Biocontrol characteristics of the fruit fly pupal parasitoid Trichopria drosophilae (Hymenoptera: Diapriidae) emerging from different hosts

Trichopria drosophilae (Hymenoptera: Diapriidae) is an important pupal endoparasitoid of Drosophila melanogaster Meigen (Diptera: Drosophilidae) and some other fruit fly species, such as D. suzukii, a very important invasive and economic pest. Studies of T. drosophilae suggest that this could be a good biological control agent for fruit fly pests. In this research, we compared the parasitic characteristics of T. drosophilae reared in D. melanogaster (TDm) with those reared in D. hydei (TDh). TDh had a larger size than TDm. The number of maximum mature eggs of a female TDh was 133.6 ± 6.9, compared with the significantly lower value of 104.8 ± 11.4 for TDm. Mated TDh female wasp continuously produced female offspring up to 6 days after mating, compared with only 3 days for TDm. In addition, the offspring female ratio of TDh, i.e., 82.32%, was significantly higher than that of TDm, i.e., 61.37%. Under starvation treatment, TDh survived longer than TDm. TDh also survived longer than TDm at high temperatures, such as 37 °C, although they both survived well at low temperatures, such as 18 °C and 4 °C. Old-age TDh females maintained a high parasitism rate and offspring female ratio, while they were declined in old-age TDm. Overall, TDh had an advantage in terms of body size, fecundity, stress resistance ability and the parasitism rate compared with TDm. Therefore, T. drosophilae from D. hydei could improve biocontrol efficacy with enormous economic benefits in the field, especially in the control of many frugivorous Drosophilidae species worldwide.

biological characteristics of T. drosophilae have been well studied by several groups. In 2012, Chabert et al. found that T. drosophilae was effective against many fruit fly species, including D. suzukii, a well-known invasive pest 12 . Female T. drosophilae emerged with a relatively high number of mature eggs, and the egg numbers increased during their first four days after eclosion. This indicates that T. drosophilae might maximize reproduction during early adult life 14 . Moreover, the parasitism rate of T. drosophilae is higher than that of another well-known cosmopolitan pupal parasitoid, Pachycrepoideus vindemmiae (Diptera: Pteromalidae) 13 . Although T. drosophilae is reported to be effective against Drosophila species under laboratory conditions, it is necessary to find the parasitoids that have the highest parasitism rate, highest female offspring numbers and longest adult longevity and which are resistant to certain stress conditions, such as food deprivation and extreme weather conditions, for the biological control purpose of augmentative release in the field.
To increase the effectiveness of parasitoids as natural enemies, female adult wasps are supplied with extra nutrient sources, such as sugars, to enhance their longevity and fecundity and subsequently, the biocontrol efficacy 15,16 . However, host quality can also have a major influence on the fitness and parasitic efficiency of offspring 17 . Lampson et al. found that different sizes of the same parasitoid had an effect on several biological characteristics, suggesting that larger parasitoids have a longer life span and greater competitiveness 18 . Another comparative study on the parasitism of P. vindemmiae hatching from housefly and fruit fly pupae showed a positive correlation between the size of the host and the size of the emerged offspring, as well as the longevity, the oviposition duration and other parasitic attributes 19 .
Based on the results of previous studies 14 , T. drosophilae reared on a larger sized host could be more advantageous for further biological control. Here, we used D. hydei as a substitute host, of which the pupae are significantly larger than those of D. melanogaster. Then, we compared the body size, fecundity, stress resistance ability and parasitism efficiency between the two parasitoid populations that emerged from the different hosts.

Results
The parasitoid and host size measurements. The respective pupal length and width were 4.05 ± 0.13 mm and 1.27 ± 0.04 mm for D. hydei (n = 18) and 2.93 ± 0.14 mm and 0.99 ± 0.06 mm for D. melanogaster (n = 37). The size of D. hydei was significantly larger than that of D. melanogaster (length: t = 28.57, df = 53, P < 0.01, width: t = 18.68, df = 53, P < 0.01). To investigate whether there was a correlation between the size of the hosts and their offspring, T. drosophilae was used to parasitize D. melanogaster and D. hydei pupae. The measurements indicated that the body length of TD h was significantly longer than that of TD m , in both females and males ( Fig. 1A-C). The body length of female TD h was 2.41 ± 0.12 mm (n = 10), compared with 2.12 ± 0.11 mm (n = 12) for female TD m (t = 5.50, df = 20, P < 0.01). The length of male TD h was 2.21 ± 0.07 mm (n = 16), compared with 1.92 ± 0.11 mm (n = 10) for male TD m (t = 7.94, df = 24, P < 0.01). These results showed that the size of TD h was much larger than that of TD m .
Parasitism rate and offspring female ratio comparison. The results showed that this local 4-day old T. drosophilae females had an extremely high parasitism rate. Approximately 97% of the D. melanogaster pupae were successfully parasitized by TD m , and no significant difference in the parasitism rate was found between TD h and TD m (t = 1.67, df = 4, P > 0.05) females. However, the offspring female ratio of TD h , which averaged 82.32%, was significantly higher than that of TD m , which averaged 61.37% (t = 8.96, df = 4, P < 0.01) ( Table 1).
The fecundity of T. drosophilae. The number of mature eggs in the ovaries of TD h and TD m females was compared among different ages (Fig. 2). The results showed that the number of mature eggs was affected by the female age for TD h (X 2 = 69.06, df = 7, P < 0.01) and TD m (X 2 = 51.84, df = 7, P < 0.01). The mean number was further compared among different age classes using ANOVA between two T. drosophilae groups. Interestingly, the number of mature eggs of female TD h and TD m increased until the TD m females were 96 hours old, whereas this increase persisted for an additional 48 hours for TD h . Thus, the maximum number of mature eggs of TD h (133.60 ± 6.87) was observed 144 h after emergence, while that of TD m (104.80 ± 11.44) was observed 96 h after emergence (t = 4.279, df = 7, P < 0.01; Fig. 2).
The stress resistance ability of T. drosophilae. To determine T. drosophilae stress resistance ability, TD h and TD m were treated with different environmental stresses, including starvation and high and low temperatures. Under food deprivation, the starved TD m wasps had a maximum life span of 192 hours, and half of the wasps survived 120 hours, whereas the TD h wasps had a maximum life span of 288 hours, and half of the wasps could survive at least 216 hours. The TD h wasps had a longer lifespan than the TD m wasps under starvation treatment (Log-rank test X 2 = 744.30, df = 1, P < 0.01) (Fig. 4).
To determine how different temperatures affect T. drosophilae survival, we placed TD h and TD m into incubators at 4 °C, 18 °C, 25 °C and 37 °C. The results showed that almost all TD h and TD m wasps survived well at lower temperatures (4 °C and 18 °C). However, the survival rates of TD h were higher than those of TD m at 25 °C or 37 °C (25 °C: Log-rank test X 2 = 23.09, df = 1, P < 0.01; 37 °C: Log-rank test X 2 = 14.79, df = 1, P < 0.01) (Fig. 5A,B).
T. drosophilae parasitism efficiency related to age. In order to evaluate the influence of T. drosophilae age on the parasitism rate, 1-, 5-, 10-, 15-, 20-, 25-, 30-and 40-day-old wasps were used to parasitize the hosts. The results showed that both TD h and TD m had an extremely high parasitism rate at all time points; however, a significant decrease in the parasitism rate was observed for the 40-day-old TD m parasitoids compared with the 40-day-old TD h parasitoids (t = 4.94, df = 4, P < 0.01) (Fig. 6A). In accordance with the results of our fecundity experiment (Table 1, Fig. 3C), the offspring female ratio of TD h was slightly higher than that of TD m ; significant differences were found between TD h and TD m at 5 days (t = 3.32, df = 4, P < 0.05), 10 days (t = 3.43, df = 4, P < 0.05) and 40 days (t = 6.87, df = 4, P < 0.01) after eclosion (Fig. 6B).

Discussion
Assessing the capacity of the T. drosophilae parasitoid to attack Drosophilidae species and enhancing its ability to adapt to extreme environments are two of the most important steps for the release of T. drosophilae as a biological control agent. In this study, we showed that local T. drosophilae was able to successfully attack D. melanogaster and D. hydei under laboratory conditions. A previous study reported that T. drosophilae offspring reared in large hosts such as D suzukii were larger than those reared in D. melanogaster 14 . Because D. hydei had a larger size than D.   There was a significant increase in the parasitism rate for the 40-day-old TD h parasitoids compared to TD m (t = 4.94, df = 4, P < 0.01) (B) The offspring female ratio of TD h and TD m at different ages. The offspring female ratio of TD h was slightly higher than that of TD m ; however, significant differences were found between TD h and TD m at 5 days (t = 3.32, df = 4, P < 0.05), 10 days (t = 3.43, df = 4, P < 0.05) and 40 days (t = 6.87, df = 4, P < 0.01) after eclosion. Values are the means ± SEM. Significant differences based on Student's t-test at P < 0.05 are indicated by asterisks. melanogaster, we compared the offspring size that emerged from the two different hosts, and found that the size of TD h was much larger than that of TD m .
Parasitoids reared in substitute hosts would help to increase the availability of biocontrol agents [20][21][22] . It has also been proven that large parasitoids of the same species have longer life spans, and large females produce approximately twice as many eggs as small females 18 . Thus, we evaluated the different parasitic characteristics of T. drosophilae reared in D. hydei and D. melanogaster pupae. Compared to T. drosophilae populations from California 14 , TD m females in our experiments had a similar number of mature eggs, and the egg load increased during the first four days. However, the number of TD h mature eggs was significantly higher than that of TD m and increased during the first six days. Fecundity is the maximum potential reproductive output of a parasitoid female over its lifetime and represents one of the major parasitic characteristics. Under the test conditions, the daily fecundity of TD m and TD h decreased with increasing female age, and when provided only with D. melanogaster pupae, the adult female TD m only survived for 10 days, which is shorter than the reported T. drosophilae lifespan 13 . However, TD h survived for 26 days and produced more female offspring than TD m . Another interesting phenomenon was that female TD h produced female offspring for 6 days after one mating event, compared with only 3 days for TD m. T. drosophilae has a sex-determination system in which males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid 23,24 . The results suggested that size differences of T. drosophilae between males or females from different hosts may influence sperm production or storage. In mosquitos, male size does correlate with total numbers of sperm within a male and the number transferred to females 25,26 .
Stress resistance ability is an important factor in evaluating parasitoid fitness and biocontrol efficacy in the field. A larger sized host may provide more nutrients that are vital for parasitoid development, which may be the reason why TD h survived longer than TD m in the starvation experiments. Additionally, our data indicated that both TD h and TD m wasps survived for a long time at lower temperatures (4 °C and 18 °C). The reason for this is that the lower temperature will slow the metabolism of the wasps and can even extend their lifespan 27 .
During the last 10 years, D. suzukii, also known spotted wing drosophila, has become widely distributed from Asia to Europe and North and South America [28][29][30][31] . D. suzukii has spread rapidly to become a serious pest that economically damages soft and thin-skinned fruits in the major fruit production areas [32][33][34] . Extensive applications of chemical insecticides will lead to a number of problems, such as pest resistance and chemical residue. Therefore, non-toxic and environmentally friendly biological control methods are urgently needed. Some entomopathogenic nematodes and fungi have been used to kill D. suzukii adults 30,35 . However, control of D. suzukii populations is very limited. So far, 50 hymenopteran parasitoids are reported to infect various drosophila species which belong to four families including two larval parasitoids, Braconidae and Eucoilidae, and two pupal parasitoids, Pteromalidae and Diapriidae 9 . Some studies have shown that most of these larval parasitoids cannot develop in D. suzukii because of its strong immune response 12 . T. drosophilae is a highly effective pupal parasitoid that can attack D. suzukii and has been proven to be a potential agent for biological control 14,36,37 . Our study demonstrates that D. hydei-reared parasitoids show more beneficial parasitic characteristics than D. melanogaster-reared parasitoids. D. hydei has a worldwide distribution and is easy to raise in large numbers. Therefore, rearing of T. drosophilae in D. hydei pupae could be a successful biocontrol strategy, especially for the aim of reducing D. suzukii infestation. Parasitism rate and offspring female ratio comparison. To compare the parasitism rate and offspring female ratio of TD h and TD m , D. melanogaster pupae were parasitized by 4-day-old TD h and TD m similar to a previous study 14 at a wasp/host ratio of 1:10 for 24 hours. This experiment was performed three times, and 200, 120 and 120 D. melanogaster host pupae were exposed to TD h and TD m . The same approach was applied to compare TD h and TD m at different ages. After eclosion, TD h and TD m adult females were maintained on apple juice wasp food at 25 °C in an incubator without hosts. Then, 1-, 5-, 10-, 15-, 20-, 25-, 30-and 40-day-old TD h and TD m female wasps were collected to parasitize D. melanogaster pupae after fully mating with young TD h and TD m males, respectively, for 24 hours. Three replicates were performed for the experiments, and 5 females and 30 host pupae were used in each experiment. After being infected, the host pupae were kept in a 25 °C incubator until the wasps emerged. The parasitism rate and offspring female ratio of the wasps were calculated using the following formulas: parasitism rate = (the number of hosts − the number of emerged flies)/the number of hosts; offspring female ratio = the number of female parasitoids/the number of total emerged parasitoids.

Methods
The fecundity and stress resistance ability of T. drosophilae. The egg load of a female parasitoid wasp. The newly emerged male and female wasps were collected and placed in plastic bottles containing apple juice wasp food without hosts. To compare the maximum egg load between TD h and TD m , ovaries of 12-, 24-, 48-, 72-, 96-, 144-, 192-and 240-h-old female T. drosophilae adults were dissected in 1 × PBS buffer, pH 7.4. Ten female wasps for each category were dissected, and the mature eggs were counted at each time point. An egg was considered mature based on criteria used in a previous study 14 : the chorion of a mature egg is smooth, thin and transparent, and the developing embryo is visible, while immature eggs lack these characteristics and are attached to each another.
The offspring of a single female wasp. To compare the offspring numbers of TD h and TD m , a fully mated female was allowed to parasitize 150 two-day old D. melanogaster pupae for 24 hours at 25 °C. Then, the host pupae were replaced by a new batch of 150 pupae the following day until the female adult died. The total number of offspring from single females was counted as the number of emerged wasps, including males and females. In total, 8 TD h and 8 TD m female wasps were used in this experiment, respectively.
Starvation and high and low temperature tolerances. One hundred newly emerged wasps of TD h and TD m (50 females, 50 males) were reared in an empty plastic bottle without any food at 18 °C for the starvation treatment. For the high and low temperature tolerance experiment, 100 newly emerged wasps of TD h and TD m (50 females, 50 males) were reared on apple juice wasp food in incubators at 4 °C, 18 °C, 25 °C and 37 °C. The survival rate (the number of surviving wasps/100) was calculated every 12 hours for the starvation treatment and daily for the high and low temperature tolerance analysis. Three replicates were performed for each experiment.
Data analysis and statistics. The effects of female age on the number of mature eggs were analysed using a generalized linear model (GLM) and the mean number of mature eggs in different age classes were further compared using analysis of variance (ANOVA). Log-rank tests (Mantel-Cox) were performed to analyse trends in the survival rate during the environmental stresses, i.e., starvation and high and low temperatures. Student's t-test was used to compare the body length or body width of parasitoids and hosts, the parasitism rate and offspring female ratio, as well as the fecundity of female parasitoid wasps. Statistical analyses were performed using GraphPad Prism version 7.0a (Graphpad Software, San Diego, CA) and SPSS software 25.0 (SPSS Inc., Chicago, IL). Error bars indicate the standard error of the mean (SEM), and all data sets are expressed as the mean ± SEM. Significant differences between groups were determined by the P-value and are marked with one asterisk for P < 0.05 and two asterisks for P < 0.01.