Sarwar et al.

Egyptian Journal of Biological Pest Control

https://doi.org/10.1186/s41938-021-00403-8
(2021) 31:58
Egyptian Journal of
Biological Pest Control

RESEARCH Open Access

Evaluation of indigenous the

nucleopolyhedrovirus (NPV) of Spodoptera
litura (Fabricius) (Lepidoptera: Noctuidae) in
combination with chlorantraniliprole
against Spodoptera species
Ghulam Sarwar1, Naeem Arshad Maan2, Muhammad Ahsin Ayub3, Muhammad Rafiq Shahid1,
Mubasher Ahmad Malik4* and Muhammad Farooq1,5*

Abstract
Background: The armyworms, Spodoptera exigua (Hübner), and S. litura (Fabricius) (Lepidoptera: Noctuidae) are
polyphagous pests of many cash crops. Heavy crop losses have been reported for the fruit and vegetable crops
each year owing to the diverse impact on global economies. The present study was aimed to sort out a novel
method of pest control using the insect’s own nucleopolyhedrosis virus (NPV) alone and in combination with a
new chemistry insecticide chlorantraniliprole.
Results: In the study, the effect of indigenous isolated nucleopolyhedrovirus (NPV) and the chemical insecticide
(chlorantraniliprole) formulations against the 2nd and 4th larval instars of S. litura and S. exigua, collected from the
different geographical region of Punjab (Pakistan) province, was evaluated. Three concentrations of the NPV isolate,
sub-lethal (1 × 104, 6 × 104 POB ml−1), lethal (3 × 105 POB ml−1), and chlorantraniliprole 0.01 μl l−1, were applied
alone and in combination against the 2nd and 4th larval instars of both pest species. The lethal concentration of
NPV + chlorantraniliprole exhibited synergistic interaction and caused high larval mortality against both instars,
while in all other combinations, additive effect was observed. Moreover, NPV + chlorantraniliprole at lethal
concentration exhibited decreased pupation, adult emergence, and egg eclosion.
Conclusion: The implications of using NPV alone and in combination with an insecticide are discussed briefly in
this study.
Keywords: Nucleopolyhedrovirus, Chlorantraniliprole, Spodoptera exigua, S. litura, Combined effect

Background including major crops such as sugar beet, cotton, soy-

Spodoptera exigua (Hübner) and S. litura (Fabricius) bean, and potatoes (Fu et al. 2017; Luna-Espino et al.
(Lepidoptera: Noctuidae) are native to Asia (Farahani 2018). The mechanism of resistance in S. exigua has
et al. 2012). Both attack more than 90 plant species been reported against different classes of insecticides
(Ishtiaq et al. 2012). Furthermore, extensive use of syn-
* Correspondence: [email protected]; thetic insecticides has resulted in enormous environ-
[email protected]
4
mental pollution and a negative impact on non-target
Department of Entomology, University of Agriculture, Faisalabad, Pakistan
1 insect species (Ahmad et al. 2008). This situation forced
Cotton Research Station, Ayub Agricultural Research Institute, Faisalabad,
Pakistan the researchers to develop eco-friendly alternatives
Full list of author information is available at the end of the article

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Sarwar et al. Egyptian Journal of Biological Pest Control (2021) 31:58 Page 2 of 7

(Sayyed et al. 2012). Baculoviruses consist of rod-shaped, artificial diet until pupation. The F1 generation of reared
dsDNA-occluded viruses that infect insect pests belong- test insects was mass cultured and used in bioassay
ing to the orders Lepidoptera, Hymenoptera, and Dip- experiments.
tera (Williams et al. 2017). Due to their fast-killing Chlorantraniliprole is a new insecticide with a novel
nature and host specificity, some of these viruses have mode of action. The semi-viscous formulation of it is
been used as commercial bioinsecticides (Sosa-Gómez provided byDuPont™ Operations Private Limited,
2017). Baculoviruses comprise two genera, granulo- Pakistan, containing 20% W/V active ingredient Rynax-
viruses (GVs) and nucleopolyhedroviruses (NPVs). The pyr (200 ml/l), and 80% other ingredients (800 ml/l) was
S. litura multiple nucleopolyhedroviruses (SlM-NPV) used for bioassays. The formulation was applied by dis-
have been molecularly characterized, and their biological solving in distilled water at a concentration of 0.01 μl l−1
activity has been tested against S. litura populations in mixed with the diet, thoroughly mixed in an electric
Pakistan (Ahmad et al. 2018; Ali et al. 2018; Ayyub et al. shaker for 30 s.
2019) and all over the world (Laarif et al. 2011 and
Kumar et al. 2012a, 2012b). Viral isolation and suspension preparation
Chlorantraniliprole is a Ryanoid class insecticide NPV-infected larvae were macerated, and obtained poly-
(anthranilic diamide), which demonstrates maximum hedral (occlusion bodies) were passed through the
mortality against the target insect pests even at a low ap- cheesecloth to remove large debris. Semi purified occlu-
plication rate (Cordova et al. 2007). Furthermore, this in- sion bodies were centrifuged at 3000 rpm for 45 min.
secticide is compatible to use with NPV to enhance the For further purification of the occlusion bodies, the
pathogenic efficacy. Ryanodine receptors are ion chan- resulting suspension was centrifuged at a high speed (16,
nels that maintain calcium levels in the sarcoplasmic 000 rpm) for 10 min. The suspension was transferred to
reticulum of muscle cells. Chlorantraniliprole opens the glass vials and stored at 4 °C. The concentrations of
ryanodine receptor for an uncontrolled release of cal- polyhedral occlusion bodies (POBs) as a stock solution
cium from muscle cells, which reduces feeding, paralysis, (2 × 108 POB l−1, 3 × 108 POB l−1, and 4 × 108 POB l−1)
and ultimately death of target species (Lahm et al. 2007). were prepared from indigenous NPV using Neubauer
Previous studies represent the efficacy of NPVs of spe- hemocytometer (Shapiro et al. 2005). From the stock so-
cific regions, but until now, no published data is avail- lution, 1 ml suspension from each concentration was
able on the effect of a local isolate of NPV against prepared as NPV-1 (1 × 106 POB ml−1), NPV-2 (1 × 107
Spodoptera species when applied in combination with POB ml−1), and NPV3 (4 × 108 POB ml−1). For bioassay,
chlorantraniliprole. Therefore, the present study was 2 sub-lethal concentrations of NPV (1 × 104; 6 × 104)
conducted to evaluate the effect of the local isolate of S. and one lethal concentration of NPV (3 × 105 POB
litura NPV alone and in combination with chlorantrani- ml−1) were used to study the synergistic, additive, or an-
liprole against Spodoptera spp. tagonism effect against Spodoptera species (these sub-
lethal concentrations were selected from a preliminary
Methods bioassay).
Rearing of insects
About 150 larvae (3rd and 4th instars) each of S. litura Insect bioassay
and S. exigua were collected by plucking the infested The 2nd and 4th larval instars of S. litura and S. exigua
leaves from the field during the winter cropping season were tested by NPV and chlorantraniliprole alone and in
of 2018–2019 to establish insect cultures. The larvae combination. The desired concentration of NPV from
along with the leaves were carried in plastic jars and the stock solution was mixed in the artificial diet for
shifted to the rearing cages in the laboratory with the even distribution. All the bioassays were conducted
help of a camel hairbrush. Pest species were identified under Biosafety Level 2 to avoid any source of contamin-
based on the morphological characters traced with the ation or leakage of the pathogens. The experiments were
help of a taxonomic key. The larvae were mass-reared conducted in a plastic vial (base radius of 2.4 cm ×
on artificial diets at 25 ± 2 °C, 70 ± 5% RH and a photo- height 6 cm). Sub-lethal concentrations of NPV-1 (1 ×
period of 14:10 h (L:D). The standard established labora- 104 POB/larva) and NPV-2 (6 × 104 POB/larva) and le-
tory protocol was followed (Ahmad et al. 2020) The thal concentration of NPV-3 (3 × 105 POB/larva) were
larvae were pupated in vials containing the artificial diet. used to calculate the percentage mortality, pupation,
The emerged adults (approximately 120 males and fe- adult emergence, and egg eclosion from the 2nd and 4th
males) were shifted from vials to plastic cages/boxes for larval instars of S. litura and S. exigua. Each plastic vial
mating and egg-laying purposes. The adults were pro- contained a 2-mm cube of diet previously soaked in re-
vided with 10% honey solution in cages. After hatching, spective NPV and chlorantraniliprole concentration.
the neonate larvae were shifted to vials containing the Twenty pre-starved (24 h) larvae of both instars of S.
Sarwar et al. Egyptian Journal of Biological Pest Control (2021) 31:58 Page 3 of 7

litura and S. exigua were allowed to feed the artificial 60.48% mortality, respectively. A combination of the le-
diet until complete consumption in the individual con- thal concentration of NPV (3 × 105 POB ml−1) with a
tainer. After being fed, the larvae were removed and sub-lethal concentration of chlorantraniliprole (0.01
then released in plastic vials containing an artificial diet ppm) showed a synergistic action (CTF ≥ 20) with a
until the larvae died or pupated. The bioassay was con- mortality of 89.35%, while the rest of the combination
ducted at 25 ± 2 °C, 70 ± 5% RH and L16:D8 h photo- exhibited an independent effect (CTF ≤ 20) (Table 1).
period. Each treatment was replicated 3 times, and each Similarly, integration of a sub-lethal + lethal combin-
bioassay was repeated thrice independently. The number ation of NPV on the 4th instar larvae showed the highest
of dead insects was recorded daily, and surviving insects level of mortality in the 4th instar larvae than the chlor-
were monitored for % pupation and adult emergence. antraniliprole alone treatment. The combined treatment
Larvae were considered dead when they were unable to of NPV + chlorantraniliprole interaction produced an
move (Ma et al. 2008). Emerged adults from each treat- antagonistic or additive effect while combing chlorantra-
ment were shifted from vials to plastic cages/boxes for niliprole with NPV at 3 × 105 POB ml−1 showed a mor-
mating and egg-laying purposes. tality rate of 75.70 ± 1.76% (Table 1).

Statistical analysis
The mortality rate of targeted insects was corrected Mortality of S. exigua
using Abbott’s formula (Abbott 1925). The co-toxicity Sub-lethal effect of chlorantraniliprole (0.01 ppm)
factor (CTF), observed mortality (Oc), and expected against the 2nd instar larvae showed 33.66% mortality
mortality (Oe) were calculated using the equation (Man- rate, whereas three concentrations of NPV (sub-lethal 1
sour et al. 1966) CTF = (Oc − Oe)/Oe × 100 to study × 104, sub-lethal 6 × 102 POB ml−1, and lethal 3 × 105
the synergistic (cytotoxicity factor above 20), additive POB ml−1) produced 30.48, 42.70, and 54.92% mortality,
(cytotoxicity factor between 20 and − 20), and antagonis- respectively. Simultaneous action of NPV (3 × 105 POB
tic (cytotoxicity factor − 20 or above) interaction be- ml−1) with a sub-lethal concentration of chlorantranili-
tween the treatments. The means were compared by prole (0.01 ppm) showed a synergistic action (CTF ≥ 20)
using the Tukey-Kramer HSD test at P = 0.05 (Sokal with a mortality of 84.02%, while the rest of the combin-
and Rohlf 1995). ation exhibited an independent effect of each other
(CTF ≤ 20) (Table 2). Similarly, the integration of a sub-
Results lethal + lethal combination of NPV on the 4th instar lar-
Mortality of S. litura vae showed the highest level of mortality in the 4th in-
The 2nd instar larvae showed a 38.10% mortality rate star larvae than the chlorantraniliprole alone treatment.
when exposed to a sub-lethal concentration of chloran- In the combined treatment of NPV + chlorantraniliprole,
traniliprole (0.01 ppm), while the larvae treated with 2 interactions produced an additive effect, while combin-
NPV (sub-lethal 1 × 104, sub-lethal 6 × 104 POB ml−1, ing chlorantraniliprole with NPV at 3 × 105 POB ml−1
and lethal 3 × 105 POB ml−1) showed 34.02, 48.41, and caused enhanced mortality of 57.30% (Table 2).

Table 1 Mortality percentage of the 2nd and 4th instars of Spodoptera litura larvae treated with nucleopolyhedrovirus alone and in
combination with chlorantraniliprole
Treatments 2nd instar larvae (n = 20) 4th instar larvae (n = 20)
(POB ml−1)
Mortality% Expected mortality CTF Interaction Mortality % Expected mortality CTF Interaction
NPV-1 (1 × 104) 34.02 20.43
NPV-2 (6 × 104) 48.41 32.69
NPV-3 (3 × 105) 60.48 42.29
Chl 38.10 24.43
NPV-1+Chl 74.13 75.58 − 6.63 Additive 42.52 43.61 − 0.85 Additive
NPV-2+Chl 85.56 75.67 14.54 Additive 55.53 54.17 12.14 Additive
Npv-3+Chl 89.35 79.42 20.16 Synergistic 75.70 65.91 19.13 Synergistic
Control 2.38 1.59
F 251 275
DF 7,71 7,62
P < 0.01 < 0.01
CFT co-toxicity factor
Sarwar et al. Egyptian Journal of Biological Pest Control (2021) 31:58 Page 4 of 7

Table 2 Mortality percentage of the 2nd and 4th instars of Spodoptera exigua larvae treated with nucleopolyhedrovirus alone and in
combination with chlorantraniliprole
Treatments 2nd instar larvae (n = 20) 4th instar larvae (n = 20)
(POB ml−1)
Mortality% Expected mortality CTF Interaction Mortality % Expected mortality CTF Interaction
NPV-1 (1 × 104) 30.48 15.89
NPV-2 (6 × 104) 42.70 25.51
5
NPV-3 (3 × 10 ) 54.92 38.68
Chl 33.66 20.56
NPV-1+Chl 65.90 60.25 1.78 Additive 37.59 31.12 10.14 Additive
NPV-2+Chl 71.53 68.24 3.74 Additive 46.67 38.31 17.21 Additive
Npv-3+Chl 84.02 70.13 26.22 Synergistic 57.30 45.12 21.57 Synergistic
Control 3.17 3.18
F 271 117
DF 7,71 7,62
P < 0.01 < 0.01
CFT co-toxicity factor

Pupation percentage of S. litura instar F7,71 = 221, p ≤ 0.01, 4th instar F7,71 = 342, p ≤
A significant interaction was found between NPV and 0.01). The lethal combination effect of NPV with chlor-
chlorantraniliprole in terms of the number of larvae that antraniliprole was synergistic with a high rate of mortal-
enter the pupation stage (2nd instar F7,71 = 257, p ≤ ity in the 2nd and 4th larval instars of S. exigua and a
0.01, 4th instar F7,71 = 319, p ≤ 0.01). The integration low percentage of pupation, while the rest of the combi-
effect of both agents was lethal as compared to their sole nations of lethal concentration of NPV with chlorantra-
applications. The combined lethal concentration of NPV niliprole produced synergistic interaction with
with chlorantraniliprole against the 2nd and 4th larval decreasing percentages of pupation (5.33 and 12.307%,
instars of S. litura produced synergistic interaction with respectively). On the other hand, a low concentration of
a decreasing percentage of pupation (3.11 and 4.15%, re- chlorantraniliprole produced the highest percentage of
spectively). In contrast, the highest percentage of pupa- pupation (51.89 and 41.22%, respectively) (Table 4).
tion (40.67 and 36.26%, respectively) was observed at a
low concentration of chlorantraniliprole (Table 3). Adult emergence and egg eclosion percentage of S. litura
Adult emergence rate was inversely proportional to all
Pupation percentage of S. exigua the treatments of NPV and chlorantraniliprole (Table 3).
Among treatment of NPV and chlorantraniliprole, the The lowest adult emergence (8.22 and 1.00%) and egg
interaction was significant for pupation percentage (2nd eclosion (2.94 and 4.32%) were observed when a high

Table 3 The mean pupation, adult emergence, and egg eclosion (% ± SE) of the 2nd and 4th instars (n = 20) of Spodoptera litura
larvae treated with nucleopolyhedrovirus alone and in combination with chlorantraniliprole
Treatments Pupation Adult emergence Egg eclosion
(POB ml−1)
2nd instar 4th instar 2nd instar 4th instar 2nd instar 4th instar
NPV-1 (1 × 104) 71.81 ± 2.22 74.63 ± 2.23 71.11 ± 2.73 64.44 ± 1.93 67.21 ± 2.70 76.63 ± 3.12
NPV-2 (6 × 104) 61.01 ± 2.57 65.81 ± 1.93 60.15 ± 2.74 52.96 ± 2.11 54.99 ± 3.84 65.65 ± 3.10
NPV-3 (3 × 105) 54.67 ± 1.47 50.52 ± 2.18 50.00 ± 1.96 48.52 ± 1.97 42.38 ± 2.69 49.33 ± 2.61
Chl 40.67 ± 2.05 36.26 ± 2.14 47.78 ± 2.51 31.85 ± 2.83 35.00 ± 3.45 38.04 ± 3.91
NPV-1+Chl 25.15 ± 2.41 24.44 ± 1.27 47.41 ± 1.77 22.16 ± 1.19 24.93 ± 2.58 29.29 ± 2.56
NPV-2+Chl 2.63 ± 2.18 13.67 ± 2.11 27.96 ± 2.55 6.30 ± 2.63 11.37 ± 3.62 18.54 ± 3.76
NPV-3+Chl 03.11 ± 0.00 4.15 ± 1.87 8.22 ± 1.58 01.00 ± 0.10 2.94 ± 2.35 4.32 ± 2.38
Control 96.41 ± 2.98 97.5 ± 2.37 94.85 ± 2.15 95.07 ± 1.34 87.37 ± 4.63 92.74 ± 4.02
F 257 319 291 366 44 53
Df 7,71 7,71 7,71 7,71 7,71 7,71
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
Sarwar et al. Egyptian Journal of Biological Pest Control (2021) 31:58 Page 5 of 7

Table 4 The mean pupation, adult emergence, and egg eclosion (% ± SE) of the 2nd and 4th instars (n = 20) of Spodoptera exigua
larvae treated with nucleopolyhedrovirus alone and in combination with chlorantraniliprole
Treatments Pupation Adult emergence Egg eclosion
(POB ml−1)
2nd instar 4th instar 2nd instar 4th instar 2nd instar 4th instar
NPV-1 (1 × 104) 82.52 ± 1.43 78.59 ± 2.58 69.89 ± 1.75 79.26 ± 1.88 74.94 ± 3.40 87.23 ± 5.55
NPV-2 (6 × 104) 72.70 ± 1.14 71.30 ± 2.54 64.44 ± 2.65 75.56 ± 2.12 62.72 ± 2.60 77.54 ± 4.25
5
NPV-3 (3 × 10 ) 60.44 ± 1.59 65.52 ± 2.14 52.22 ± 3.14 65.93 ± 2.17 49.53 ± 3.17 58.08 ± 3.99
Chl 51.89 ± 2.45 41.22 ± 1.93 44.07 ± 1.17 51.74 ± 2.38 40.68 ± 5.26 49.29 ± 5.07
NPV-1+ Chl 30.37 ± 0.00 34.07 ± 2.24 36.67 ± 3.06 50.37 ± 1.91 33.38 ± 2.17 42.14 ± 2.98
NPV-2+Chl 21.59 ± 1.85 20.56 ± 1.48 12.00 ± 2.66 32.44 ± 1.82 20.99 ± 2.10 25.07 ± 4.88
NPV-3+Chl 5.33 ± 1.37 12.30 ± 3.17 4.15 ± 2.74 17.59 ± 1.54 5.50 ± 1.23 9.55 ± 2.26
Control 97.29 ± 2.58 96.63 ± 2.65 95.56 ± 1.12 96.69 ± 2.65 90.63 ± 2.71 93.89 ± 8.56
F 221 342 168 219 36 26
Df 7,71 7,71 7,70 7,71 7,71 7,71
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

concentration of NPV (3 × 105) was combined with ornamental, and field crops have been shown more vul-
chlorantraniliprole against the 2nd and 4th larval instars, nerability to the pathogenicity of NPV infection (Rios-
respectively. Adult emergence and egg eclosion rates Velasco et al. 2011). Early instar larvae were more suc-
were high at the sole application of chlorantraniliprole. cumb to pathogenic infection than older larvae because
The combined application of microbes produced fatal ef- early ones usually consumed more surface of viral
fects on adult emergence and egg eclosion as compared treated leaves (Gothama et al. 1995). It is likely due to
to their sole application. more deposition of cuticular melanism in the oldest lar-
vae which prevents the entrance of pathogens (Wilson
Adult emergence and egg eclosion percentage of S. et al. 2001).
exigua Chlorantraniliprole is a novel insecticide (Cordova
The relationship between adult emergence rate and bio- et al. 2006). In the present study, it showed a good con-
assay treatment was inversely proportion (Table 4). The trol against both larval instars of Spodoptera spp., but
percentage of adult emergence (4.15 and 17.59%) and the 2nd instar larvae showed high susceptibility. Low
egg eclosion (5.50 and 9.55%) of the 2nd and 4th larval concentrations of chlorantraniliprole gave fair control
instars, respectively, were the lowest when a lethal con- against lepidopteron insect pests (Cordova et al. 2006;
centration of NPV (3 × 105 POB ml−1) was combined Wakil et al. 2013; Wang et al. 2013). Synergistic inter-
with chlorantraniliprole. Sole application of both agents action was produced in the present study when a high
produced a high percentage of adult emergence and egg concentration of NPV was combined with chlorantranili-
eclosion. The integration effect of biorational insecti- prole. Similar synergistic interaction was reported by
cides was toxic for adult emergence and egg eclosion Wakil et al. (2012) for Bacillus thuringiensis + chloran-
than that of their sole application. traniliprole. On the other hand, additive or synergistic
effects have been observed at high concentrations of
Discussion NPV + biorational insecticides (Wakil et al. 2013). A
The practical use of microbial insecticides is limited due possible reason for NPV-Ch synergism is that the selec-
to host specificity and slow in action. Combing biora- tion pressure of insecticides makes them more vulner-
tional insecticides with microbes is a premixing strategy able to viral occlusion bodies (OB). Exposure to
to manage insecticide overuse (Kumar et al. 2012a, chemical insecticide increases the developmental period,
2012b). Biological insecticides have been used to control and this allows the viral infection to develop in the in-
lepidopteran insect pests of agricultural, horticultural, sect (Kumar et al. 2008). The antagonistic or additive
and forest importance (Nathan and Kalaivani 2006). interaction in the present study might be due to a de-
A molecularly identified isolate of NPVs has been used crease in normal feeding on treatment or a change of
to control Spodoptera spp. in Pakistan (Ahmad et al. gut pH (El-Helaly and El-Bendary 2013). The results of
2018; Ali et al. 2018) and worldwide (Luna-Espino et al. the present study showed that the sub-lethal concentra-
2018). In the present study, after the viral infection, Spo- tion of chlorantraniliprole + high concentration of NPV
doptera spp. showed stunted growth and prolonged lar- could be a better strategy to manage insecticidal
val and pupal duration. Insect pests of vegetable, resistance.
Sarwar et al. Egyptian Journal of Biological Pest Control (2021) 31:58 Page 6 of 7

Conclusion Ahmad JN, Mushtaq R, Ahmad SJN, Maqsood S, Ahuja I, Bones AM (2018)
The obtained data showed that the lethal concentration Molecular identification and pathological characteristics of NPV isolated from
Spodoptera litura (Fabricius) in Pakistan. Pak J Zool 50(6):2229–2237
of NPV in combination with chlorantraniliprole caused Ahmad M, Sayyed AH, Saleem MA, Ahmad M (2008) Evidence for field evolved
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boratory conditions and could be suggested as an effect- from Pakistan. Crop Protection 27(10):1367–1372. https://doi.org/10.1016/j.
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but further experiments under field conditions would be Pakistani isolate of Spodoptera litura nucleopolyhedrovirus. Biocontrol Sci
very helpful for the integration of NPV biopesticides Technol 28(1):20–33. https://doi.org/10.1080/09583157.2017.1409339
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polyhedrosis virus; RH: Relative humidity; L:D: Light:dark; W/V: Weight by (2006) Anthranilic diamides: a new class of insecticides with a novel mode of
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