Performance of the parasitoid species Aphelinus asychis Walker (Hymenoptera: Aphelinidae), Aphidius ervi (Haliday) (Hymenoptera: Braconidae) and Diaeretiella rapae (McIntosh) (Hymenoptera: Braconidae), using Myzus persicae (Sulzer) (Hemiptera: Aphididae) as host

Three parasitoid species viz. Aphelinus asychis Walker (Hymenoptera: Aphelinidae), Aphidius ervi (Haliday) (Hymenoptera: Braconidae) and Diaeretiella rapae (McIntosh) (Hymenoptera: Braconidae) parasitizing the aphid species Myzus persicae (Sulzer) (Hemiptera: Aphididae) in mid-hills of north India were studied. At different locations and times of the year, the parasitization by A. asychis, A. ervi, and D. rapae ranged from 7.53 to 37.58, 4.26 to 80.45, and 74.25 to 80.48%, respectively. All the 3 parasitoids successfully completed their development on different nymphal instars of the aphid host and the total developmental duration of A. asychis, A. ervi, and D. rapae ranged 10.4–14.6, 24.2–29.6, and 10.2–15.2 days, respectively. It was significantly longer on the 1st nymphal instar of the host. The longevity of the female parasitoids was significantly longer than their counterparts. Differences in host age significantly influenced the longevity of female parasitoids and it was more on 1 to 2-day-old nymphs than that on 4 to 5-day-old nymphs. Fecundity and ovipositional periods of the parasitoids on younger (1–2 days old) host age group were considerably prolonged than on the older ages of the aphid. Average total fecundity of A. asychis and D. rapae was significantly higher when parasitizing 1–2-day-old nymphs. In A. asychis, host feeding behavior was also observed by a total host feeding of 89.2 aphids (1–2 days old) and 43.4 aphids (4–5 days old) during its life span. It is concluded that A. asychis, A. ervi, and D. rapae can be mass reared using M. persicae as host and can be utilized successfully in augmentative biological control program.


Background
Myzus persicae (Sulzer) (Hemiptera: Aphididae) feeds on over 500 host plants belonging to 40 unlike families and various chief agricultural crops cultivated in natural fields and protected environments (Byeon et al. 2011). It is an important pest of cucumber, capsicum, carnation, and gerbera grown under protected environment and oilseed brassicas under open field conditions (Sanchez et al. 2010 andGavkare 2014). Among various management practices, the use of chemical insecticides is common among farmers. However, the extensive use of these chemicals has ensued in several problems like insecticide resistance, resurgence of minor insect pests, and mortality of beneficial organisms. All these harmful consequences of injudicious use of pesticides have guided to the evolution of integrated pest management, wherein biological control is a very important component. Many biocontrol agents (parasitoids, predators, and entomopathogens) associated with M. persicae have been reported from distinct parts of the world (van Emden et al. 1969 andPerdikis et al. 2008).
In the present study, main biological parameters of 3 predominant parasitoid species namely A. asychis, A. ervi, and D. rapae were estimated when parasitizing the aphid species, M. persicae.  (30°, 86,004′ N, 77°, 17.303′ E) under humid subtropical mid-hills of Himachal Pradesh, India. Ten samples of aphid-infested leaves of C. annuum were collected from each of the 5 polyhouses selected at different locations of each district and also from Brassica oilseed crops from open fields. Fifty samples were collected from each district under polyhouse and open fields. The aphid infested leaves of C. annuum and apical twigs of Brassica oilseed plants were transferred to the laboratory in paper bags. The samples were examined under stereo-zoom microscope to count total numbers of healthy and mummified aphids. From these counts, percent parasitization was estimated, using the formula of Walton (1986)

Insect rearing
The stock culture of M. persicae was reared on C. annuum in an insectary at 25 ± 1°C, 70 ± 5% RH, and a L16:D8 photoperiod as per the method adopted by Kumar et al. (2019). Mummies of the 2 parasitoid species A. asychis and A. ervi were collected from the polyhouses at different locations, whereas the ones of D. rapae was obtained from Brassica oilseed farms in the locality. The stock cultures of parasitoids were reared on M. persicae maintained on C. annuum under caged conditions in small plastic containers (50 mm × 80 mm) as per the method of Sengonca et al. (2008). The identity of parasitoid was ascertained by comparing its morphological attributes with taxonomic keys (Takada 2002 andRakhshani et al. 2015) under stereo-zoom microscope in the laboratory. Fresh parasitoids individuals were used in experimentation. Plastic containers having mummified aphids were examined daily for the emergence of adult parasitoids. The emerged adults were separated by sex and the females were allowed to mate for 6 h in glass containers before initiating the experiments. The mated female wasps were transferred to plastic pots contained aphid colony. As a food source to the adult parasitoids, cotton swabs saturated with honey-water solution (30%) were provided.

Developmental duration
Potted plants of C. annuum infested with M. persicae (n = 150) were exposed to mated female parasitoids (n = 50) for oviposition. After 24 h, the plants were placed in the insectary for further development. After emergence, the parasitoids were collected and reared in separate cages on the respective day. One leaf of capsicum, with aphid nymphs (n = 10) of each instar, was offered to one mated female in separate glass vials (15 × 150 mm). The vials were examined at 24 h interval from oviposition till mummification.

Forewing width of the parasitoids
Forewings of males (n = 10) and females (n = 10) of the parasitoids viz. A. asychis and A. ervi and D. rapae were mounted on slides using a Canada balsam. The prepared slides were then placed in the oven for 4-5 days at 25°C . The slides were examined under a stereo-zoom microscope fitted with imaging software to measure the forewing widths.

Longevity of male and female parasitoids
After mating (for 6 h), the emerged adult parasitoids were separated into males and females. Twenty nymphs of each host age group were offered daily to each mated male (n = 10) and female (n = 10) (1 day old) in individual rearing glass jars throughout their life span. The male parasitoids were removed on 3rd day, while the females were placed daily into new glass jars with fresh aphids (from their stock culture) until death. Longevity of adult males and females of the parasitoids were estimated.
Age-specific fecundity and ovipositional periods of the parasitoids These parameters of female parasitoids were studied in 2 different age groups (1-2 and 4-5 days old) of M. persicae as per the method of Sengonca et al. (2008). Ten mated females of the parasitoid provided daily, until their death, to 20 nymphs of aphid of each host age group. After 12 h exposure, the female parasitoids were daily transferred into glass containers having fresh aphids. After 3-4 days, parasitized aphids were dissected and the number of parasitoid larvae was counted. Mummified aphids in the old jars were observed for adult emergence. In case of A. asychis, data on age-specific host feeding was also recorded by counting the number of aphids killed by the parasitoid (shrunken and decapacitated aphids). This behavior of host feeding was observed in case of the A. asychis females on both the host age groups of the aphid and the data were recorded throughout the entire life span of the parasitoid. The males did not show host feeding behavior like females. All the experiments were repeated 10 times.

Statistical analysis
Data collected were subjected to completely randomize one-way ANOVA at 5% level of significance following the method of Gomez and Gomez (1984). Data on the natural parasitism were subjected to arcsine transformation before ANOVA. The means that differed significantly were separated by least significant difference (LSD) at p = 0.05. Differences among the means of ovipositional, post ovipositional period, fecundity, and host feeding (in A. asychis) of the parasitoids on 2 different host age groups were compared using t test (p ≤ 0.05). Two-way ANOVA was used to analyze the effect of parasitoid species on mean longevity of male and female parasitoids and mean longevity of female parasitoids parasitizing different age groups of M. persicae.
Sampling for the parasitoids was carried out during December and March and high parasitization by A. ervi and  Similarly, in D. rapae, the developmental duration from oviposition to mummification was significantly longer in younger instars (first and second) than that in later instars (third and fourth) (LSD = 0.90, F 3, 16 = 5.44, p = 0.009). The developmental duration from mummification to adult emergence ranged from 4.60 ± 1.11 to 8.00 ± 0.88 days, and the differences amongst the instars were significant (LSD = 0.92, F 3, 16 = 22.18, p < 0.00001). Total developmental duration from oviposition to adult emergence was longer in 1st instar (15.20 ± 1.36 days) than in all other instars of the aphid (LSD = 1.24, F 3, 16 = 28.00, p < 0.00001).
The present investigation gave important information on biological attributes of native strains of A. asychis, A. ervi, and D. rapae using M. persicae as host. Selecting excellent bio-control agent is critical for any successful bio-control program. The choice of suitable aphid instar   can affect considerably the population development of both host and parasitoid. Developmental period is important for mass rearing of the parasitoids. From these results, it can be concluded that the developmental duration of the parasitoids affected with the host age as it was significantly longer on early instars than in the later ones. Prior studies showed that the competence of the parasitoid was influenced by the host age, hence, can be enhanced by choosing the most preferred stage for augmentative biological control (Li et al. 2006). Sengonca et al. (2008) also observed significantly shorter developmental duration of A. asychis on the older instars of Aphis gossypii (Glov.). The present findings also corroborate with Guigo et al. (2012) who reported that the developmental duration of D. rapae varied from 15.65 to 16.41 days on M. persicae reared on Brassica species. Host age is important for optimizing the mass rearing; therefore, these results are also consistent with the findings of Schirmer et al. (2008) who reported that under the same climatic conditions, A. asychis completed its development in a significantly shorter period when parasitizing 4-5-day-old rather than 1-2-day-old A. gossypii nymphs.

Forewing width of the emerging parasitoids
The mean forewing width of A. asychis, A. ervi, and D. rapae females was measured as 0.32, 0.86, and 0.67 mm, while for males, it was 0.25, 0.77, and 0.59 mm, respectively (Table 3). Based on two-way ANOVA (Table 6), the forewings of females were wider than those of the males (df = 1,59; F = 22.49; p = 1.58281E−05). Among the parasitoids, the forewing width of A. ervi was greater than that of A. asychis and D. rapae (df = 2,59; F = 347.33; p = 1.476E−31). Further, analysis showed no interaction between forewing width of parasitoid species and sex (df = 2,59; F = 0.28; p = 0.75). Tatsumi and Takada (2005) measured the forewing width of A. asychis and A. albipodus emerging from different aphids viz. A. gossypii, M. persicae, and M. euphorbiae and found that the females of both parasitoids had broader forewing width than the males. He and Wang (2008) observed that the females of A. ervi preferred to oviposit fertilized eggs in large hosts, while unfertilized eggs in the smaller host, which resulted in the large-sized females and smaller sized males, respectively. This behavior of females directly affects the forewing width of the parasitoids.
The longevity of A. asychis females was significantly longer (25.8 ± 3.96 days), whereas the longevity of A. ervi  and D. rapae females did not differ significantly, while parasitizing 1-2-day-old nymphs (Table 5) (LSD = 2.04, F 2, 27 = 162.14, p < 0.00001). In case of 4-5-day-old aphid hosts, significant differences in the longevity of female parasitoids were observed (LSD = 1.37, F 2, 27 = 296.88, p < 0.00001). Results of ANOVA (Table 6) showed a significant variation in mean longevity of the parasitoid between different age groups of the aphid (df = 1,59; F = 83.22; p = 1.59E−12), but showed no interaction between parasitoid species and mean longevity of parasitoids parasitizing different aphid age groups (df = 2,59; F = 0.91; p = 0.41). It is clear from these results that the mean longevity of female parasitoids was longer, while parasitizing nymphs of younger age groups than the older age groups. Nutritional and physiological attributes of the host significantly influence the development, mortality, longevity, and fertility of parasitoids (Roitberg et al. 2001). Sengonca et al. (2008) made similar observations on the longevity of female parasitoids on different host ages and reported that the mean longevity of A. asychis females, when parasitizing and host feeding on 1-2-day-old nymphs of A. gossypii, was 32.8 days, while on host age groups of 4-5-day-old nymphs and adults the obtained values were 25.2 and 24.2 days, respectively.
Ovipositional period and fecundity of the parasitoids A. asychis when parasitizing younger nymphs (1-2 day old) of M. persicae had a mean ovipositional period of 19.00 ± 0.45 days and total fecundity of 138.60 ± 7.40 eggs (Table 7). On an average, a single female of the parasitoid was capable of host feeding 4.0 ± 1.53 nymphs (1-2 days old) of the aphid. The parasitoid exhibited its host feeding behavior from the very 1st day of its emergence and continued until its death. During its entire average life span of 25.8 days as an adult, the parasitoid killed 89.2 aphids by its host feeding behavior. On 4-5day-old nymphs, the parasitoid killed 2.3 ± 0.65 nymphs per day by host feeding and a single parasitoid killed 43.4 nymphs in about 20 days of its adult life span. It is clear from the results that the ovipositional period (t = 19.88, p < 0.00001, df = 18), post ovipositional period (t = 3.21, p < 0.00001, df = 18), and daily fecundity (t = 3.80, p < 0.00001, df = 18) of the parasitoid were greater when parasitizing 1-2-day-old nymphs of M. persicae.
D. rapae when parasitizing 1-2-day-old nymphs of the M. persicae had a mean oviposition period of 8.40 ± 0.84 days and a total fecundity of 54.10 ± 3.17 eggs (Table 9). In case of 4-5-day-old nymphs, the ovipositional period of female parasitoids was 7.80 ± 1.05 days. The ovipositional period (t = 27.16, p < 0.00001, df =  The ovipositional period and fecundity of the parasitoids were less when parasitizing 4-to 5-day-old nymphs than the 1-to 2-day-old nymphs of M. persicae. Observations on the ovipositional behavior of A. asychis revealed that the older nymphs (4-5 days old) could defend themselves against ovipositing females of the parasitoid more effectively than the younger nymphs (1-2 days old). Therefore, it can be inferred that the younger instars of M. persicae were the most suitable age for oviposition to produce the fittest progeny of the parasitoid. Environmental factors like temperature, day length, and size of the adult female also affects the fecundity of the parasitoids (Stary 1988 andHagvar andHofsvang 1991). Sengonca et al. (2008) observed longer ovipositional period and high fecundity of A. asychis on younger nymphs (1-2 days old) of A. gossypii than those on the older nymphs (4-5 days old and adults) of the aphid. Similarly, host feeding by A. asychis was significantly more on the younger instars than on the older instars of M. persicae. Tatsumi and Takada (2005) found similar results on the cotton aphid, A. gossypii, where highly significant differences in host feeding behavior of A. asychis were found in distinct age groups (1-2 and 4-5 days old) of the aphid. In accordance to present results, net fecundity rates of D. rapae were 40.82 eggs on B. brassicae (Hosseini-Gharalari et al. 2003) and 238.7 eggs on M. persicae (Fukui and Takada 1988). The differences between the fecundity rates in previous and present studies could be attributed to the difference in the host's and parasitoid strain, host plants, temperature, and photoperiod conditions (Reed et al. 1992 andBernal andGonzalez 1993).

Conclusions
Selection of a suitable host age is critical for mass rearing protocols of parasitoids and their utilization in augmentative biological control programs. Biological parameters of the parasitoids were better when parasitized younger aphids. This information can be useful for standardizing the mass rearing and release protocols of these parasitoids to supplement the biological control programs of M. persicae in India.