Indian Journal of Exprimental Biology Vol. 52, April 2014, pp. 369-374
Efficacy of Spodoptera litura multiple nucleopolyhedrovirus after serial passage through the homologous insect larval host Mudasir Gani*, R K Gupta & K Bali Division of Entomology, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu, Chatha, India 180 009 Received 9 August 2013; revised 28 October 2013 An originally isolated baculovirus, Spodoptera litura multiple nucleopolyhedrovirus (SpltMNPV) was serially passed through the S. litura larvae for upto four generations to determine the mean number of occlusion bodies (OBs) harvested per larva and their efficacy in terms of infectivity, feeding cessation and speed of kill of host larvae. The results revealed that the mean number of OBs harvested per larva increased significantly with increase in the dose of SpltMNPV at each passage and the yield was significantly lower in original stock wild-type SpltMNPV (P0) as compared to serially passed SpltMNPV (P1, P2, P3 and P4). Laboratory bioassays indicate that median lethal doses (LD50), median times to feeding cessation (FT50) and median survival times (ST50) of P0, P1, P2, P3 and P4 were significantly different from each other. The OBs of each passage when tested for their cross-infectivity against Spodoptera exigua and Spilarctia obliqua revealed significant reduction in their mortality. These results indicate that serially passed SpltMNPV is more host specific and more effective biocontrol agent than the original stock wild-type virus and can be adopted for mass production as a viral pesticide for control of the S. litura. Keywords: Baculoviruses, Bioinsecticides, Entomopathogens, Noctuidae
Despite decades of warnings, the inappropriate use of chemical pesticides continues to pose threats to the environment and human health. There have been massive upsurges in pesticide use in recent years1 and their use is increasingly associated with negative impacts to both human health and the environment. In addition their surging costs are beyond the reach of resource-poor farmers in developing countries. Under these circumstances utilization of natural occurring insect pathogens may prove worthy for insect-pest control. With a rich biodiversity of several entomopathogens, the biocontrol workers in India are developing safe, economical and reliable control of crop pests. Improved access to locally produced, and therefore affordable, biopesticides is a key component of a national Integrated Pest Management (IPM) policy. In this direction, many baculoviruses have been now a day successfully used as biopesticides on a large scale2 to regulate insect-pest populations owing to their strong pathogenecity, persistence and ecological safety. An important feature of baculoviruses is their capacity to replicate in —————— *Correspondent author Telephone: 0191-2262119, Ext 2452 Fax: 0191-22262845 E-mail:
[email protected]
and spread through host populations under field conditions by horizontal and vertical transmission. The polyhedra which are the form of virus used for insect control are stable, can survive for years if they are in the favourable conditions and cause lethal epizootics in the field which provide added control of pests in nature3. Although, SpltMNPV has been found very effective against Spodoptera litura (Fab.) (Lepidoptera: Noctuidae), greater amount of variation in its efficacy hindered its practical utility in the field conditions4. If produced, formulated and applied in appropriate ways, it can provide ecological and effective solutions to pest problems. For instance, in India, virus production by small farmers or village based biopesticide units is straight forward with the nucleus stock of virus obtained from infected larvae under field conditions. Unfortunately, variation within baculoviruses isolates may influence some biological properties, such as the virulence to their specific host as well as its host range. Serial passage of baculoviruses through the homologous larval host drives the evolution of virulence as the pathogen strain with the highest replication rate achieves the greatest numerical representation in the inoculum5. A thorough understanding of the interactions of
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nucleopolyhedroviruses (NPVs) with the homologous and heterologous insect hosts is therefore necessary to facilitate their development as biopesticides in order to seek better investment prospects from the public sector to the multinational private sector6. In depth investigations were therefore undertaken to assess the pattern of virus production and efficacy of SpltMNPV after serial passage through the homologous insect larval host, S. litura, and to check the cross-infectivity against the heterologous insect larval hosts, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) and Spilosoma obliqua (Walker) (Lepidoptera: Arctiidae) which is an issue of direct relevance to the environmental assessment studies on the stability of baculoviruses. Materials and Methods Host insect Spodoptera litura—The S. litura larvae used in this study was obtained from a laboratory colony, established from a larval population of the insect collected in the region of Jammu during 2011. The culture was maintained at 28±2 °C, 60% RH, and 16L:8D photoperiod on the artificial diet as suggested by Shorey and Hale7 with certain modifications proposed by Gupta et al8. Egg masses were collected and surface-sterilized with 0.01% sodium hypochlorite. The larvae were reared in a plastic container (30 × 20 × 10 cm) lined with a paper towel at the bottom. The diet and the paper were changed every 24 h. A laboratory cohort of S. litura larvae were randomly selected and reared on artificial diet as already described. This was termed as parental group. From this group, 50 larvae were allowed to complete their development and the rest were used in the experiment at 2nd or 3rd instar as desired. At adult emergence, ten randomly selected females were mated to an equal number of males separately in glass jars (30 × 20 × 10 cm) and their resulting progeny were reared for four generations. In each generation, a group of 1000 larvae were randomly selected and used in the experiments. Virus preparation—The multiple nucleopolyhedrovirus used in this study was originally isolated from naturally field-infected S. litura larvae collected from tomato fields in Jammu, India9. Viral occlusion bodies (OBs) were extracted by homogenizing virus-killed larvae in 0.1% sodium dodecyl sulphate (SDS), followed by filtration through muslin cloth and subsequent pelleting through continuous sucrose gradient centrifugation for 1 h at 50,000 g10.
After several washes in TE (10 mm Tris-HCl, pH 8, 115 1 mm EDTA) the OBs were resuspended in 0.75 mL of distilled water and stored in aliquots at −20 °C. The virus was quantified using a haemocytometer and phase contrast microscope at ×1000 magnification. Effect on polyhedra production of SpltMNPV after serial passage through the S. litura larvae—To determine the polyhedra production of SpltMNPV after serial passage through the S. litura larvae, five different doses of SpltMNPV viz 3 × 103, 1.5 × 103, 7.5 × 102, 3.75 × 102, 1.87 × 102 OBs/larva were used in the study at each passage. The polyhedra production of SpltMNPV was compared between the passage zero (P0) and passage through S. litura larvae (P1-P4) in the subsequent four generations. The original stock polyhedra were termed herein as passage zero OBs (P0). To obtain the passage one OBs (P1), each 3rd instar S. litura larvae were placed in individual Petri dish (80 mm × 15 mm) and were offered a diet plug contaminated with above described virus-doses. For each dose 10µL of viral suspension were pipetted on each diet plug. Larvae, having eaten the entire diet plug within 24 h, were transferred to fresh uncontaminated diet and reared at 28±2 ºC, 60% RH, and 16L:8D photoperiod. Larvae that did not consume the entire diet plug were discarded. Similarly, to obtain occlusion bodies (OBs) in subsequent passages viz P2, P3 and P4, the 3rd instar S. litura were offered diet plug contaminated with virus inoculum of preceding passage i.e. P1, P2 and P3, respectively. In all passages viral OBs were extracted by homogenizing each individual virus-killed larvae in 0.1% sodium dodecyl sulphate (SDS) and were purified as per Monobrullah et al9. The OBs were counted using a haemocytometer and phase contrast microscope at ×1000 magnification. The purified OBs were stored at −20 °C until further use. The viral doses and a corresponding experimental control group of each passage was replicated three times with 50 larvae per replicate. Control consisted of larvae that were fed on a diet plug inoculated with distilled water. Efficacy of SpltMNPV after serial passage through the S. litura larvae—The OBs harvested at each passage were used to determine the efficacy of SpltMNPV after serial passage through the S. litura larvae by per os bioassay. The intrinsic biological activities medial lethal dose (LD50s), median time to feeding cessation (FT50s) and median survival time
GANI et al.: EFFICACY OF SPODOPTERA LITURA MULTIPLE NUCLEOPOLYHEDROVIRUS
(ST50s) of SpltMNPV was compared between the passage zero (P0) and passage through S. litura larvae (P1-P4) in the subsequent four generations. In each passage freshly moulted second-instar larvae were starved for 24 h and then inoculated with five doses of SpltMNPV viz 3 × 103, 1.5 × 103, 7.5 × 102, 3.75 × 102, 1.87 × 102 OBs/larva using artificial diet described earlier. For each dose 10 µL of viral suspension were pipetted on each diet plug. Diet plugs were air-dried and then placed individually in an acrylic compartment insect rearing system designed by National Bureau of Agriculturally Important Insects (NBAII) Bangalore, India. One larva was placed in each compartment (2 × 2 cm). Larvae, having eaten the entire diet plug within 24 h, were transferred to fresh uncontaminated diet with smooth surface and reared at 28±2 ºC, 60% RH, and 16L:8D photoperiod. Larvae that did not consume the entire plug were discarded. For FT50 determination lack of feeding marks within 24 h was considered as feeding cessation. For time response the virus doses were first standardized to produce similar mortality rates in order to facilitate comparisons among the passages. Equally effective doses causing less than 50% mortality were chosen, because the ST50 generated from these data are relatively insensitive to dose differences. The survival time and time to feeding cessation for each larva were taken as the midpoint of the interval in which they died or stopped feeding. Mortality was recorded daily for upto 10 days period. Mortality caused by virus was diagnosed from typical virus disease characteristics (soft, flaccid body) and was confirmed by microscopic examination through giemsa staining11. Control consisted of larvae that were fed on a diet plug inoculated with distilled water. Bioassay of each viral dose and a corresponding experimental control group was replicated three times with 50 larvae per replicate, for a total of 150 larvae per dose. Cross infectivity of SpltMNPV against S. exigua and S. obliqua larvae—The cross infectivity of original stock wild-type (P0) and serially passed SpltMNPV (P1, P2, P3, P4) was checked against 2nd instar S. exigua and S. obliqua larvae by per os bioassay. Freshly moulted second-instar larvae of both species were starved for 24 h and then inoculated with a dose of 1.00 × 105 OBs/larva using artificial diet as described earlier. In each passage 10 µL of viral suspension were pipetted on each diet plug. Diet plugs were air-dried and then placed individually in
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an acrylic compartment insect rearing system and the experimental set up and conditions were similar as mentioned in the earlier section. Mortality was recorded daily for upto 10 days period and was confirmed by the methods described earlier. Statistical analyses—All analyses were performed utilizing SPSS version 16. Data on mean number of OBs harvested per larva and their infectivity (cumulative mortality percentage) at each passage were analyzed through nonparametric Kruskal-Wallis H test. Probit analyses were performed for the calculation of median lethal doses (LD50 ± 95% C.L.). Median times to feeding cessation (FT50s), median survival times (ST50s) and their 95% confidence limits were calculated using log rank test under Kaplan-Meier analyses. Differences of LD50s, FT50s and ST50s among the passages were analysed by Univariate Analysis of Variance. Results For field application of baculoviruses it is necessary to understand the interactive behaviour of virus and host in relation to polyhedra production and their efficacy as affected by passage through the homologous insect larval host. In this study the mean number of OBs harvested per larva and their efficacy by per os bioassay was examined with five different doses of SpltMNPV at each passage. The results revealed that the mean no. of OBs harvested per larva increases significantly with increase in the dose of SpltMNPV at each passage. Regardless of the dose, the mean number of OBs harvested per larva was significantly lower in P0 as compared to P1, P2, P3 and P4 (Table 1). No OBs were detected in the larvae that were fed on a diet plug inoculated with distilled water. The OBs harvested per larva in each dose at each passage were tested for their actual pathogenecity by per os bioassay to 2nd instar S. litura larvae using five different doses. The results revealed significant increase in the cumulative mortality percentage of 2nd instar S. litura larvae with an increase in the viral dose at each passage, with a significant positive correlation (r values; P0 = 0.770; P1 = 0.752; P2 = 0.740; P3 = 0.712; P4 = 0.832) between dose and mortality. Further, at each dose a gradual increase in the cumulative mortality percentage was observed over successive passages (Table 2). Probit analysis of the mortality data showed median lethal dose (LD50) estimates of 2.33 × 102, 2.07 × 102, 1.78 × 102, 1.32 × 102 and 1.02 × 102
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OBs/larvae at P0, P1, P2, P3 and P4, respectively (Table 3). At P0 the LD50 of SpltMNPV was significantly higher (F = 7.362; df = 4, 70; P < 0.001) than LD50 at successive passages i.e. P1, P2, P3 and
P4. It was found that the serially passed SpltMNPV was 11-56 times more infective than the original stock virus. The median time to feeding cessation (FT50) values at P0, P1, P2, P3 and P4 were 97, 89, 78,
Table 1—OBs harvested per larva at different doses of SpltMNPV after serial passage through the S. litura larvae for upto four generations (n = 150 per treatment) OBs/larva
Dose of Splt MNPV
P0
P1
P2
1.87×102
3.18×102 1.51×105 2.34×105 2 3 (0.07×10 ) (4.04×10 ) (4.94×103) 2 3 5 3.75×10 7.18×10 1.73×10 2.28×106 (0.66×102) (4.56×103) (5.80×104) 7.50×102 1.72×104 1.82×105 2.37×106 2 3 (1.97×10 ) (1.80×10 ) (1.12×105) 3 5 6 1.50×10 1.58×10 1.22×10 2.81×107 4 4 (1.30×10 ) (4.51×10 ) (4.30×105) 3 5 6 3.00×10 2.65×10 1.42×10 2.81×108 (2.16×103) (3.30×104) (4.91×106) *Kruskal-Wallis H test; P < 0.001; Values in parenthesis are SE
P3
P4
2.70×106 (5.29×104) 2.90×106 (4.53×104) 3.14×106 (6.77×104) 3.14×107 (2.03×105) 3.34×108 (1.42×106)
2.18×107 (1.73×105) 2.39×107 (4.55×105) 2.80×108 (4.30×106) 3.88×109 (4.62×107) 4.36×109 (4.21×107)
χ2-value*
P value*
21.086
< 0.001
19.095
< 0.001
21.011
< 0.001
20.104
< 0.001
23.104
< 0.001
Table 2—Cumulative mortality percentage of 2nd instar S. litura larvae with the OBs harvested after serial passage of SpltMNPV through S. litura larvae for upto four generations [Values are mean ± SE from 150 observations per treatment] OBs/larva
Cumulative mortality percentage (mean ± SE) P0
P1
P2
2
1.87×10 40.60 ± 1.53 47.80 ± 1.57 51.80 ± 1.53 3.75×102 55.00 ± 1.59 63.20 ± 1.62 68.80 ± 1.68 2 7.50×10 66.80 ± 1.65 74.20 ± 1.75 77.00 ± 1.79 1.50×103 74.80 ± 1.76 76.80 ± 1.81 84.20 ± 1.90 3.00×103 81.80 ± 1.87 84.20 ± 1.91 90.40 ± 1.95 *Kruskal-Wallis H test; P < 0.001; SE= standard error of means
P3
P4
57.80 ± 1.60 74.40 ± 1.74 82.20 ± 1.87 89.20 ± 1.90 92.20 ± 1.97
64.20 ± 1.64 78.80 ± 1.81 89.00 ± 1.89 92.00 ± 1.94 94.80 ± 2.01
χ2-value*
P value*
20.87 21.04 19.64 23.06 21.76
< 0.001 < 0.001 < 0.001 < 0.001 < 0.001
Table 3—Median lethal dose (LD50; OBs/larva), median time to feeding cessation (FT50; h.p.i.) and median survival time (ST50; h.p.i.) and associated statistics of S. litura larvae infected with different doses of original stock wild-type and serially passed SpltMNPV Passage
95 % confidence limits Lower Upper *
P0 P1
P2
P3
P4
LD50 FT50 † ST50 * LD50 † FT50 † ST50 * LD50 † FT50 † ST50 * LD50 † FT50 † ST50 * LD50 † FT50 † ST50 †
2.33 × 102 97.00 185.00 2.07 × 102 89.00 169.00 1.78 × 102 78.00 153.00 1.32 × 102 70.00 142.00 1.02 × 102 65.00 130.00
*Probit analyses Kaplan-Meier analyses; h p i = hours post inoculation
†
203 94.16 179.32 192 85.21 167.11 159 77.11 149.21 137 68.74 140.11 158 63.51 124.70
307 99.84 190.68 299 92.79 170.89 278 78.90 156.79 289 71.26 143.89 299 66.49 135.30
χ2/df 10.08/13 18.84/4 3.94/4 15.03/13 3.46/4 5.35/4 15.24/13 6.05/4 4.01/4 18.95/13 6.87/4 14.35/4 21.95/13 8.47/4 19.62/4
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Table 4—Cross infectivity of original stock wild-type (P0) and serially passed SpltMNPV (P1, P2, P3, P4) against 2nd instar S. exigua and S. obliqua larvae (n = 150 per treatment) [Values are mean ± from 150 observations per treatment] Cumulative mortality percentage Host
P0 S. exigua 63.32 ± 2.43 S. obliqua 58.70 ± 2.19 *Kruskal-Wallis H test; P < 0.001
P1 55.85 ± 2.03 50.20 ± 1.82
P2 40.22 ± 1.53 42.20 ± 1.65
70, and 65 hours post inoculation (h p i) respectively. The feeding time of S. litura larvae infected with original stock wild-type virus was significantly higher (F=5.963; df = 4, 70; P < 0.001) than those infected with serially passed virus. The FT50 of serially passed virus was 8-32 times less than the original stock wild-type virus. The ST50 values at P0, P1, P2, P3 and P4 were 185, 169, 153, 142 and 130 h p i., respectively and show a significant decrease (F=6.432; df = 4, 70; P < 0.001) with the successive passages. Regardless of dose the speed of kill of serially passed virus was more than the original stock wild-type virus. The ST50 of serially passed virus was 8-29 times less than the original stock wild-type virus. These results indicate that the efficacy of SpltMNPV is enhanced after serial passage through the S. litura larvae. The results on the cross infectivity of SpltMNPV against S. exigua (χ2=17.77, P < 0.001) and S. obliqua (χ2 = 16.34, P < 0.001 ) revealed that larval mortality decreases significantly with the successive passages of SpltMNPV through the S. litura larvae (Table 4). From this study it became evident that S. exigua and S. obliqua are susceptible to SpltMNPV, but serial passage of this virus through its natural host, S. litura makes it more host specific and limits its host range. Discussion The baculoviruses infect host cells, exploit what is there to replicate within them, produce polyhedra, and get transmitted from infected to healthy individuals (horizontal transmission), and also from parents to offspring’s (vertical transmission). They are effective and environmentally benign insect control agents and their transmission is a key to their persistence in the environment12. The SpltMNPV is used on a large scale in different parts of the country for management of the polyphagous defoliator, Spodoptera litura13 but, greater amount of variation in its efficacy has been reported14. In the present study the serially passed SpltMNPV
P3 30.00 ± 1.10 24.30 ± 1.05
P4 18.27 ± 0.74 15.75 ± 0.63
χ2-value*
P value*
17.77 16.34
< 0.001 < 0.001
was generated and tested through the homologous insect larval host. It showed a significant improvement in insecticidal properties in laboratory per os bioassays as compared to original stock wild-type virus. Mean number of OBs harvested per larva increased significantly in the successive passages. When tested for its actual pathogenecity, it was found that the serially passed SpltMNPV was 11-56 times more infective than the original stock wild-type virus. The harvested OBs resulted in a quicker cessation of feeding and the FT50 of serially passed virus was 8-32 times less than the original stock virus. This finding is of practical utility since prevention of feeding damage is of primary importance. Further, onset of death in 2nd instar S. litura larvae was quicker as the ST50 of serially passed virus was 8-29 times less than the original stock wild-type virus. The increase in the OBs yield of a serially passed baculovirus is of great importance for the in vivo industrial production of baculoviruses and for evaluating the logistics and economics of virus production in virus factories. The original stock wild-type (P0) and serially passed SpltMNPV (P1, P2, P3, P4) when tested for their cross infectivity against S. exigua and S. obliqua revealed that larval mortality decreased significantly with the successive passages of SpltMNPV through the S. litura larvae. These results indicate that the S. exigua and S. obliqua are susceptible to SpltMNPV, but serial passage of this virus through its natural host, S. litura makes it more host specific and limits its host range. Since, baculoviruses consist of a mixture of different, often very similar genotypes these variations may influence some biological properties, such as the virulence to their specific target host. The composition of this mixture depends among other factors on the genotype of the host used to multiply the baculovirus. Although such variations during serial passage experiments were noticed by many workers it is assumed that increase in the polyhedra production in successive passages increased the infectivity, feeding cessation and speed
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of kill of host larvae. These findings are of great utility, as secondary transmission under field conditions is important for the success of the program for baculoviruses use as biopesticides. It is proposed that for in vivo production systems the nucleus stock of all baculoviruses must be serially passed through the homologous insect larval host for at least 4 generations for greater efficacy and ecological specificity. Acknowledgement Thanks are due to Dr. R. J. Rabindra for suggestions and guidance, to the Director of Research, SKUAST-Jammu for support, sincere thanks are also due to the Department of Science and Technology, Government of India for financial assistance, and to the National Bureau of Agriculturally Important Insects (NBAII) for encouragement and co-operation.
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