effects on fitness, usually occurs within two closely related species which sharing overlapping ecological niches. Our preliminary experiments showed D. suzukii population decreased when reared with D. melanogaster. However, the explanation of this phenomenon is not much clear. Based on the overlapping ecological niches and courtship behaviours of D. suzukii and D. melanogaster, we deduced that reproductive interference was the dominant reason. We found that the proportion of heterospecific orientation time is significantly greater in male D. suzukii (7.5 ± 4.183 %) than in male D. melanogaster (4.439 ± 4.936 %, U=194.5, Z=2.223, P=0.026). The successful mating rate of D. suzukii was significantly lower when the species was reared with D. melanogaster (F1,31=4.303, p=0.046). The egg production of D. suzukii was significantly lower in the combined rearing condition (F1,343=98.623, p<0.001), for D. melanogaster, the egg production in combined condition was significantly greater than in single rearing conditions (F1,354=4.202, p=0.041). The reproductive interference exerted by D. melanogaster increased the heterospecific courtship behaviours of male D. suzukii, causing the successful mating rate to decline and ultimately reducing the number of eggs.

Methods

Male conspecific and heterospecific courting behaviours

To ensure that the flies used for courting observations were unmated, we placed each pupa into 2-ml Eppendorf tubes for emergence. All tubes were maintained in incubators with a light:dark photoperiod of 14:10 h, temperature of 25±1°C, and humidity of 60±5%. All flies were kept in Eppendorf tubes for 48 h from eclosion until observation started. By comparing the male courtship behaviours of D. suzukii and D. melanogaster (Revadi et al. 2015; Spieth 1974), we selected seven specific behaviours—orientation, tapping, wing scissoring and spreading, circling, successful mating, heterospecific mating attempts and premating duration—for observation (Table 2). Proportion of misdirected courting time to total courting time in the two files were also recorded. Based on the rearing conditions, D. suzukii and D. melanogaster were divided into three groups (Figure 1): (1) D. suzukii single rearing (DSS): two pairs of D. suzukii; (2) D. melanogaster single rearing (DMS): two pairs of D. melanogaster; (3) D. suzukii and D. melanogaster combined rearing (DC):one pair of D. suzukii and one pair of D. melanogaster. Drosophila were transferred from Eppendorf tubes into perforated Petri dishes (355 mm) containing food, using a custom-made Drosophila aspirator. We recorded a 40-min video of courting behaviour with a microscope (VHX-5000, Osaka Japan, Keyence Corporation) for 20 replicates. During the data collection process, courtship time or frequency was calculated starting from the occurrence of the first courtship behaviour and ending with either mating occurrence or the specified time limit.

Successful mating rate

For obtaining virgin D. suzukii and D. melanogaster, the method is the same as described in the previous section. Based on the rearing conditions, D. suzukii and D. melanogaster were divided into four groups (Figure 1): (1) D. suzukii single rearing (DSS): two pairs of D. suzukii; (2) D. melanogaster single rearing (DMS): two pairs of D. melanogaster; (3) D. suzukii and D. melanogaster combined rearing (DC):one pair of D. suzukii and one pair of D. melanogaster; (4) D. suzukii and D. melanogaster combined rearing in dark (DCD): one pair of D. suzukii and one pair of D. melanogaster were combined rearing under red lights (wavelength:620-630 nm). Drosophila were transferred from Eppendorf tubes into perforated Petri dishes (355 mm) containing food, using a custom-made Drosophila aspirator. We then recorded 40-min videos (VHX-5000, Osaka Japan, Keyence Corporation) of the two flies under the two rearing conditions with a microscope, and 20 replicates were performed. Frequency of heterospecific mating attempts, rate of successful mating and rate of successful mating in dark of each pair involved were recorded.

Number of eggs

For obtaining virgin D. suzukii and D. melanogaster, the method is the same as described in the second section of Methods. Based on the rearing conditions, D. suzukii and D. melanogaster were divided into three groups (Figure 1): (1) D. suzukii single rearing (DSS): two pairs of D. suzukii; (2) D. melanogaster single rearing (DMS): two pairs of D. melanogaster; (3) D. suzukii and D. melanogaster combined rearing (DC):one pair of D. suzukii and one pair of D. melanogaster. Drosophila were transferred from Eppendorf tubes into Drosophila tube containing food, using a custom-made Drosophila aspirator. The next day, the adult flies in each tube were transferred to another clear tube with fresh solid food and maintained for ten days. Because D. melanogaster larvae have a deterring effect on D. suzukii oviposition (Tungadi et al., 2022), the number of eggs was counted daily starting from the egg stage. Twenty replicates of each fly species and rearing type were established.