Effect of Prey Species and Prey Densities on the Performance of Adult Coenosia attenuata

Simple Summary The predaceous fly Coenosia attenuata Stein has received attention because of its ability to effectively suppress a wide range of agricultural pests, such as fungus gnats, whiteflies and leaf miners. An effective level of control requires large numbers of C. attenuata to be available at low cost for release. Adult fungus gnats and drosophilids are now the main prey used to rear C. attenuata adults. However, previous studies showed C. attenuata fertility is lower when fed drosophilids compared to fungus gnats. The current study investigated the performance of C. attenuata adults when reared on different densities of adult Drosophila melanogaster Meigen or Bradysia impatiens (Johannsem). Results showed that the optimal prey density in the mass rearing of adult C. attenuata was 12–24 adult B. impatiens daily per predator. Additionally, C. attenuata adults suffered more wing damage, at some of the prey densities, when reared on D. melanogaster compared to B. impatiens. This information will be used to optimize rearing methods and decrease the cost of mass rearing in C. attenuata. Abstract Mass production of Coenosia attenuata Stein at low cost is very important for their use as a biological control agent. The present study reports the performance of C. attenuata adults when reared on Drosophila melanogaster Meigen or Bradysia impatiens (Johannsem). Different densities (6, 9, 15, 24 and 36 adults per predator) of D. melanogaster or (6, 12, 24, 36 and 48 adults per predator) of B. impatiens were used at 26 ± 1 °C, 14:10 (L:D) and 70 ± 5% RH. The results concluded that C. attenuata adults had higher fecundity, longer longevity and less wing damage when reared on B. impatiens adults compared to D. melanogaster adults. Additionally, C. attenuata adults demonstrated greater difficulty catching and carrying heavier D. melanogaster adults than lighter B. impatiens adults. In this case, 12 to 24 adults of B. impatiens daily per predator were considered optimal prey density in the mass rearing of adult C. attenuata.


Fungus Gnats
A colony of fungus gnats was initiated with about 400 B. impatiens adults captured from a greenhouse at Wuqing Experiment Station (Tianjin, China). Fungus gnats were reared using the method very similar to that reported by Zou et al. (2021) [39]. Modifications were made to simplify and improve the processes of rearing and collecting fungus gnats for use in bioassays. Briefly, 300 mL of black peat (Lvdimeijing Science and Technology Co., Ltd., Beijing, China) and 55 to 60 g of dry kidney bean powder were placed in an open plastic box (25.5 × 19 × 7.8 cm 3 ). The mix was then moistened with 250 mL of tap water and 0.2-cm thick layer of moist coir (Shanghai Galuku Agricultural Science and Technology Co., Ltd. Shanghai, China; desalted, EC = 0.5, family pack, common grade) was placed on the top of the mix. Then the open plastic box was placed in a tissue bag (50 × 35 cm 2 , 0.4-mm mesh openings). In this case, 400 to 500 newly emerged adult fungus gnats were placed in the tissue bag and closed with a binder clip. Fresh rearing medium was prepared daily and new cultures were set up daily.
The new fungus gnat adults deposited eggs on the media consisted of black peat, tap water and kidney bean powder. Newly hatched larvae fed on the media and adults emerged after 18-22 d. The colony was maintained in a laboratory incubator and held at 26 ± 1 • C, 14:10 (L:D) and 70 ± 5% RH.

Tiger-Fly
The C. attenuata used to establish a laboratory colony in this study were collected at Leizhuangzi flower farm of Tianjin, China. Adults were provided an oviposition tissue cage (60 × 55 × 50 cm 3 ), in which an open plastic box (29 × 20 × 7 cm 3 ) containing black peat, tap water, kidney bean powder and eggs of B. impatiens. A 0.5-cm thick layer of moist coir was placed on the top of the rearing media and used for oviposition. B. impatiens and D. melanogaster adults were supplied as prey daily. Five to 6 days later, the plastic boxes containing eggs of C. attenuata and rearing media were removed to another cage and second and third instar larvae of B. impatiens were added to the box to feed larvae of C. attenuata. Distilled water was added to the box when the media became dry. About 20 to 21 days later, adults of C. attenuata emerged. The colony was maintained in an artificial climate chamber and held at 26 ± 1 • C, 14:10 (L:D) and 70 ± 5% RH.

Performance of Tiger-Fly Adults Reared on Different Prey Species at Different Prey Densities
Five female/male pairs of newly emerged adults of C. attenuata (<24-h-old), in the first generation that originated from field-caught adults, were transferred into tissue cages (60 × 55 × 50 cm 3 ) containing an open 90-mm-diameter Petri dish containing of 0.7-cm thick layer of moist coir for oviposition. The moist coir was replenished after collecting egg daily. Two 110 cm strings were hung inside each cage and served as a perch for adult predators. In this case, 6, 9, 15, 24 and 36 adults of D. melanogaster and 6, 12, 24, 36 and 48 adults of B. impatiens (<24-h-old) were provided daily per predator adult. Prey were used only once and fresh prey were added daily. Five female/male pairs of C. attenuata adults (in one cage) were tested until death in each treatment and replicated 9 times. In total, 45 pairs of C. attenuata adults were tested in each treatment within a phytotron held at 26 ± 1 • C, 14:10 (L:D) and 70 ± 5% RH. Wing damage in C. attenuata adults was measured daily using a Mitutoyo 500-196-30 digital caliper (Mitutoyo, Kawasaki, Japan). Wing damage occurred along the long axis of wing. The extent of damage was calculated as a percentage using the length of damaged wing/the total length of wing × 100%. Both wings were assessed and the average was taken. The numbers of surviving and killed prey were recorded daily. Eggs deposited in the moist coir were collected and counted from each cage daily using a 00-sized paintbrush. They were then placed in 60-mm-diameter Petri dishes containing a single 55-mm-diameter filter paper, moistened with distilled water, sealed with Parafilm and inverted to keep the eggs moist. Hatch occurred ca. 6 days after oviposition and egg viability was calculated. Distilled water was sprayed to each tissue cage two times in the morning and afternoon per day. Tiger-flies were maintained in this manner until death.

Comparation of Body Weight and Body Length in C. attenuata, D. Melanogaster and B. Impatiens
The adults of C. attenuata caught prey in flight. So carrying prey with different body weights may cause different levels of wing damage for adults of C. attenuata. Adults of C. attenuata (<24-h-old) and D. melanogaster (<24-h-old) were placed in tissue bags (40 × 30 cm 2 ) and held in a Siemens BCD-501W fridge (Siemens, Nanjing, China) at −20 • C for 2 min before taking body length and weight measurements. Adults of B. impatiens (<24-h-old) were handled in the same way with C. attenuata and D. melanogaster and held at −20 • C for 3 min before using. Adult body length was measured (in resting position) from the apex of the head to the wing tip for C. attenuata and D. melanogaster. For B. impatiens, body length was measured (in resting position) from the apex of the head to the abdomen tip. Body length measurements were made using a Mitutoyo 500-196-30 digital caliper (Mitutoyo, Kawasaki, Japan). Weight measurements were made using a Sartorius BP 211D (Sartorius AG, Göttingen, Germany) balance. In this case, 30 females and 30 males were measured for each species.

Statistical Analyses
One-way ANOVA with subsequent Tukey's HSD test at α = 0.05 was used to compare the proportion of damaged wing, number of prey killed, preovipositional period, total fecundity between different prey densities, body weight and body length between insect species. To avoid possible mistakes due to multiple testing of the same data base, the p-values were Bonferroni corrected. Two sample t-tests for means were used to compare proportion of damaged wing, preovipositional period and total fecundity between prey species. These comparisons were carried out on day 4 and the last day. The proportion of eggs successfully hatched was compared between treatments by the Chi-square test at α = 0.01. All the statistical tests were carried out using SAS version 9.4.

Comparison of Wing Damage of C. attenuata When Reared on Different Prey at Different Prey Densities
The mean proportion of damaged wings of C. attenuata females fed on D. melanogaster and males fed on B. impatiens or D. melanogaster did not differ significantly between prey densities at early ages (day 4) (F 4, 220 = 1.27, p = 0.2826; F 4, 220 = 1.55, p = 0.1886; F 4, 220 = 2.4, p = 0.0513, respectively) ( Figure 1B-D). However, C. attenuata females fed 48 adults of B. impatiens daily lost more wings compared with those fed 12 adults of B. impatiens daily on day 4 (F 1, 88 = 9.39, p = 0.0029, Bonferroni-corrected p = 0.005) ( Figure 1A). The wing damage increased with age in every case, but at different rates depending on prey species and density. When fed with B. impatiens, C. attenuata females showed significant differences at late age (31 days) between prey densities of 12 and 24 (F 1, 88 = 13.15, p = 0.0004, Bonferroni-corrected p = 0.005), or between prey densities of 36 and 48 (F 1, 88 = 10.16, p = 0.002, Bonferroni-corrected p = 0.005) ( Figure 1A). For C. attenuata males, there were no significant differences between prey densities at late age (19 days) (F 4, 220 = 0.63, p = 0.6425) ( Figure 1B). When fed with D. melanogaster, both females and males of C. coenosia did not show significantly different wing damage between prey densities at later age (8 days) (F 4, 220 = 2.08, p = 0.0848; F 4, 220 = 2.07, p = 0.0856, respectively) ( Figure 1C,D). Females and males of C. attenuata had much shorter longevity when fed D. melanogaster adults compared to those fed B. impatiens adults (maximum age of 12 for D. melanogaster and of 46 for B. impatiens in females; maximum age of 10 for D. melanogaster and of 35 for B. impatiens in males). Mean proportion of wing damage in flies fed with B. impatiens at age 8 ranged from 3.25 to 22.42% in females and from 4.68 to 9.58% in males, while in flies fed with D. melanogaster it ranged from 12.28 to 21.56% in females and from 9.16 to 19.77% in males ( Figure 1).
C. attenuata females fed 6 adults of D. melanogaster daily lost significantly more wings than those fed 6 adults of B. impatiens daily on day 4 (t = −2.935, df = 88, p = 0.0043) ( Figure 1A,C). However, there was no significant difference in wing damage for C. attenuata females fed 24 adults of D. melanogaster compared with those fed 24 adults of B. impatiens daily, or for C. attenuata females fed 36 adults of D. melanogaster compared with those fed 36 adults of B. impatiens daily on day 4 (t = −1.934, df = 88, p = 0.0564; t = −0.447, df = 88, p = 0.6561, respectively) ( Figure 1A,C). C. attenuata females lost significantly more wings when fed adults of D. melanogaster daily compared to B. impatiens at the prey density of 6 and 36 on day 8 (t = −4.556, df = 88, p < 0.0001; t = 2.803, df = 88, p = 0.0062, respectively). However, there was no significant difference in wing damage for C. attenuata females when fed 24 adults of D. melanogaster compared with those fed 24 adults of B. impatiens on day 8 (t = −0.874, df = 88, p = 0.3847) ( Figure 1A,C).
There was no significant difference in wing damage for C. attenuata males fed 6 adults of D. melanogaster compared with those fed 6 adults of B. impatiens, or for C. attenuata  C. attenuata adults killed 3.17 to 4.52 D. melanogaster adults when fed 6 prey adults daily per predator adult ( Figure 3). The number of prey killed by C. attenuata adults fluctuated when fed 9, 15, 24 and 36 prey daily per predator adult. The numbers of prey killed daily per predator were 4.27 to 6.54, 4.37 to 7.29, 7.57 to 11.98 and 9.58 to 15.55 for C. attenuata adults fed 9, 15, 24 and 36 prey daily per predator adult, respectively. The number of prey killed per predator daily decreased in the last few days with the increase of age and the proportion of broken wings for C. attenuata adults in all treatments. The number of D. melanogaster adults killed daily per predator adult differed significantly with prey densities of 6 vs. 9 (F 1, 16 = 10.64, p = 0.0049, Bonferroni-corrected p = 0.005), 9 vs. 36 (F 1, 16 = 17.21, p = 0.0008, Bonferroni-corrected p = 0.005) and with the prey densities of 6 vs. 15, 6 vs. 24, 6 vs. 36 and 15 vs. 36 (all p < 0.0001) at early ages (day 4). No significant differences were found in the other 4 comparisons at early ages (day 4) (Figure 3). Additionally, no significant differences were found in all 10 comparisons at later age (day 8) (Figure 3).

Preovipositional Period of C. attenuata Female Reared on Different Prey at Different Prey Densities
There were no significant differences in preovipositional period in any of the treatments of C. attenuata females when fed 6,9,15,24 (Figure 4). The preovipositional periods were significantly longer for C. attenuata females fed 24 adults of D. melanogaster prey than of C. attenuata females fed 24 adults of B. impatiens prey. These differences, although statistically significant, were small (t = −2.132, df = 16, p = 0.0489) (Figure 4).

Comparation of Body Weight and Body Length in C. attenuata, D. melanogaster and B. impatiens
Adult females of C. attenuata were significantly longer than those of D. melanogaster and B. impatiens (F 2, 87 = 1335.80, p < 0.0001) ( Table 1). Adult females of D. melanogaster were significantly longer than those of B. impatiens (F 2, 87 = 1335.80, p < 0.0001). Similar, adult males of C. attenuata were significantly longer than those of D. melanogaster and B. impatiens (F 2, 87 = 2101.27, p < 0.0001) and the body length was significantly longer for adult males of D. melanogaster than for adult males of B. impatiens (F 2, 87 = 2101.27, p < 0.0001). Adult females of C. attenuata were significantly heavier than those of D. melanogaster and B. impatiens and there was a significant difference in the body weight of adult females between D. melanogaster and B. impatiens (F 2, 87 = 2188.57, p < 0.0001). Similar, adult males of C. attenuata were significantly heavier than those of D. melanogaster and B. impatiens and there was a significant difference in the body weight of adult males between D. melanogaster and B. impatiens (F 2, 87 = 1164.34, p < 0.0001) ( Table 1).

Discussion
The flight of C. attenuata individuals was affected by environmental factors and was increased in response to increases in the number of prey flights [37]. Bonsignore (2016) found that predatory flights of adult C. attenuata comprised a small percentage (ca. 6%) of the total flights, with a predation success rate of 61% [37]. In our study, the mean proportion of damaged wings of C. attenuata females when fed 6, 12, 24 and 36 adults of B. impatiens daily per predator adult was increased in response to increases in the number of prey densities. However, the mean proportion of wing damage in C. attenuata females was lower for prey densities of 48 adults of B. impatiens than for prey densities of 36. The high density of 48 adults of B. impatiens probably increased the predation success rate and thereby decreased the mean proportion of damaged wings of C. attenuata female although the tiger-fly is regarded to have predation instinct [40,41]. Damaged wings of C. attenuata males fed on B. impatiens continued to increase with an increase in age of C. attenuata. However, the mean proportions of damaged wings in C. attenuata males were not consistent with those of females. Prey density did not cause significant effect on wing damage for C. attenuata males, which suggests prey density was not the only factor affecting wing damage in C. attenuata males. Being attacked by female C. attenuata and attempting to mate with female C. attenuata could also influence wing damage. Additionally, male adults required less prey compared to female adults, which means low prey densities could increase the predation success rate and thereby decreased the proportion of damaged wings of C. attenuata males. According to the damaged wings and longevity of C. attenuata adults, prey densities of 12 to 24 should be optimal density for mass rearing of adult C. attenuata.
Prey density of vinegar fly did not cause a significant effect on the mean proportion of damaged wings in both female and male adults of C. attenuata. It seems reasonable to conclude that the short lifespan of the tiger-fly was too short to manifest an effect of D. melanogaster prey density on wing damage of C. attenuata adults. C. attenuata adults fed D. melanogaster prey daily lost more wings compared to those fed B. impatiens prey at the same age for some prey density. This may be related to the increased difficulty in carrying heavier D. melanogaster adults than B. impatiens adults.
Bonsignore (2016) sorted adult C. attenuata flights into three groups, movement flights, territory defense flights and predatory flights in greenhouse [37]. However, we observed that there should be another type of flight, escape flights in cage. We hypothesize that adult C. attenuata want to escape from the cage when encountering high density of prey, resulting in less wing damage. Escaping from the environment with high prey density may be a self-protection response for adult C. attenuata.
We found that females lived longer than males, as reported by Kühne et al. (1997) [23]. However, these authors record 38 days and 33 days as the maximum female and male longevity, respectively, under laboratory conditions (25 • C and 50-60% RH) and an estimated longevity of eight weeks under greenhouse conditions. Predators fed B. impatiens adults in our study lived 46 days and 35 days as the maximum female and male longevity, respectively, possibly because they had a better food supply. However, in our study, predators fed adult D. melanogaster flies lived only 12 days and 10 days as the maximum female and male longevity, respectively. We speculate that C. attenuata adults were able to more easily capture lighter B. impatiens adults than heavier adult D. melanogaster. Additionally, adult C. attenuata were able to attack adult B. impatiens on the bottom of cage when they could only jump or crawl because of damaged wings. However, it is difficult for C. attenuata with damaged wings to capture adult D. melanogaster.
Female adult C. attenuata were found to exhibit a type I functional response to adult sciarid flies, which was conducted in glass vials 8 cm long and 8 cm in diameter at 25 • C at 60-80% RH, with a 16L:8D photoperiod. Sciarids were consumed in significantly different numbers at densities from 5 to 20 individuals (the number of killed flies changed from 2.90 to 8.4, respectively). However, increasing prey availability beyond 20 individuals resulted in no substantial increase in predation [30]. However, female adult C. attenuata were found to exhibit a type II functional response to adult D. melanogaster flies, which was conducted in Plexiglas cages with a dimension of 25 by 25 by 25 cm at 30 • C at 65 ± 5% RH, with a 12L:12D photoperiod. D. melanogaster flies were consumed in significantly different numbers at densities from 5 to 55 individuals (the number of attacked flies changed from 3.50 to 5.67, respectively) [42]. Kühne (2000) states that each adult C. attenuata needs either 1.5 adults of D. melanogaster or 6.9 adults of B. impatiens per day [25]. We did not analyze the functional response to adult B. impatiens or D. melanogaster flies because more than one factor affected functional response, such as intraspecific competition and predation. The number of killed prey in our study was more than those mentioned above, which was probably caused by intraspecific competition and predation instinct resulting from cage and space differences. The flight ability of adult B. impatiens is weak and often some of them stayed on the bottom of cage which made it more convenient for adult C. attenuata without flight ability, because of damaged wings, to catch the adult B. impatiens. In contrast, the flight ability of adult D. melanogaster is strong and it is more difficult for adult C. attenuata with weakened flight ability to catch adult D. melanogaster, although adult C. attenuata has been proved to be more efficient in information sampling and processing than adult D. melanogaster [43,44].
The preoviposition period of C. attenuata is approximately 4 days [23]. Our reports showed similar preoviposition periods when fed adults of B. impatiens with 3.89-4.67 days and adults of D. melanogaster with 4.22-5.22 days. Prey density, prey species and damaged wings did not cause negative effects on the preoviposition period of C. attenuata. Sanderson et al. (2009) found the tiger-flies laid more eggs with fungus gnat prey than shore fly prey [27]. We found that the tiger-flies laid much more eggs with fungus gnat prey than vinegar fly prey. However, the total fecundity per female of C. attenuata did not continue to increase with an increase in prey density. Shorter life span probably cause the lower fecundity for C. attenuata female when fed adult D. melanogaster compared to adult B. impatiens. Martins et al. (2015) presented an optimized method for mass rearing C. attenuata with fungus gnats and Drosophilids as prey, where the number of adults that emerged per parental pair ranged from 1.8 to 9.0 (= per pair progeny production, or the number of adult offspring that emerged in each cage divided by the number of parental pairs) [38]. In our study, the number of adult offspring that emerged ranged from 4.96 to 7.64 and 21.16 to 39.27 at least for per parental pair when fed adult D. melanogaster and B. impatiens, respectively according to survival rates of larvae, percentages of pupation and adult emergence in our previous reports [17,39]. The proportion of eggs that successfully hatched was much higher for C. attenuata adults fed B. impatiens adults than for those fed D. melanogaster adults at the same prey densities. Longer longevity in male C. attenuata and lighter body weight in B. impatiens prey correlated to increased proportion of eggs that successfully hatched in C. attenuata.
Predation by adult C. attenuata is rapid, and adults take off as soon as they observe their prey in flight, although they do not know the absolute size of the potential prey prior to the flight [45]. One important physical factor affecting predator responses is prey size [46]. Body length and body weight of adult C. attenuata, D. melanogaster and B. impatiens were analyzed in our study to better understand the complexity of predation. We report body length and body weight of adult C. attenuata from Tianjin to be similar to those reported by us previously [17,39] and to those reported in Uruguay where the C. attenuata flies measured approximately 2.5-5.00 mm in length [47]. Body weight of adult D. melanogaster measured in this study is similar to those reported by Chen et al. (2019) [48]. Body length of adult B. impatiens analyzed in this study is similar to those reported by Wilkinson and Daugherty (1970) [49]. Obviously, it is more difficult for adult C. attenuata to catch and carry heavier adult D. melanogaster than lighter adult B. impatiens. Most importantly, it is more difficult for male adult C. attenuata to catch and carry female adult D. melanogaster, that are 74.15% weight of male adult of C. attenuata than to catch adult female B. impatiens that only weigh 28.57% of their weight.
In our study, we demonstrated that adult B. impatiens was an optimal prey in the mass rearing of adult C. attenuata although rearing drosophilids is quick, easy and not particularly expensive. In addition, we provide evidence that damage to wings of adult C. attenuata when fed adult D. melanogaster vs. B. impatiens is an important consideration for prey selection. We conclude a prey density of 12-24 adult fungus gnats daily per adult predator as optimal for mass rearing of adult C. attenuata. Rearing cost, nutritional difference, digestion efficiency, chemical, morphological and behavioral defense mechanisms of a prey will be explored in future studies.

Conclusions
We present the first report of wing damage for C. attenuata adults when reared on different prey. The results indicate that C. attenuata adults had higher fecundity, longer longevity and generally less wing damage when reared on B. impatiens compared to D. melanogaster. Lighter body weight and weaker flight ability in adult B. impatiens prey likely contributed to prolonged longevity and increased fecundity in adult C. attenuata. In this case, 12 to 24 adults of B. impatiens daily per predator were considered optimal prey density in the mass rearing of adult C. attenuata adult.