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Effect of age dependent cold storage of factitious host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) for their continuous production and Trichogramma chilonis (Ishii) (Hymenoptera: Trichogrammatidae) rearing
Journal of Asia-Pacific Entomology 20 (2017) 928–934
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Effect of age dependent cold storage of factitious host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) for their continuous production and Trichogramma chilonis (Ishii) (Hymenoptera: Trichogrammatidae) rearing
MARK
Enakshi Ghosha,b,⁎, Chandish R. Ballala a b
ICAR-National Bureau of Agricultural Insect Resources, Post Bag No. 2491, H.A. Farm Post, Bellary Road, Bangalore–24, India Department of Biotechnology, Center for Post Graduate Studies, Jain University, Jayanagar, Bengaluru 560011, Karnataka, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichogramma chilonis Parasitism Age of host eggs UV irradiation
In Asia Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) is widely used as a factitious host for the mass rearing of several bio-control agents viz. chrysopids, braconids, trichogrammatids and anthocorids. The age dependent cold storage amenability of C. cephalonica eggs was evaluated. At 5 ± 1 °C with a 24 h of scotophase, 0 to 22 h old eggs of C. cephalonica could be stored for up to 5 days resulting in 83 to 94% hatching. Eggs of C. cephalonica are UV irradiated for mass rearing of T. chilonis. Four, eight, sixteen and twenty four-hour-old C. cephalonica eggs were stored at 5 ± 1 °C for different durations prior to and post-UV irradiation and tested for their amenability to parasitism by T. chilonis. Adult females of T. chilonis could parasitise 67–78% of eggs stored for up to 10 days prior to UV treatment, while 74–78.2% parasitism was recorded in the case of eggs stored for 10 days post-UV. The host eggs of all age groups stored for 20 days (10 days pre-UV and 10 days post-UV) were effectively parasitised providing 83.8–88.4% parasitism. Eight and sixteen hour-old old host eggs stored for 30 days (15 days of pre-UV and 15 days of post-UV) recorded 70–80% parasitism. The host egg storage technique developed through this study would be beneficial for insectaries to stock the host eggs, which can lead to uninterrupted production and supply of ‘Tricho-cards’, especially during seasonal high demand.
Introduction Addressing pest problems by adopting integrated pest management strategy requires mass multiplication of several natural enemies. Trichogrammatids are important egg parasitoids used against lepidopteran pests- especially since 1926 when a suitable mass rearing strategy was developed (Flanders, 1929). The biology of Trichogramma spp. has been studied extensively (Nagaraja, 1973; Strand, 1986).The egg period of Trichogrammatids lasts 16 to 24 h, larval period 2 to 3 days, pre-pupal period 2 days and pupal period 2 to 3 days. The total development is completed in 8 to 10 days during summer and 9 to 12 days during winter. Different species of Trichogramma proved to be effective against different pests infesting various crops in different ecosystems (Li et al., 1994). Among all the identified species, Trichogramma chilonis (Ishii) is extensively used in India, China, Japan, Nepal, Pakistan, Taiwan, Reunion Island, Kenya, Australia and South Africa due to its wide host acceptability and high survival capacity (Jalali et al., 2006). Trichogramma production has gained a lot of attention recently among government and private insectaries. One of the major obstacles preventing the successful production and utilization of this ⁎
endo-parasitoid is the availability and constant supply of host eggs. Majority of the laboratories in Asia mass produce Trichogramma species on factitious host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). Sitotroga cerealella (Oliver) (Lepidoptera: Gelechiidae) and Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) are used in other countries (Consoli et al., 1999). Corcyra cephalonica eggs are subjected to UV irradiation to arrest the growth of the developing host embryo prior to exposing them to Trichogramma adults for parasitism in order to prevent hatching of larvae from these eggs (Hamed et al., 2009). This further prevents predation of the parasitized eggs by the freshly hatched C. cephalonica larvae, while ensuring nutrient availability for developing Trichogramma embryo. A recent study has proved that two species of Trichogramma could also be reared using eggs of eri silk worm Samia cynthia (Lalitha et al., 2013). Corcyra cephalonica can also be used to rear other natural enemies like green lacewings, braconid parasitoids, anthocorid predators and entomopathogenic nematodes (Li et al., 1994; Jalali et al., 2003; Ballal and Yamada, 2016). The short shelf-life of C. cephalonica eggs is a limitation for production and supply of Trichogrammatids. Development of efficient storage technique for fresh eggs is important for continuous host rearing, while that for UV irradiated C.
Corresponding author at: ICAR-National Bureau of Agricultural Insect Resources, Post Bag No. 2491, H.A. Farm Post, Bellary Road, Bangalore–24, India. E-mail address: [email protected] (E. Ghosh).
http://dx.doi.org/10.1016/j.aspen.2017.06.013 Received 12 February 2017; Received in revised form 17 June 2017; Accepted 26 June 2017 Available online 29 June 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Journal of Asia-Pacific Entomology 20 (2017) 928–934
E. Ghosh, C.R. Ballal
70 ± 10% of RH. Five replications were maintained for each treatment and control. The stored eggs were UV irradiated for 60 min. UV treated cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in a walk-in chamber set at 26 ± 1 °C with RH 70 ± 5%. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
cephalonica eggs is important for Trichogramma rearing. This will enable the availability of Trichogrammatids during pest outbreaks and satisfy seasonal demands. Storage of UV irradiated C. cephalonica eggs at − 6 ± 1 °C for > 50 days adversely affected the fitness attributes of Trichogramma spp. reared on them (Das and Ram, 1983), while storage at 0 and 9 °C for 3and 15 days, respectively, provided positive results for Trichogramma production (Balasubramanian et al., 1991). Vacuum packed UV irradiated C. cephalonica eggs stored at 8 °C for 7 to 42 days resulted in > 70% parasitism by T. chilonis (Jalali et al., 2007). Previous studies have not evaluated the effect of the age of live and UV irradiated eggs on their amenability to storage. Our aim was to extend the shelf-life of fresh C. cephalonica eggs for continuous host rearing and UV irradiated eggs for continuous production of Trichogrammatids by identifying the right combination of the egg age and storage durations.
Post-UV storage (Fig. 1.Bpost) Corcyra cephalonica eggs of 0–2 h were UV irradiated for 60 min and then incubated at room temperature for 4, 8, 16 and 24 h to form different experimental age groups: 4–6 h, 8–10 h, 16–18 h and 24–26 (referred same as pre-UV storage). From each age group, 100 paper cards (measuring 2 × 1 cm) which held one hundred eggs, after which they were placed in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH. From the storage unit, five cards from each age group were removed at 24 h intervals and this was continued till 20 days. One un-stored batch for each age group was maintained as control. Five replications were maintained for each treatment and control. The post-UV stored cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were placed in a walk-in chamber set at 26 ± 1 °C, RH 70 ± 5% with L:D-12:12. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
Materials and methods Maintenance of the cultures Corcyra cephalonica (National Accession No: NBAII-MP-PYR-01) is reared continuously in the live insect repository of ICAR-NBAIR, Bangalore. These are reared on a medium comprising of insecticide free sorghum, groundnut seeds, yeast and streptomycin sulphate (Lalitha and Ballal, 2015). The eggs of C. cephalonica are exposed to UV irradiation by placing the eggs at a distance of 2 ft from the UV tube light for 45 min. These UV irradiated eggs are glued on thick paper cards measuring 12 × 10 cm. These cards are exposed to T. chilonis (continuously lab reared) adults in the ratio of 40:1 (eggs:female) until mortality. The cultures were maintained at 25 ± 4 °C, 65 ± 10% RH with L:D-12:12.
Combined pre and post-UV storage (Fig. 1.Bcombined) Corcyra cephalonica eggs of 0–2 h old were incubated at room temperature for 4, 8, 16 and 24 h to arrive at different experimental age groups: 4–6 h, 8–10 h, 16–18 h and 24–26 h (referred same as pre-UV storage). Forty four sets of hundred eggs pasted on thick paper card (measuring 2 × 1 cm) from each age group were stored in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH for different durations (2, 4, 6, 8 and 10 days) and UV irradiated for 60 min and further placed at 5 ± 1 °C. These UV irradiated stored cards were removed at 24 h intervals (0 days to 10th day) and exposed to healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in walk-in chamber set at (26 ± 1 °C, RH 70 ± 1% with L:D-12:12) and checked for percent parasitization.
Experimental setup The overall protocol of the experimental set up for the four experiments is depicted in Fig. 1. Storage of C. cephalonica eggs for continuous host rearing (Fig. 1.A) Corcyra cephalonica eggs of 0–2 h old were collected from the rearing units and incubated at room temperature (25 ± 3 °C) (for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 h) to arrive at the following age groups: 0–2 h, 2–4 h, 4–6 h, 6–8 h, 8–10 h, 10–12 h, 12–14 h, 14–16 h, 16–18 h, 18–20 h, 20–22 h, 22–24 h. Approximately one 100 eggs from each age group were glued on a thick paper card (measuring 2 × 1 cm) and kept in a BOD (biological oxygen demand) (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C, 65 ± 10% RH and L:D-0:24 for different experimental durations viz. 5 days, 10 days, 15 days, after which they were placed in a walk-in chamber set at 26 ± 1 °C with 70 ± 5% RH. Each treatment was replicated five times. The batches were regularly checked for hatching, incubation period and percent hatching were recorded.
Combined pre and post-UV storage (beyond 20 days) An additional experiment was conducted to test if further combined pre- and post-UV storage beyond 20 days would be feasible for utilization in Trichogramma production. Egg cards of each age group, with 100 eggs on each card were stored at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH (Remi cooling incubator - Cl-12S model) for 10 to 20 days (e.g. 10 days pre-UV + 10 days post-UV, 11 days pre-UV + 11 days post-UV up-to 20 days pre-UV + 20 days post-UV). Four cards from each age group were removed at 24 h intervals and subjected to UV irradiation for 60 min. Post-UV irradiation, the stored UV treated cards of different age groups were further stored for 10 to 20 days. Four cards were removed from the storage unit at every 24 h interval for up-to 20 days. These cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in a walk-in chamber set at 26 ± 1 °C with RH 70 ± 5%. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
Storage of C. cephalonica eggs for T. chilonis production Pre-UV storage (Fig. 1.Bpre) Corcyra cephalonica eggs of 0–2 h old were incubated at room temperature for 4, 8, 16 and 24 h to form four different experimental age groups viz. 4–6 h (referred as 4 h), 8–10 h (referred as 8 h), 16–18 h (referred as 16 h) and 24–26 h (referred as 24 h). From each age group, 100 paper cards (measuring 2 × 1 cm) each holding hundred eggs were placed in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH. From the storage unit, five cards from each age group were removed at 24 h intervals and this was continued till 20 days. One un-stored batch for each age group was maintained as control in a walk-in chamber set at 26 ± 2 °C with
Statistical analysis Treatment effects on percent hatching and percent parasitism were evaluated using one way analysis of variance (ANOVA) (SPSS 16.0). When ANOVA was significant, comparison of relevant means were made using Turkey's post hoc test at a significant level of 5%. Data recorded on percent hatching of host eggs were subjected to angular 929
Journal of Asia-Pacific Entomology 20 (2017) 928–934
E. Ghosh, C.R. Ballal
Fig. 1. Overall outline of the experimental protocol followed.
gradually decreased with an increase in storage period. Four and 24 h old eggs were significantly inferior to control (F = 12.7; df = 16, 68; P ≤ 0.0001) (F = 160; df = 16, 68; P ≤ 0.0001). Percent parasitism of 8 h old eggs 16 h old eggs stored for 4 days were statistically on par with the respective control batches (Fig. 3) (F = 22.19; df = 16, 68; P ≤ 0.0001) (F = 170.9; df = 16, 68; P ≤ 0.0001). > 70% parasitism was recorded in 4,8,16 and 24 h old eggs after storage at 5 °C for a duration of 11, 9, 9 and 10 days, respectively. Based on two way ANOVA, among the experimental host age groups, 4 h old eggs were found to be statistically superior (F = 4.73; df = 3, 13; P ≤ 0.0001).
transformation before statistical analysis. Percent parasitism data were also subjected to two way analysis of variance. The aim was to investigate the effect of main treatments (i.e., age of the host eggs and the duration of storage) on parasitoid performance and also to check if an interaction existed between them. Where significant values were obtained, the results were interpreted at 5% level of significance based on LSD test. Results Effect of low temperature storage on hatching of C. cephalonica eggs
Parasitism of C. cephalonica eggs stored post-UV irradiation The current laboratory experiments indicated that live C. cephalonica eggs stored at 5 °C for 10 and 15 days resulted in 0 to 20% hatching. Hence only the data on percent hatching from eggs (belonging to different age groups) when stored for 5 days at 5 °C were subjected to statistical analysis. Eggs belonging to all other age groups (except 22 to 24 h) could be stored for 5 days resulting in 83.4–94.2% hatching (F = 43.92; df = 12,52; P ≤ 0.0001) (Fig. 2). The incubation period of stored eggs ranged between 9.2 and 10.2 days, while in control it was 6 to 7 days. The total incubation period (including the storage period) ranged between 13.2 and 14.6 days and thus, the incubation period could be staggered by one week.
It was observed that parasitism rates of 8 and 16 h old eggs after 4 and 5 days of storage respectively, were statistically on par with their respective control (Fig. 4) (F = 22.16; df = 16, 68; P ≤ 0.0001) (F = 94.7; df = 16, 68; P ≤ 0.0001). In case of 4 and 24 h old eggs the parasitism rates were significantly reduced after 1 or 2 days of storage in comparison to their control (F = 305.8; df = 16, 68; P ≤ 0.0001) (F = 95.8; df = 16, 68; P ≤ 0.0001). > 70% parasitism could be recorded in 4, 8, 16 and 24 h old eggs after storage at 5 °C for up-to 10, 10, 11 and 9 days, respectively (Fig. 4). Parasitism of C. cephalonica eggs after pre- plus post-UV combined storage
Parasitism of C. cephalonica eggs stored prior to UV irradiation Corcyra cephalonica eggs of different ages stored at 5 °C for a total of 20 days (10 days of pre- plus 10 days of post-UV storage) when exposed
Parasitism rate of un-stored host eggs was high while the rate 930
Journal of Asia-Pacific Entomology 20 (2017) 928–934
E. Ghosh, C.R. Ballal
Fig. 2. Percent hatching (mean ± SE) of different ages of Corcyra eggs after storage at 5 °C (L:D-0:24, RH-70 ± 10%) for 5 days.
120 100
93.2a
92ab
87.2ab
91.2ab 85b
Percent hatching
83.4b
94.2a 83.4b
90.4ab 88.4ab 88.8ab
84.2b
80 60 40 20 0.8c .
0 C
0-2
2-4
4-6
6-8 8-10 10-12 12-14 14-16 16-18 18-20 20-22 22-24 Age of Corcyra eggs (hours)
most suitable age amenable for storage irrespective of storage duration was found out to be 8 h (Table 1).
to T. chilonis, the percent parasitism values of 88.4, 84.8, 87 and 83.8, respectively were recorded, all being statistically on par (F = 1.24; df = 3,16; P = 0.32) (Fig. 5).
Discussion Combined pre- and post-UV storage (beyond 20 days) A successful bio-control strategy utilizing endo-parasitoids like Trichogramma is dependent on the availability of the parasitoids in large numbers for field releases. Storing the host eggs by staggering their development to improve continuous host egg production and also improving their viability for Trichogramma rearing can ultimately help synchronize their mass production during high seasonal demands. The current study revealed that 0–22 h old C. cephalonica eggs can be safely stored at 5 °C for 5 days, and incubation period could be staggered by a week, with hatching rate maintained between 83 and 94%. However,
To achieve a minimum of 70% parasitism, 8 and 16 h old eggs could be stored for up to 30 days (15 days pre- + 15 days post-UV). However, 24 h old eggs could be stored for only 24 days (12 days pre- + 12 days post-UV), 4 h old eggs could not be stored beyond 20 days (10 days pre- + 10 days post-UV). Statistical analysis indicated that all the age groups, beyond 20 days, parasitism rate decreased drastically, however, in the case of 16 h old eggs up to 28 days (14 days pre- + 14 days postUV) the parasitism rate was on par with the 20 days storage batch. The 100
100
94.2a 86.6bc 81.8c
90 80
83.4bc
88b 82.6bc
82c
80.2c
71d
50
47f
Percent parasitism
60.6e
38.6g
40 41.4fg
30 26.2h
20
83.2ab
69.8d 65.8d
60
54.2e 53e
50 40 32.4f
30 26.2g
20
18h
0C*1 1 2 2 3 3 4 4 5 5 66 77 88 9 10 11 13 14 14 15 9 10 11 12 12 13 15 16 16 17
56 67 7 8 8 9 9 1010111112121313 14 14 15 15 16 16 17 17
No. of days of storage
No. of days of storage 120
120
96a
16h 93a
86.2b
88.2ab
100 88.4ab 83.2bc 79c
86b
24h 89.6b 89.4b
81.2c
Percent parasitism
Percent parasitism
81.2b
0 1C* 2 1 32 43 54
72d 71.6d 65.8e 64e
60
72.6cd
10
10
80
8h 76c
83.6ab
72d
60
100
83.6ab
70
70 Percent parasitism
83.4ab 80.8b
80
78c
84.8bc
86.8a
90
4h
82c
68.8de
55.4f
52.2f
40 29.4g
20
80
85.8bc
82.4c
80.6c
79.8c 73.4d
80.2c 74d
58.8d
60
48.2e
40
40.6f 38f
20
20.2g
16h 12.4h
12.8h
0
0
0 C*1 12 2 3 3 4 4 5 5 6 6 77 88 99 10 13 14 14 15 16 17 17 10 11 11 12 12 13 15 16 No. of days of storage
0C* 11 22 33 44 55 66 77 88 99 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 No. of days of storage
Fig. 3. Effect of storage duration of C. cephalonica eggs of different ages [4 h, 8 h, 16 h and 24 h] pre-UV irradiation on percent parasitism (mean ± SE) by T. chilonis. C⁎-control.
931
Journal of Asia-Pacific Entomology 20 (2017) 928–934
E. Ghosh, C.R. Ballal
110
100 95.2a
100
83.2c
80.4cd
80.4cd
88.6a
85ab
84.6ab
90 80
78.2d
89.8b
85.2ab
85.2ab
82.8b
84.2b
8h
81.8b 76.4c
82.4b
80.8b
70
82.6cd 81.2cd
70
79.8cd
79d
Percent parasitism
Percent parasitism
90 80
4h
87.6b
61.6e
60
54.6f 49.6g
50 40
35.6h
30
67d
60
55.8e
50
44.2f 36.6g
40 30
31.6h
20
31.6i
16.4i
10
20 20.8j
0
10 0C* 11 22
0C* 11 22 33 44 55 66 77 88 99 10 10 11 11 12 1213 131414 15 15161617
3 44 55 66 7 7 8 8 9 910 10 1111 12 12 13 13 14 14 15 15 16 16 17 17
No. of days of storage
No. of days of storage 110
100 90
84.2ab
16h
84.2ab 80.4b
Percent parasitism
80
100
86.8a
70.4c
76.4b 77.8b
70
89ab
60
80
60.2d 53e
50
44.2f
40
37.2g
30
83.2bc
90
76b 83.8ab 82.6ab
24h
93a
77.6b
Percent parasitism
86.2a
81.2c 87b
74d 83.4bc
77cd
70
72.2
60
21.2h
63.8e 57.6f
50
45g
43g
40 41.8gh
30
20
74d
82.4bc
37.2h
20
10
10 0C* 1 1 22 33 44 55 66 77 88 9 9 1010111112121313141415151616 17 17
0C* 11 22 33 4
5 66 77 8 8 9 9 101011111212131314141515161617
No. of days of storage
No. of days of storage
Fig. 4. Effect of storage duration C. cephalonica eggs of different ages [4 h, 8 h, 16 h and 24 h] post-UV irradiation on percent parasitism (mean ± SE) by T. chilonis. C⁎-control.
Acceptance of host by any parasitoid depends on many factors: 1) chemical signals emanating from the host, 2) strength of the parasitoid ovipositor and 3) tolerance to change in physical stimuli such as color, size and shape of the host eggs (Stoepler et al., 2011). Upon storage many of the chemical signals, physical stimuli and the nutritional quality of the host eggs get deteriorated (Filho et al., 2014). In our study, we tested the interaction of host egg age and duration of storage by storing them before UV, after UV and a suitable combination of preand post-UV duration. However, we could observe that 8 and 16 h old C. cephalonica eggs could be stored for a maximum duration of 30 days viz. 15 days pre- and 15 days post-UV irradiation to achieve a minimum parasitism rate of 70%. In the case of only pre-UV or post-UV storage, the duration could not be prolonged for > 10 days (irrespective of the host egg age) as parasitism recorded was < 70%. This could due to the fact that longer storage durations might have led to depletion in nutrient quality by deterioration of yolk as repeated by Pratissoli et al. (2003). Prolonged storage period also may have altered physical and chemical stimuli due to which often female parasitoids reject their hosts (Soares et al., 2009; Goubault et al., 2011). In the combined storage experiment UV irradiation after 15 days of pre-UV storage could have arrested the embryonic development at the right time which might have enabled the egg quality to be retained at for a further period of 15 days under post-UV storage. The chorion is known to be responsible for maintaining the shape of the eggs by providing air tightness (Jalali et al., 2007). In the current study, only 8 and 16 h old eggs were amenable for extended storage, probably because UV irradiation at the right age/stage might have helped to retain the natural rigidity of the chorion further promoting optimum parasitism. Further studies are required to understand the physiology behind the age dependent
100
Percent parasitism by T. chilonis
95 90
87
88.4 84.8
83.8
85 80
75 70 65 60
55 50 4
8 16 Age of Corcyra eggs (hours)
Fig. 5. Effect of storage for 10 days pre- and post-UV irradiation of Corcyra eggs of its different ages on mean ± SE percent parasitism by T. chilonis.
22 to 24 h old eggs were not suitable for storage as they shrunk when they were removed from storage. The capability of cold tolerance depends on many factors like genotypic variability, laboratory adaptation and phenotypic plasticity (Colinet and Boivin, 2011). Not much is known about the level of cold tolerance in C. cephalonica. Eggs used in these experiments are from the insectary, where they are being continuously reared. Laboratory adaptations or bio-deterioration could have occurred due to continuous rearing. However, this can be ascertained only through comparison with the cold tolerance of a fresh warehouse population of C. cephalonica. 932
Journal of Asia-Pacific Entomology 20 (2017) 928–934 (F = 201.7; df = 3172; P ≤ 0.0001) (F = 256.6; df = 10,165; P ≤ 0.0001) (F = 18.77; df = 43,132; P ≤ 0.0001) 1.9 3.1 6.2
LSD at 5%
Age Duration Age × duration
storage suitability of C. cephalonica eggs. Previous studies have reported that E. kuehniella eggs could be stored for 24 days to achieve a maximum parasitism of 65% (Filho et al., 2014; Ozder, 2004) while through our study the duration of storage could be extended with a much higher parasitism rate. Ramos and Jimenez (1993) reported that at 7 °C vacuum packed eggs of S. cerealella could be stored for up-to 35 days and Jalali et al. (2007) could store C. cephalonica eggs for 7 to 42 days to achieve75 to 100% parasitism rate by T. chilonis. However, storage technique developed through current study could be adopted by insectaries even with most basic storage facilities. The storage of live C. cephalonica eggs for their continuous production has not been explored previously. Through this study we could stagger the incubation period by 13 to 14 days with minimum 70% larval hatching. This finding would help in planning shipments of C. cephalonica eggs and to avoid hatching in transit. We could identify the right age and the optimum duration of storage of C. cephalonica eggs for maximum parasitism by Trichogramma.
Mean figures with the same letter in the last column and row are not significantly different. Mean figures in the 2nd to 12th columns with the same letter are not significantly different.
0.0 ± 0.0h 40.3 ± 2.1de 0.0 ± 0.0h 0.0 ± 0.0h 13I 7.5 ± 2.5 g 67.5 ± 1.4cd 0.0 ± 0.0h 0.0 ± 0.h 19.5H 1.4g 6.0cd 3.2fg 1.4g ± ± ± ± 12.5 51.3 17.5 12.5 28G 1.4f 6.6cd 3.2e 2.4f 22.5 ± 53.5 ± 32.5 ± 21.3 ± 34.6F 4.5ef 5.4d 5.0cd 3.2ef 30.0 ± 50.0 ± 58.8 ± 27.5 ± 40.4E 6.7e 2.2b 4.7bc 5.6cd 33.3 ± 74.3 ± 70.3 ± 57.0 ± 50.5D 2.2e 5.0b 1.7ab 4.4b 35.8 ± 74.0 ± 76.3 ± 71.0 ± 54CD 2.1ef 1.2ab 1.2ab 2.3bc ± ± ± ± 27.5 78.3 78.5 66.3 53C 3.2c 2.4b 4.0ab 3.3ab 60.8 ± 74.8 ± 79.5 ± 75.5 ± 59.1BC 4.3d 2.5ab 1.6ab 4.8b 47.3 ± 75.3 ± 76.8 ± 74.5 ± 56.6B 1.6ab 2.1a 0.9a 2.9a 77.3 ± 83.5 ± 84.0 ± 83.3 ± 65.4A 4 8 16 24 Mean
(20 + 20) (19 + 19) (18 + 18) (17 + 17) (11 + 11) (10 + 10)
(12 + 12)
(13 + 13)
(14 + 14)
(15 + 15)
(16 + 16)
38 34 28 26 24 22 20
Percent parasitism at storage duration (pre + post-UV in days)
Age of Corcyra eggs (h)
Table 1 Mean ± SE parasitism rate of T. chilonis after pre- and post-UV combined storage at 5 °C (L:D-0:24, RH-70 ± 10%).
30
32
36
40
Mean
35.0D 47.81A 44.8B 39.8C
E. Ghosh, C.R. Ballal
Acknowledgments The funding provided by Department of Biotechnology, Govt. of India (Grant no. BT/PR3339/AGR/5/558/2011) for this research project is gratefully acknowledged. The authors thank the Director, ICARNBAIR, Bangalore for the facilities provided and Dr. Y. Lalitha for technical support. References Balasubramanian, S., Bishnoi, A.L., Pawar, A.D., 1991. Results of laboratory trials on rate of parasitism of the stored eggs of Corcyra cephalonica (Stainton) under low temperature by the parasite Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Plant Prot. Bull. Faridabad 43, 4–5. Ballal, C.R., Yamada, K., 2016. Anthocorid predators. In: Omkar (Ed.), Ecofriendly Pest Management for Food Security. Elsevier Inc, Academic Press, pp. 183–216 (Chapter 6). Colinet, H., Boivin, G., 2011. Insect parasitoids cold storage: a comprehensive review of factors of variability and consequences. Biol. Control 58, 83–95. Consoli, F.L., Kitajima, E.W., Parra, J.R.P., 1999. Ultrastructure of the natural and factitious host eggs of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Int. J. Insect Morphol. Embryol. 28, 211–231. Das, R., Ram, A., 1983. Effect of frozen eggs of Corcyra cephalonica Stainton (Pyralidae: Lepidoptera) on parasitism of Trichogramma exiguum (Pinto and Platner) (Trichogrammatidae: Hymenoptera). Indian J. Entomol. 45, 345–347. Filho, S.C.R.P., Leite, D.L.G., Soares, A.M., Alvarenga, C.A., Paulo, D.P., Satos, T.D.L., 2014. Effects of duration of cold storage of host eggs on percent parasitism and adult emergence of each of the ten Trichogrammatidae (Hymenopteran) species. Fla. Entomol. 97 (1), 14–21. Flanders, S.E., 1929. The mass production of Trichogramma minutum Riley and observations on the natural and artificial parasitism of the codling moth egg. In: Proc. Fourth Internl. Cong. Entomol. 2, pp. 110–130. Goubault, M., Cortesero, A.M., Paty, C., Fourrier, J., Dourlot, S., Leralec, A., 2011. Abdominal sensory equipment involved in external host discrimination in a solitary parasitoid wasp. Microsc. Res. Tech. 74, 1145–1153. Hamed, M., Nadeem, S., Riaz, A., 2009. Use of gamma radiation for improving the mass production of Trichogramma chilonis and Crysoperla carnea. Biocontrol Sci. Tech. 19, 43–48. Jalali, S.K., Rabindra, R.J., Rao, N.S., Dasan, C.B., 2003. Mass Production of Trichogrammatids and Chrysopids. Project Directorate of Biological Control, Bangalore 560 024, India (16 pp). Jalali, S.K., Singh, S.P., Venkatesan, T., Moorthy, K.S., Lalitha, Y., 2006. Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera, Trichogrammatidae). Indian J. Exp. Biol. 44, 584–590. Jalali, S.K., Venkatesan, T., Moorthy, K.S., Rabindra, R.J., Lalitha, Y., 2007. Vacuum packaging of Corcyra cephalonica (Stainton) eggs to enhance shelf-life for parasitization by the egg parasitoid Trichogramma chilonis. Biol. Control 41 (1), 64–67. Lalitha, Y., Ballal, C.R., 2015. Influence of seasons and inoculum dosages on the production efficiency of Corcyra cephalonica Stainton. J. Biol. Control. 29 (1), 25–30. Lalitha, Y., Jalali, S.K., Venkatesan, T., Sriram, S., 2013. Production attributes of Trichogramma reared on Eri silk worm eggs vis-à-vis Corcyra eggs and economics of the rearing system. In: 12th Workshop of the Arthropod Mass Rearing and Quality Control Working Group of the IOBC-Blueprint of the Future of Arthropod Rearing and Quality Assurance. Li, L.Y., Wajenberg, E., Hassan, H.A. (Eds.), 1994. Biological Control With Egg Parasitoids. CAB International, Oxon, UK, pp. 37–53pp. Nagaraja, H., 1973. On new species of Indian Trichogramma (Hymenoptera: Trichogramatoidea). Orient. Insects 12, 489–529. Ozder, Nihal, 2004. Effect of different cold storage periods on parasitization performance
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19, 64–71. Soares, M.A., Torres-Gutierrez, C., Zanuncio, J.C., Pedrosa, A.R.P., Lorenzona, S., 2009. Super-parasitismo de Palmistic huselaeisis (Hymenoptera: Eulophidae) y comportamiento de defensa de dos hospederos. Rev. Colomb. Entomol. 35, 62–65. Stoepler, T.M., Lill, J.T., Murphy, S.M., 2011. Cascading effects of host size and host plant species on parasitoid resource allocation. Ecol. Entomol. 36, 724–735. Strand, M.R., 1986. Physiological interactions of parasitoids and hosts. In: Waage, J., Greathead, D. (Eds.), Insect Parasitoids. Academic Press, London, pp. 109–118.
of Trichogramma cacoeciae (Hymenoptera, Trichogrammatidae) on eggs of Ephestia kuehniella (Lepidoptera, Pyralidae). Biocontrol Sci. Tech. 14 (5), 441–447. Pratissoli, D., Vianna, U.R., Oliveira, H.N., Pereira, F.F., 2003. Efeito do armazenamento de ovos de Anagasta kuehniella,(Lepidoptera: Pyrali-dae) nascaracterísticas biológicasde trêsespécies de Trichogramma (Hymenoptera: Trichogrammatidae). Rev. Ceres 50, 95–105. Ramos, J.P., Jimenez, V.J., 1993. Cold preservation of eggs of Sitotroga cerealella (Olivier) in refrigeration and vacuum packing at different pressures. Rev. Colomb. Entomol.
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Effect of age dependent cold storage of factitious host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) for their continuous production and Trichogramma chilonis (Ishii) (Hymenoptera: Trichogrammatidae) rearing
MARK
Enakshi Ghosha,b,⁎, Chandish R. Ballala a b
ICAR-National Bureau of Agricultural Insect Resources, Post Bag No. 2491, H.A. Farm Post, Bellary Road, Bangalore–24, India Department of Biotechnology, Center for Post Graduate Studies, Jain University, Jayanagar, Bengaluru 560011, Karnataka, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichogramma chilonis Parasitism Age of host eggs UV irradiation
In Asia Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) is widely used as a factitious host for the mass rearing of several bio-control agents viz. chrysopids, braconids, trichogrammatids and anthocorids. The age dependent cold storage amenability of C. cephalonica eggs was evaluated. At 5 ± 1 °C with a 24 h of scotophase, 0 to 22 h old eggs of C. cephalonica could be stored for up to 5 days resulting in 83 to 94% hatching. Eggs of C. cephalonica are UV irradiated for mass rearing of T. chilonis. Four, eight, sixteen and twenty four-hour-old C. cephalonica eggs were stored at 5 ± 1 °C for different durations prior to and post-UV irradiation and tested for their amenability to parasitism by T. chilonis. Adult females of T. chilonis could parasitise 67–78% of eggs stored for up to 10 days prior to UV treatment, while 74–78.2% parasitism was recorded in the case of eggs stored for 10 days post-UV. The host eggs of all age groups stored for 20 days (10 days pre-UV and 10 days post-UV) were effectively parasitised providing 83.8–88.4% parasitism. Eight and sixteen hour-old old host eggs stored for 30 days (15 days of pre-UV and 15 days of post-UV) recorded 70–80% parasitism. The host egg storage technique developed through this study would be beneficial for insectaries to stock the host eggs, which can lead to uninterrupted production and supply of ‘Tricho-cards’, especially during seasonal high demand.
Introduction Addressing pest problems by adopting integrated pest management strategy requires mass multiplication of several natural enemies. Trichogrammatids are important egg parasitoids used against lepidopteran pests- especially since 1926 when a suitable mass rearing strategy was developed (Flanders, 1929). The biology of Trichogramma spp. has been studied extensively (Nagaraja, 1973; Strand, 1986).The egg period of Trichogrammatids lasts 16 to 24 h, larval period 2 to 3 days, pre-pupal period 2 days and pupal period 2 to 3 days. The total development is completed in 8 to 10 days during summer and 9 to 12 days during winter. Different species of Trichogramma proved to be effective against different pests infesting various crops in different ecosystems (Li et al., 1994). Among all the identified species, Trichogramma chilonis (Ishii) is extensively used in India, China, Japan, Nepal, Pakistan, Taiwan, Reunion Island, Kenya, Australia and South Africa due to its wide host acceptability and high survival capacity (Jalali et al., 2006). Trichogramma production has gained a lot of attention recently among government and private insectaries. One of the major obstacles preventing the successful production and utilization of this ⁎
endo-parasitoid is the availability and constant supply of host eggs. Majority of the laboratories in Asia mass produce Trichogramma species on factitious host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). Sitotroga cerealella (Oliver) (Lepidoptera: Gelechiidae) and Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) are used in other countries (Consoli et al., 1999). Corcyra cephalonica eggs are subjected to UV irradiation to arrest the growth of the developing host embryo prior to exposing them to Trichogramma adults for parasitism in order to prevent hatching of larvae from these eggs (Hamed et al., 2009). This further prevents predation of the parasitized eggs by the freshly hatched C. cephalonica larvae, while ensuring nutrient availability for developing Trichogramma embryo. A recent study has proved that two species of Trichogramma could also be reared using eggs of eri silk worm Samia cynthia (Lalitha et al., 2013). Corcyra cephalonica can also be used to rear other natural enemies like green lacewings, braconid parasitoids, anthocorid predators and entomopathogenic nematodes (Li et al., 1994; Jalali et al., 2003; Ballal and Yamada, 2016). The short shelf-life of C. cephalonica eggs is a limitation for production and supply of Trichogrammatids. Development of efficient storage technique for fresh eggs is important for continuous host rearing, while that for UV irradiated C.
Corresponding author at: ICAR-National Bureau of Agricultural Insect Resources, Post Bag No. 2491, H.A. Farm Post, Bellary Road, Bangalore–24, India. E-mail address: [email protected] (E. Ghosh).
http://dx.doi.org/10.1016/j.aspen.2017.06.013 Received 12 February 2017; Received in revised form 17 June 2017; Accepted 26 June 2017 Available online 29 June 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
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70 ± 10% of RH. Five replications were maintained for each treatment and control. The stored eggs were UV irradiated for 60 min. UV treated cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in a walk-in chamber set at 26 ± 1 °C with RH 70 ± 5%. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
cephalonica eggs is important for Trichogramma rearing. This will enable the availability of Trichogrammatids during pest outbreaks and satisfy seasonal demands. Storage of UV irradiated C. cephalonica eggs at − 6 ± 1 °C for > 50 days adversely affected the fitness attributes of Trichogramma spp. reared on them (Das and Ram, 1983), while storage at 0 and 9 °C for 3and 15 days, respectively, provided positive results for Trichogramma production (Balasubramanian et al., 1991). Vacuum packed UV irradiated C. cephalonica eggs stored at 8 °C for 7 to 42 days resulted in > 70% parasitism by T. chilonis (Jalali et al., 2007). Previous studies have not evaluated the effect of the age of live and UV irradiated eggs on their amenability to storage. Our aim was to extend the shelf-life of fresh C. cephalonica eggs for continuous host rearing and UV irradiated eggs for continuous production of Trichogrammatids by identifying the right combination of the egg age and storage durations.
Post-UV storage (Fig. 1.Bpost) Corcyra cephalonica eggs of 0–2 h were UV irradiated for 60 min and then incubated at room temperature for 4, 8, 16 and 24 h to form different experimental age groups: 4–6 h, 8–10 h, 16–18 h and 24–26 (referred same as pre-UV storage). From each age group, 100 paper cards (measuring 2 × 1 cm) which held one hundred eggs, after which they were placed in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH. From the storage unit, five cards from each age group were removed at 24 h intervals and this was continued till 20 days. One un-stored batch for each age group was maintained as control. Five replications were maintained for each treatment and control. The post-UV stored cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were placed in a walk-in chamber set at 26 ± 1 °C, RH 70 ± 5% with L:D-12:12. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
Materials and methods Maintenance of the cultures Corcyra cephalonica (National Accession No: NBAII-MP-PYR-01) is reared continuously in the live insect repository of ICAR-NBAIR, Bangalore. These are reared on a medium comprising of insecticide free sorghum, groundnut seeds, yeast and streptomycin sulphate (Lalitha and Ballal, 2015). The eggs of C. cephalonica are exposed to UV irradiation by placing the eggs at a distance of 2 ft from the UV tube light for 45 min. These UV irradiated eggs are glued on thick paper cards measuring 12 × 10 cm. These cards are exposed to T. chilonis (continuously lab reared) adults in the ratio of 40:1 (eggs:female) until mortality. The cultures were maintained at 25 ± 4 °C, 65 ± 10% RH with L:D-12:12.
Combined pre and post-UV storage (Fig. 1.Bcombined) Corcyra cephalonica eggs of 0–2 h old were incubated at room temperature for 4, 8, 16 and 24 h to arrive at different experimental age groups: 4–6 h, 8–10 h, 16–18 h and 24–26 h (referred same as pre-UV storage). Forty four sets of hundred eggs pasted on thick paper card (measuring 2 × 1 cm) from each age group were stored in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH for different durations (2, 4, 6, 8 and 10 days) and UV irradiated for 60 min and further placed at 5 ± 1 °C. These UV irradiated stored cards were removed at 24 h intervals (0 days to 10th day) and exposed to healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in walk-in chamber set at (26 ± 1 °C, RH 70 ± 1% with L:D-12:12) and checked for percent parasitization.
Experimental setup The overall protocol of the experimental set up for the four experiments is depicted in Fig. 1. Storage of C. cephalonica eggs for continuous host rearing (Fig. 1.A) Corcyra cephalonica eggs of 0–2 h old were collected from the rearing units and incubated at room temperature (25 ± 3 °C) (for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 h) to arrive at the following age groups: 0–2 h, 2–4 h, 4–6 h, 6–8 h, 8–10 h, 10–12 h, 12–14 h, 14–16 h, 16–18 h, 18–20 h, 20–22 h, 22–24 h. Approximately one 100 eggs from each age group were glued on a thick paper card (measuring 2 × 1 cm) and kept in a BOD (biological oxygen demand) (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C, 65 ± 10% RH and L:D-0:24 for different experimental durations viz. 5 days, 10 days, 15 days, after which they were placed in a walk-in chamber set at 26 ± 1 °C with 70 ± 5% RH. Each treatment was replicated five times. The batches were regularly checked for hatching, incubation period and percent hatching were recorded.
Combined pre and post-UV storage (beyond 20 days) An additional experiment was conducted to test if further combined pre- and post-UV storage beyond 20 days would be feasible for utilization in Trichogramma production. Egg cards of each age group, with 100 eggs on each card were stored at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH (Remi cooling incubator - Cl-12S model) for 10 to 20 days (e.g. 10 days pre-UV + 10 days post-UV, 11 days pre-UV + 11 days post-UV up-to 20 days pre-UV + 20 days post-UV). Four cards from each age group were removed at 24 h intervals and subjected to UV irradiation for 60 min. Post-UV irradiation, the stored UV treated cards of different age groups were further stored for 10 to 20 days. Four cards were removed from the storage unit at every 24 h interval for up-to 20 days. These cards were exposed to 1 to 2 days old healthy T. chilonis females for 24 h in 1:30 (female:host eggs) ratio. These exposed cards were kept in a walk-in chamber set at 26 ± 1 °C with RH 70 ± 5%. Percent parasitism was calculated based on the number of blackened eggs out of the total number of eggs on each card.
Storage of C. cephalonica eggs for T. chilonis production Pre-UV storage (Fig. 1.Bpre) Corcyra cephalonica eggs of 0–2 h old were incubated at room temperature for 4, 8, 16 and 24 h to form four different experimental age groups viz. 4–6 h (referred as 4 h), 8–10 h (referred as 8 h), 16–18 h (referred as 16 h) and 24–26 h (referred as 24 h). From each age group, 100 paper cards (measuring 2 × 1 cm) each holding hundred eggs were placed in a BOD (Remi cooling incubator - Cl-12S model) set at 5 ± 1 °C with L:D-0:24 and 65 ± 10% RH. From the storage unit, five cards from each age group were removed at 24 h intervals and this was continued till 20 days. One un-stored batch for each age group was maintained as control in a walk-in chamber set at 26 ± 2 °C with
Statistical analysis Treatment effects on percent hatching and percent parasitism were evaluated using one way analysis of variance (ANOVA) (SPSS 16.0). When ANOVA was significant, comparison of relevant means were made using Turkey's post hoc test at a significant level of 5%. Data recorded on percent hatching of host eggs were subjected to angular 929
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Fig. 1. Overall outline of the experimental protocol followed.
gradually decreased with an increase in storage period. Four and 24 h old eggs were significantly inferior to control (F = 12.7; df = 16, 68; P ≤ 0.0001) (F = 160; df = 16, 68; P ≤ 0.0001). Percent parasitism of 8 h old eggs 16 h old eggs stored for 4 days were statistically on par with the respective control batches (Fig. 3) (F = 22.19; df = 16, 68; P ≤ 0.0001) (F = 170.9; df = 16, 68; P ≤ 0.0001). > 70% parasitism was recorded in 4,8,16 and 24 h old eggs after storage at 5 °C for a duration of 11, 9, 9 and 10 days, respectively. Based on two way ANOVA, among the experimental host age groups, 4 h old eggs were found to be statistically superior (F = 4.73; df = 3, 13; P ≤ 0.0001).
transformation before statistical analysis. Percent parasitism data were also subjected to two way analysis of variance. The aim was to investigate the effect of main treatments (i.e., age of the host eggs and the duration of storage) on parasitoid performance and also to check if an interaction existed between them. Where significant values were obtained, the results were interpreted at 5% level of significance based on LSD test. Results Effect of low temperature storage on hatching of C. cephalonica eggs
Parasitism of C. cephalonica eggs stored post-UV irradiation The current laboratory experiments indicated that live C. cephalonica eggs stored at 5 °C for 10 and 15 days resulted in 0 to 20% hatching. Hence only the data on percent hatching from eggs (belonging to different age groups) when stored for 5 days at 5 °C were subjected to statistical analysis. Eggs belonging to all other age groups (except 22 to 24 h) could be stored for 5 days resulting in 83.4–94.2% hatching (F = 43.92; df = 12,52; P ≤ 0.0001) (Fig. 2). The incubation period of stored eggs ranged between 9.2 and 10.2 days, while in control it was 6 to 7 days. The total incubation period (including the storage period) ranged between 13.2 and 14.6 days and thus, the incubation period could be staggered by one week.
It was observed that parasitism rates of 8 and 16 h old eggs after 4 and 5 days of storage respectively, were statistically on par with their respective control (Fig. 4) (F = 22.16; df = 16, 68; P ≤ 0.0001) (F = 94.7; df = 16, 68; P ≤ 0.0001). In case of 4 and 24 h old eggs the parasitism rates were significantly reduced after 1 or 2 days of storage in comparison to their control (F = 305.8; df = 16, 68; P ≤ 0.0001) (F = 95.8; df = 16, 68; P ≤ 0.0001). > 70% parasitism could be recorded in 4, 8, 16 and 24 h old eggs after storage at 5 °C for up-to 10, 10, 11 and 9 days, respectively (Fig. 4). Parasitism of C. cephalonica eggs after pre- plus post-UV combined storage
Parasitism of C. cephalonica eggs stored prior to UV irradiation Corcyra cephalonica eggs of different ages stored at 5 °C for a total of 20 days (10 days of pre- plus 10 days of post-UV storage) when exposed
Parasitism rate of un-stored host eggs was high while the rate 930
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Fig. 2. Percent hatching (mean ± SE) of different ages of Corcyra eggs after storage at 5 °C (L:D-0:24, RH-70 ± 10%) for 5 days.
120 100
93.2a
92ab
87.2ab
91.2ab 85b
Percent hatching
83.4b
94.2a 83.4b
90.4ab 88.4ab 88.8ab
84.2b
80 60 40 20 0.8c .
0 C
0-2
2-4
4-6
6-8 8-10 10-12 12-14 14-16 16-18 18-20 20-22 22-24 Age of Corcyra eggs (hours)
most suitable age amenable for storage irrespective of storage duration was found out to be 8 h (Table 1).
to T. chilonis, the percent parasitism values of 88.4, 84.8, 87 and 83.8, respectively were recorded, all being statistically on par (F = 1.24; df = 3,16; P = 0.32) (Fig. 5).
Discussion Combined pre- and post-UV storage (beyond 20 days) A successful bio-control strategy utilizing endo-parasitoids like Trichogramma is dependent on the availability of the parasitoids in large numbers for field releases. Storing the host eggs by staggering their development to improve continuous host egg production and also improving their viability for Trichogramma rearing can ultimately help synchronize their mass production during high seasonal demands. The current study revealed that 0–22 h old C. cephalonica eggs can be safely stored at 5 °C for 5 days, and incubation period could be staggered by a week, with hatching rate maintained between 83 and 94%. However,
To achieve a minimum of 70% parasitism, 8 and 16 h old eggs could be stored for up to 30 days (15 days pre- + 15 days post-UV). However, 24 h old eggs could be stored for only 24 days (12 days pre- + 12 days post-UV), 4 h old eggs could not be stored beyond 20 days (10 days pre- + 10 days post-UV). Statistical analysis indicated that all the age groups, beyond 20 days, parasitism rate decreased drastically, however, in the case of 16 h old eggs up to 28 days (14 days pre- + 14 days postUV) the parasitism rate was on par with the 20 days storage batch. The 100
100
94.2a 86.6bc 81.8c
90 80
83.4bc
88b 82.6bc
82c
80.2c
71d
50
47f
Percent parasitism
60.6e
38.6g
40 41.4fg
30 26.2h
20
83.2ab
69.8d 65.8d
60
54.2e 53e
50 40 32.4f
30 26.2g
20
18h
0C*1 1 2 2 3 3 4 4 5 5 66 77 88 9 10 11 13 14 14 15 9 10 11 12 12 13 15 16 16 17
56 67 7 8 8 9 9 1010111112121313 14 14 15 15 16 16 17 17
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No. of days of storage 120
120
96a
16h 93a
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100 88.4ab 83.2bc 79c
86b
24h 89.6b 89.4b
81.2c
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Percent parasitism
81.2b
0 1C* 2 1 32 43 54
72d 71.6d 65.8e 64e
60
72.6cd
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10
80
8h 76c
83.6ab
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100
83.6ab
70
70 Percent parasitism
83.4ab 80.8b
80
78c
84.8bc
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4h
82c
68.8de
55.4f
52.2f
40 29.4g
20
80
85.8bc
82.4c
80.6c
79.8c 73.4d
80.2c 74d
58.8d
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48.2e
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40.6f 38f
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16h 12.4h
12.8h
0
0
0 C*1 12 2 3 3 4 4 5 5 6 6 77 88 99 10 13 14 14 15 16 17 17 10 11 11 12 12 13 15 16 No. of days of storage
0C* 11 22 33 44 55 66 77 88 99 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 No. of days of storage
Fig. 3. Effect of storage duration of C. cephalonica eggs of different ages [4 h, 8 h, 16 h and 24 h] pre-UV irradiation on percent parasitism (mean ± SE) by T. chilonis. C⁎-control.
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110
100 95.2a
100
83.2c
80.4cd
80.4cd
88.6a
85ab
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90 80
78.2d
89.8b
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81.8b 76.4c
82.4b
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70
82.6cd 81.2cd
70
79.8cd
79d
Percent parasitism
Percent parasitism
90 80
4h
87.6b
61.6e
60
54.6f 49.6g
50 40
35.6h
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67d
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40 30
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16.4i
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20 20.8j
0
10 0C* 11 22
0C* 11 22 33 44 55 66 77 88 99 10 10 11 11 12 1213 131414 15 15161617
3 44 55 66 7 7 8 8 9 910 10 1111 12 12 13 13 14 14 15 15 16 16 17 17
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No. of days of storage 110
100 90
84.2ab
16h
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86.8a
70.4c
76.4b 77.8b
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83.2bc
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76b 83.8ab 82.6ab
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93a
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86.2a
81.2c 87b
74d 83.4bc
77cd
70
72.2
60
21.2h
63.8e 57.6f
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45g
43g
40 41.8gh
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10 0C* 1 1 22 33 44 55 66 77 88 9 9 1010111112121313141415151616 17 17
0C* 11 22 33 4
5 66 77 8 8 9 9 101011111212131314141515161617
No. of days of storage
No. of days of storage
Fig. 4. Effect of storage duration C. cephalonica eggs of different ages [4 h, 8 h, 16 h and 24 h] post-UV irradiation on percent parasitism (mean ± SE) by T. chilonis. C⁎-control.
Acceptance of host by any parasitoid depends on many factors: 1) chemical signals emanating from the host, 2) strength of the parasitoid ovipositor and 3) tolerance to change in physical stimuli such as color, size and shape of the host eggs (Stoepler et al., 2011). Upon storage many of the chemical signals, physical stimuli and the nutritional quality of the host eggs get deteriorated (Filho et al., 2014). In our study, we tested the interaction of host egg age and duration of storage by storing them before UV, after UV and a suitable combination of preand post-UV duration. However, we could observe that 8 and 16 h old C. cephalonica eggs could be stored for a maximum duration of 30 days viz. 15 days pre- and 15 days post-UV irradiation to achieve a minimum parasitism rate of 70%. In the case of only pre-UV or post-UV storage, the duration could not be prolonged for > 10 days (irrespective of the host egg age) as parasitism recorded was < 70%. This could due to the fact that longer storage durations might have led to depletion in nutrient quality by deterioration of yolk as repeated by Pratissoli et al. (2003). Prolonged storage period also may have altered physical and chemical stimuli due to which often female parasitoids reject their hosts (Soares et al., 2009; Goubault et al., 2011). In the combined storage experiment UV irradiation after 15 days of pre-UV storage could have arrested the embryonic development at the right time which might have enabled the egg quality to be retained at for a further period of 15 days under post-UV storage. The chorion is known to be responsible for maintaining the shape of the eggs by providing air tightness (Jalali et al., 2007). In the current study, only 8 and 16 h old eggs were amenable for extended storage, probably because UV irradiation at the right age/stage might have helped to retain the natural rigidity of the chorion further promoting optimum parasitism. Further studies are required to understand the physiology behind the age dependent
100
Percent parasitism by T. chilonis
95 90
87
88.4 84.8
83.8
85 80
75 70 65 60
55 50 4
8 16 Age of Corcyra eggs (hours)
Fig. 5. Effect of storage for 10 days pre- and post-UV irradiation of Corcyra eggs of its different ages on mean ± SE percent parasitism by T. chilonis.
22 to 24 h old eggs were not suitable for storage as they shrunk when they were removed from storage. The capability of cold tolerance depends on many factors like genotypic variability, laboratory adaptation and phenotypic plasticity (Colinet and Boivin, 2011). Not much is known about the level of cold tolerance in C. cephalonica. Eggs used in these experiments are from the insectary, where they are being continuously reared. Laboratory adaptations or bio-deterioration could have occurred due to continuous rearing. However, this can be ascertained only through comparison with the cold tolerance of a fresh warehouse population of C. cephalonica. 932
Journal of Asia-Pacific Entomology 20 (2017) 928–934 (F = 201.7; df = 3172; P ≤ 0.0001) (F = 256.6; df = 10,165; P ≤ 0.0001) (F = 18.77; df = 43,132; P ≤ 0.0001) 1.9 3.1 6.2
LSD at 5%
Age Duration Age × duration
storage suitability of C. cephalonica eggs. Previous studies have reported that E. kuehniella eggs could be stored for 24 days to achieve a maximum parasitism of 65% (Filho et al., 2014; Ozder, 2004) while through our study the duration of storage could be extended with a much higher parasitism rate. Ramos and Jimenez (1993) reported that at 7 °C vacuum packed eggs of S. cerealella could be stored for up-to 35 days and Jalali et al. (2007) could store C. cephalonica eggs for 7 to 42 days to achieve75 to 100% parasitism rate by T. chilonis. However, storage technique developed through current study could be adopted by insectaries even with most basic storage facilities. The storage of live C. cephalonica eggs for their continuous production has not been explored previously. Through this study we could stagger the incubation period by 13 to 14 days with minimum 70% larval hatching. This finding would help in planning shipments of C. cephalonica eggs and to avoid hatching in transit. We could identify the right age and the optimum duration of storage of C. cephalonica eggs for maximum parasitism by Trichogramma.
Mean figures with the same letter in the last column and row are not significantly different. Mean figures in the 2nd to 12th columns with the same letter are not significantly different.
0.0 ± 0.0h 40.3 ± 2.1de 0.0 ± 0.0h 0.0 ± 0.0h 13I 7.5 ± 2.5 g 67.5 ± 1.4cd 0.0 ± 0.0h 0.0 ± 0.h 19.5H 1.4g 6.0cd 3.2fg 1.4g ± ± ± ± 12.5 51.3 17.5 12.5 28G 1.4f 6.6cd 3.2e 2.4f 22.5 ± 53.5 ± 32.5 ± 21.3 ± 34.6F 4.5ef 5.4d 5.0cd 3.2ef 30.0 ± 50.0 ± 58.8 ± 27.5 ± 40.4E 6.7e 2.2b 4.7bc 5.6cd 33.3 ± 74.3 ± 70.3 ± 57.0 ± 50.5D 2.2e 5.0b 1.7ab 4.4b 35.8 ± 74.0 ± 76.3 ± 71.0 ± 54CD 2.1ef 1.2ab 1.2ab 2.3bc ± ± ± ± 27.5 78.3 78.5 66.3 53C 3.2c 2.4b 4.0ab 3.3ab 60.8 ± 74.8 ± 79.5 ± 75.5 ± 59.1BC 4.3d 2.5ab 1.6ab 4.8b 47.3 ± 75.3 ± 76.8 ± 74.5 ± 56.6B 1.6ab 2.1a 0.9a 2.9a 77.3 ± 83.5 ± 84.0 ± 83.3 ± 65.4A 4 8 16 24 Mean
(20 + 20) (19 + 19) (18 + 18) (17 + 17) (11 + 11) (10 + 10)
(12 + 12)
(13 + 13)
(14 + 14)
(15 + 15)
(16 + 16)
38 34 28 26 24 22 20
Percent parasitism at storage duration (pre + post-UV in days)
Age of Corcyra eggs (h)
Table 1 Mean ± SE parasitism rate of T. chilonis after pre- and post-UV combined storage at 5 °C (L:D-0:24, RH-70 ± 10%).
30
32
36
40
Mean
35.0D 47.81A 44.8B 39.8C
E. Ghosh, C.R. Ballal
Acknowledgments The funding provided by Department of Biotechnology, Govt. of India (Grant no. BT/PR3339/AGR/5/558/2011) for this research project is gratefully acknowledged. The authors thank the Director, ICARNBAIR, Bangalore for the facilities provided and Dr. Y. Lalitha for technical support. References Balasubramanian, S., Bishnoi, A.L., Pawar, A.D., 1991. Results of laboratory trials on rate of parasitism of the stored eggs of Corcyra cephalonica (Stainton) under low temperature by the parasite Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Plant Prot. Bull. Faridabad 43, 4–5. Ballal, C.R., Yamada, K., 2016. Anthocorid predators. In: Omkar (Ed.), Ecofriendly Pest Management for Food Security. Elsevier Inc, Academic Press, pp. 183–216 (Chapter 6). Colinet, H., Boivin, G., 2011. Insect parasitoids cold storage: a comprehensive review of factors of variability and consequences. Biol. Control 58, 83–95. Consoli, F.L., Kitajima, E.W., Parra, J.R.P., 1999. Ultrastructure of the natural and factitious host eggs of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Int. J. Insect Morphol. Embryol. 28, 211–231. Das, R., Ram, A., 1983. Effect of frozen eggs of Corcyra cephalonica Stainton (Pyralidae: Lepidoptera) on parasitism of Trichogramma exiguum (Pinto and Platner) (Trichogrammatidae: Hymenoptera). Indian J. Entomol. 45, 345–347. Filho, S.C.R.P., Leite, D.L.G., Soares, A.M., Alvarenga, C.A., Paulo, D.P., Satos, T.D.L., 2014. Effects of duration of cold storage of host eggs on percent parasitism and adult emergence of each of the ten Trichogrammatidae (Hymenopteran) species. Fla. Entomol. 97 (1), 14–21. Flanders, S.E., 1929. The mass production of Trichogramma minutum Riley and observations on the natural and artificial parasitism of the codling moth egg. In: Proc. Fourth Internl. Cong. Entomol. 2, pp. 110–130. Goubault, M., Cortesero, A.M., Paty, C., Fourrier, J., Dourlot, S., Leralec, A., 2011. Abdominal sensory equipment involved in external host discrimination in a solitary parasitoid wasp. Microsc. Res. Tech. 74, 1145–1153. Hamed, M., Nadeem, S., Riaz, A., 2009. Use of gamma radiation for improving the mass production of Trichogramma chilonis and Crysoperla carnea. Biocontrol Sci. Tech. 19, 43–48. Jalali, S.K., Rabindra, R.J., Rao, N.S., Dasan, C.B., 2003. Mass Production of Trichogrammatids and Chrysopids. Project Directorate of Biological Control, Bangalore 560 024, India (16 pp). Jalali, S.K., Singh, S.P., Venkatesan, T., Moorthy, K.S., Lalitha, Y., 2006. Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera, Trichogrammatidae). Indian J. Exp. Biol. 44, 584–590. Jalali, S.K., Venkatesan, T., Moorthy, K.S., Rabindra, R.J., Lalitha, Y., 2007. Vacuum packaging of Corcyra cephalonica (Stainton) eggs to enhance shelf-life for parasitization by the egg parasitoid Trichogramma chilonis. Biol. Control 41 (1), 64–67. Lalitha, Y., Ballal, C.R., 2015. Influence of seasons and inoculum dosages on the production efficiency of Corcyra cephalonica Stainton. J. Biol. Control. 29 (1), 25–30. Lalitha, Y., Jalali, S.K., Venkatesan, T., Sriram, S., 2013. Production attributes of Trichogramma reared on Eri silk worm eggs vis-à-vis Corcyra eggs and economics of the rearing system. In: 12th Workshop of the Arthropod Mass Rearing and Quality Control Working Group of the IOBC-Blueprint of the Future of Arthropod Rearing and Quality Assurance. Li, L.Y., Wajenberg, E., Hassan, H.A. (Eds.), 1994. Biological Control With Egg Parasitoids. CAB International, Oxon, UK, pp. 37–53pp. Nagaraja, H., 1973. On new species of Indian Trichogramma (Hymenoptera: Trichogramatoidea). Orient. Insects 12, 489–529. Ozder, Nihal, 2004. Effect of different cold storage periods on parasitization performance
933
Journal of Asia-Pacific Entomology 20 (2017) 928–934
E. Ghosh, C.R. Ballal
19, 64–71. Soares, M.A., Torres-Gutierrez, C., Zanuncio, J.C., Pedrosa, A.R.P., Lorenzona, S., 2009. Super-parasitismo de Palmistic huselaeisis (Hymenoptera: Eulophidae) y comportamiento de defensa de dos hospederos. Rev. Colomb. Entomol. 35, 62–65. Stoepler, T.M., Lill, J.T., Murphy, S.M., 2011. Cascading effects of host size and host plant species on parasitoid resource allocation. Ecol. Entomol. 36, 724–735. Strand, M.R., 1986. Physiological interactions of parasitoids and hosts. In: Waage, J., Greathead, D. (Eds.), Insect Parasitoids. Academic Press, London, pp. 109–118.
of Trichogramma cacoeciae (Hymenoptera, Trichogrammatidae) on eggs of Ephestia kuehniella (Lepidoptera, Pyralidae). Biocontrol Sci. Tech. 14 (5), 441–447. Pratissoli, D., Vianna, U.R., Oliveira, H.N., Pereira, F.F., 2003. Efeito do armazenamento de ovos de Anagasta kuehniella,(Lepidoptera: Pyrali-dae) nascaracterísticas biológicasde trêsespécies de Trichogramma (Hymenoptera: Trichogrammatidae). Rev. Ceres 50, 95–105. Ramos, J.P., Jimenez, V.J., 1993. Cold preservation of eggs of Sitotroga cerealella (Olivier) in refrigeration and vacuum packing at different pressures. Rev. Colomb. Entomol.
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