Trichopria drosophilae is a globally distributed pupal parasitoid that targets various species within the Drosophilidae family, including the invasive Drosophila suzukii. The mass rearing of T. drosophilae is a crucial step in ensuring their successful application for field D. suzukii. control. The pupae of Drosophila melanogaster are currently used as a host for the mass rearing of T. drosophilae. The impact of utilizing irradiated pupae on T. drosophilae was investigated. Our results showed that irradiated pupae had a significant impact on the parasitism rate, offspring eclosion rate, offspring number, and female body size of F1 T. drosophilae, all of which were significantly lower than those in the normal group. However, there was no significant difference in parasitism rate, body size, offspring eclosion rate, offspring number, and offspring sex ratio between F2 T. drosophilae emerging from the two types of Drosophila pupae. Compared to F2, F1 had a significantly higher net reproductive rate (R₀, t₄₉ = 19.07, P < 0.01), mean generation time (T, t₄₉ = 146.26, P < 0.01), and doubling time (DT, Z = -6.20, P < 0.01), while the intrinsic rate of increase (r, Z = -6.28, P < 0.01) was significantly lower. Using irradiated D. melanogaster pupae provides an efficient method for the mass rearing of T. drosophilae and offers valuable insights into its potential effectiveness in field D. suzukii control.

Insects

Laboratory colonies of D. melanogaster and T. drosophilae were collected from blueberry orchards in Huangtai Village, Wuhu, Anhui Province, P.R. China. All collected insects were reared in controlled environments within artificial climate chambers (25 ± 1℃, 65 ± 5% RH, 12:12 h (L/D)). Drosophila melanogaster were collected from Anhui Normal University, Anhui Province, China, in 2010 (31.33°N, 118.37°E), reared in nylon cube cages (35×35×35 cm). Drosophila melanogaster were fed a 240-ml plastic cup containing 80 ml of solid food (water: 1000 ml, corn flour: 44.67 g, sucrose: 71.33 g, agar: 16.00 g, yeast: 25.00 g, propionic acid: 6.30 ml, 100% ethanol: 22.30 ml). The plastic cup containing food is replaced daily, and the removed cups are kept in the climate chamber. Once the larvae pupate on the walls of the plastic cups, the cup walls are removed and provided for parasitism by T. drosophilae. T. drosophilae were reared in plastic insect rearing containers (diameter: 10 cm, height: 8 cm) are fitted with nylon mesh on the top, which ensures adequate ventilation while effectively preventing the escape of the parasitoids. Fresh 2-day-old D. melanogaster pupae are provided to the T. drosophilae at a ratio of 1:20 daily for parasitism. After 24 hours, the Drosophila pupae are removed from the rearing container and transferred to a new rearing container to develop until eclosion, where they serve as the maternal generation for the next cycle of parasitoid reproduction. Additionally, cotton soaked in a 10% honey-water solution is placed inside the container to serve as a food source for the parasitoids.

Irradiation treatment of Drosophila pupae

On day 0, the plastic cups with pupae not yet formed on the walls, are selected. On day 1 (24 hours later), Drosophila pupae of similar size are carefully selected from the cup walls and placed into 50 μL transparent thin-walled tubes. To prevent damage to the pupae during collection, a small amount of sterile water is sprayed on the cup walls, and a soft brush is used to gently sweep the pupae into the thin-walled tubes. On day 2, the 2-day-old pupae of D. melanogaster are subjected to a 1200 Gy γ-irradiation treatment using the HFY-YC γ-irradiation device from the Crop and Nuclear Technology Utilization Institute, Zhejiang Academy of Agricultural Sciences, with a dose rate of 240 Gy/h.

Utilization of irradiated D. melanogaster pupae by T. drosophilae

After 24 hours of parasitism by T. drosophilae, each D. melanogaster pupa is individually placed into a 50 μl transparent thin-walled tube. The eclosion of T. drosophilae inside the thin-walled tubes is checked daily. Newly eclosed T. drosophilae are selected, with 60 pairs chosen. After mating, they are fed a 10% honey water until they reach 4 days of age because the number of mature eggs of T. drosophilae was boosted during the first four days (Wang et al. 2016). The female T. drosophilae are placed into Drosophila tubes (24 mm in diameter, 95 mm in height). The tubes are contained with cotton balls soaked in a 10% honey-water solution to provide food. Irradiated or normal 2-day-old D. melanogaster pupae are provided as hosts, with the irradiated pupae designated as the irradiated group and the normal pupae as the normal group. After 24 hours of parasitism, the female T. drosophilae are removed, and the parasitized pupae are individually placed into thin-walled tubes for further processing. The number and sex of eclosed T. drosophilae in each group are observed and recorded daily at 10:00 AM and 8:00 PM. For Drosophila pupae that neither eclose into flies nor parasitoids, dissection is performed under a microscope 10 days after the first eclosion to determine their status (un-eclosed Drosophila, parasitized but un-eclosed parasitoids, or unknown). The eclosed T. drosophilae are recorded as F1.

Effect of parasitism on irradiated D. melanogaster pupae on parasitoid offspring reproduction

T. drosophilae eclosed from irradiated pupae will be continuously reared on normal D. melanogaster pupae for two generations, and life table experiments will be conducted. One female and one male T. drosophilae, both within 12 hours of eclosion, are placed into a Drosophila tube. The tube contains cotton soaked in 10% honey-water solution for feeding, as well as 20 normal 2-day-old D. melanogaster pupae as hosts. Every 24 hours, fresh cotton with honey-water and normal Drosophila pupae are provided. After the death of the female T. drosophilae, no additional hosts are supplied, and only the male T. drosophilae is provided with cotton soaked in honey-water for feeding. The parasitized pupae removed during replacement are placed in 3 mL sample tubes and kept for T. drosophilae eclosion. The survival status of adult T. drosophilae, as well as the number and sex of eclosed offspring, are observed and recorded daily. Ten days after the first eclosion, pupae that neither eclosed into Drosophila nor parasitoids are dissected. The T. drosophilae in the sample tubes are frozen at -20°C for preservation. Afterward, their hind tibia length is measured under a microscope (VHX-5000, Osaka Japan, Keyence Corporation).

Data analyses

In the obtained data, groups that produced only male offspring are excluded from statistical analysis. Statistical comparisons are conducted to assess the effects of using irradiated D. melanogaster pupae on parental parasitoid parasitism rate, eclosion rate, offspring sex ratio. We analyzed the differences in lifespan, body size, parasitism rate, offspring eclosion rate, offspring sex ratio, offspring number and population life table parameters when F1 and F2 generation T. drosophilae parasitized irradiated and normal pupae. Eclosion rate is calculated as the number of eclosed parasitoids divided by the sum of eclosed parasitoids and parasitized but non-eclosed individuals. Parasitism rate is calculated as the sum of eclosed parasitoids and parasitized but non-eclosed individuals divided by the total number of pupae provided. Sex ratio, is equal to the number of male parasitoids divided by the total number of offspring.

Based on the obtained data, standard life table includes four parameters: the intrinsic rate of natural increase (r), net reproductive rate (R₀), mean generation time (T), and doubling time (DT). The value of r was calculated as =1, where  is expressed as female age in days,  is expressed as the age-specific survival rate, and  is the number of daughters produced per female alive at age , R₀=, T= ln R₀/r, DT =ln (2)/r (Carey 1993).

For continuous data, we performed a Shapiro-Wilk normality test on longevity and body size. For normally distributed data, a t-test was used, while non-parametric tests were applied for skewed data. For binary data, the eclosion rate, parasitism rate and sex ratio were analyzed by a generalized linear model (GLM) as the effect using a quasibinomial error due to a large residual deviance value relative to the degrees of freedom. For count data, the number of offspring was analyzed by GLM as the effect of a quasipoisson error due to a large residual deviance value relative to the degrees of freedom. All the tests mentioned above were performed with R version 4.2.2 (R Core Team 2013).