Impact of simultaneous treatment of gamma irradiation and Bacillus thuringiensis on cotton leaf worm Spodoptera littoralis (Boisd.) (Noctuidae: Lepidoptera)

The two biological control methods, inherited sterility technique (IST) and Bacillus thuringiensis (Bt), are considered as the two promising methods for lepidopteran pest management. Simultaneous treatment with both techniques was assessed against the cotton leaf worm, Spodoptera littoralis (Boisd.) (Noctuidae: Lepidoptera) compared to individual treatment for developing the lepidopteran management program. Pupation and adult emergence were significantly lower in simultaneous treatment than irradiated insects individually. Larval mortality test estimated a LC50 of 1.6 × 104 IU/mg (Bt), in which decreased to 1.5 × 104 IU/mg (Bt and 75 Gy), and 7.7 × 103 IU/mg (Bt and 125 Gy). Insect survival was prolonged for both individual and simultaneous treatments. The ratio of males was 3.5-fold to females in 1.5 × 104 IU/mg and 75 Gy, while threefold of males to females was recorded in the treatments of 3.75 × 103 IU/mg and 75 Gy, 7.5 × 103 IU/mg and 75 Gy, 7.5 × 103 IU/mg and 125 Gy and 1.5 × 104 IU/mg and 125 Gy. Results of fecundity, fertility and sperm transfer of F1 males were significantly lowered in irradiated insect combined with Bt than individual treatment. Gamma irradiation treatment combined with Bt concentrations achieved a high reduction of S. littoralis, and the toxicity effect of Bt increased as compared with individual treatments taking advantage as compatible control tactics. Simultaneous treatment of IST and Bt could be considered as a competent approach for S. littoralis population suppression.


Background
Over the last decades, attempts to promote safe and sustainable alternative control methods have been intensively studied and interestingly engrossed, whereas the extensive use of conventional pesticides against lepidopteran insect pests has had adverse impacts on the environment and its fauna (Mulé et al. 2017). The program that incorporates various biocontrol techniques synchronously for growing healthy crops and avoiding pesticide hazardous has been approved globally, which is the concept of promising integrated pest management (Singh et al. 2019). Evidence, insect resistance against several control agents, is a critical point for the design of an effective control programmer. Simultaneous treatment protocol brings regenerative pest management a step closer for a successful program against targeted insect pests in particular that developed resistance to control agents. Sterile insect technique (SIT) is a promising genetic method for pest population suppression, based on releasing sterile insects systemically in area wide after Page 2 of 7 Sayed et al. Egyptian Journal of Biological Pest Control (2022) 32:102 being exposed to Ionizing radiation (Dyck et al. 2021). Lepidoptera are radioresistance requiring high doses to induce sterility in adult moths. These sterilizing doses cause deleterious effect of irradiated insect that reduce the efficacy of sterile moths to compete the wild type in nature (Marec and Vreysen 2019). Consequently, inherited sterility technique (IST) offers significant advantages over classical sterile release method for lepidopteran pests, and sterility in the F 1 generation is induced by irradiating the parents with sub-sterilizing doses which could be partial sterility (Bloem et al. 2003). Although IST has been reported as an effective technique for lepidopteran population suppression, it was supposed that it is a complementary component of an area-wide and sustainable long-term strategy in the pest management programs rather than a stand-alone technique (Suckling et al. 2017). Simultaneous treatment of IST with another compatible control method could improve the SIT program efficacy (Benedict 2021) that it is essential to suppress the population density of insects to a level, which makes it economically feasible to release a dominant population of the sterile insect over an extended period. Various biorational pesticides have been suggested to use in combination with SIT for pest management (Howse et al. 2007). Biocontrol methods can be stimulated as a supplementary control tool to IST with regard to an environmentally safe, effective and alternative method. Attempts have been conducted to use agents in combination with irradiation treatment such as the predator on Tuta absoluta (Cagnotti et al. 2016), the parasitoid on Spodoptera exigua (Hübner) (Carpenter and Sheehan 1996), the Baculovirus on Spodoptera littoralis (Sayed and El-Helaly 2018), the entomopathogenic nematode on Galleria mellonella (Salem et al. 2020) and the entomopathogens fungi on S. littoralis (Gabarty et al. 2019). Recently, Bacillus thuringiensis (Bt) is considered one of the most effective entomopathogenic against lepidopteran pests (Raymond et al. 2010). It is widely used in many regions over the world due to its harmless to vertebrate, non-target organism and other ecosystems (Bravo et al. 2011). Both IST and Bt offer a way to equate the two separate treatments with a simultaneous one. Both treatments could be used against lepidopteran insects effectively but are adjusted with regard to the different conditions of operation (e.g., time, doses and species). It has been found that the use of IST program resulted to suppressing resistance of pink bollworm, Pectinophora gossypiella (Saund.) to genetically engineered cotton crop contains Bt (Tabashnik et al. 2010). The advantages of this combination treatment have been demonstrated by the economic benefits achieved (Tabashnik et al. 2021). The cotton leaf S. littoralis is one of the most destructive lepidopteran pests in tropical and subtropical area that cause damage to more than 100 different crops and vegetable plants considering it as an economic pest in different countries (Sayed et al. 2021). In this context, the present trial aimed to provide assessment of the simultaneous treatment, Bt and IST as promising methods for S. littoralis management, which has a multiple regression optimization perspective, taking into account the effective dose, insect response and interaction of the two techniques.

Insect
The colony of cotton leaf worm, S. littoralis, was maintained under laboratory conditions at 25 ± 2 0 C and 65% R.H. Full-formed pupae were placed in an adult rearing cage (40 × 20 × 10 cm) with small cups, containing pieces of cotton wool soaked with 10% sugar for feeding the emergent moths. Egg masses were collected daily, and treated with a formalin (10%) for surface sterilizing against pathogens as suggested by (Connell 1981), and then, they were transferred to glass gars (1600 cc) covered with muslin cloth until hatching. The newly hatched larvae were transferred to rounded plastic bowls (70 × 15 cm) provided with semi-artificial diets that were developed by (Sayed et al. 2021) and covered with both muslin cloths until pupating.

Irradiation
The source of gamma radiation used during the present study was from a Cobalt 60 ( 60 Co) irradiator; the dose rate of irradiation source was 407.2 Gy/h. The full-grown male pupae of S. littoralis were irradiated (24-48 h before emergence of adults) with sub-sterilizing doses of gamma radiation (75 and 125 Gy) (Sayed and El-Helaly 2018). After the adult emergence, irradiated adult males were paired with untreated adult females. A group of untreated pairs were used as control. The deposited eggs were collected to continue the F 1 generation.

Bacillus thuringiensis
The commercial product of Bacillus thuringiensis, aizawai (Bta) strain (XenTari, ABTS-1857, 540 g/kg) at 15,000 international unit IU per mg was used in this study. Stock solution of the compound was prepared in tap water and then diluted by water to series of concentration solutions 3.75 × 10 3 , 7.5 × 10 3 , 1.5 × 10 4 , 3.0 × 10 4 and 6.0 × 10 4 IU/mg. Newly molted 3 rd larvae were treated with Bta by feeding on a semi-artificial diet treated with the previous concentrations. One milliliter of each concentration was spread on the surface plate (20 × 10 × 3 cm) containing 50 ml of different semiartificial diets, and each treatment was repeated in five replicates with 50 larvae each. The mortality responses of larvae were daily recorded.

Bioassay studies
The F 1 larvae from P 1 male irradiated as full-grown pupae with 75 and 125 Gy were divided into two groups; the first was treated as 3 rd larvae with different concentrations of Bta, while the second group was left without Bta treatment for comparison. Moreover, another group of un-irradiated larvae was treated by Bta only. In addition, a group of untreated control was left for comparison. Larval mortality and larval duration, pupal duration, pupation, emergence, surviving from larvae to adult emergence and sex ratio were calculated for all individual and combined treatments. Resulted male moths from each treatment were paired with un-irradiated female moths, whereas the number of eggs/female and egg hatching were recorded. Moreover, numbers of mating/ female were assessed upon death, and the female moths were dissected to estimate the number of sperm transferred (Spermatophores in the bursa copulatrix). Five replicates were used for each treatment. Each replicate consisted of 20 larvae.

Statistical analysis
Mortality response of various treatments either Bt concentrations or simultaneous treatment of Bta and gamma radiation doses (75 and 125 Gy) was tested using the Probit analysis which were expressed as a percentages, and the median lethal concentration (LC 50 ) was estimated. Analysis of variance (ANOVA) technique was used to analyzed the data of biological studies and the averages were analyzed using Tukey's range test (F-tests) (P = 0.05) (Gurvich and Naumova 2021). Data of egg hatch, pupation and emergence (%) were transformed by arcsine tables, while the means and standard errors were from original data.

Results
Mortality response assay of S. littoralis larvae Figure 1 presents the mortality responses of treated S. littoralis larvae to Bta concentrations. The LC 50 was 1.6 × 10 4 IU/mg, and this response was slightly increased for F 1 larvae irradiated with 75 Gy, where the LC 50 was 1.5 × 10 4 IU/mg, while the toxicity was highly increased for F 1 larvae irradiated with 125 Gy (twofold), since the LC 50 was 7.7 × 10 3 IU/mg. Based on these results, there was a positive interaction effect between the Bta concentrations and sub-sterilizing doses of gamma irradiation, where the F 1 larvae were more susceptible to the Bta than to the un-irradiated ones.

Bioassay studies
Regarding the mortality response studies, the Bta concentrations (3.75 × 10 3 , 7.5 × 10 3 and 1.5 × 10 4 IU/mg) were selected to further experiments because no yields (pupae and adults) were obtained at 3.0 × 10 4 and 6.0 × 10 4 IU/ mg. Survivals of remaining alive larvae of various treatments, irradiation, Bta and simultaneous treatments are listed in Fig. 2. In female line, the durations of larvae were significantly prolonged at 7.5 × 10 3 IU/mg, 1.5 × 10 4 IU/ mg, 125 Gy and all simultaneous treatments compared to 75 Gy, 3.75 × 10 3 IU/mg and control treatment (F 11,59 = 58.1, p < 0.0001). The same trend was recorded in male line, whereas the duration reached 21.06 days of Page 4 of 7 Sayed et al. Egyptian Journal of Biological Pest Control (2022)  Results of pupation and adult emergence resulted from different treatments showed that the percentages of pupation and adult emergence were reduced gradually by increasing the Bta concentration levels and higher significant reductions were caused in simultaneous treatment than the individual and control treatments. Data of the percentage of larvae to reach pupae were highly significant between the different treatments (F 11,59 = 298.5, P < 0.0001), where low pupation percentages (35.06, 56.26, 57.6 and 61.9%) were recorded in the treatments of 1.5 × 10 4 IU/mg and 125 Gy, 1.5 × 10 4 IU/ mg, 3.75 × 10 3 IU/mg and 125 Gy and 1.5 × 10 4 IU/mg and 75 Gy, respectively, as compared to the high pupation percentages ( 95.2, 80.3 and 80.2%) of control, 75 Gy and 125 Gy (Fig. 3). In the same table, adult emergence percentages were highly significant among the different treatments (F 11,59 = 561.4, P < 0.0001), where the adults were emerged in low percentage (14,9%) of 1.5 × 10 4 IU/ mg and 125 Gy as compared to (80.1,80.5,70.3 and Fig. 3 Pupation and adult emergence of Spodoptera littoralis F 1 progeny resulting from irradiated P 1 pupae with gamma radiation doses (Gy), treated with Bta concentrations (IU/mg) as larvae and simultaneous treatment of Bta concentrations and gamma radiation doses Fig. 4 Sex ratio of Spodoptera littoralis F 1 progeny resulting from irradiated P 1 pupae (a), treated with Bta concentrations as larvae (b) and simultaneous treatments of Bta concentrations and gamma radiation doses (c) Page 5 of 7 Sayed et al. Egyptian Journal of Biological Pest Control (2022) 32:102 34.4%) of control, 75 Gy, 125 Gy and 1.5 × 10 4 IU/mg, respectively. The sex ratio of males to females of F 1 adults varied in different treatments, and the males/females ratios were highly increased to males by increasing the gamma irradiation doses (Fig. 4a), respectively, and Bta concentrations (Fig. 4b), respectively, as compared to 1:1 in the control (0) treatment. Figure 4c shows that the highest ratio (3.5:1) of males/females was found in the 1.5 × 104 IU/mg and 75 Gy as compared to (3:1) of 3.75 × 10 3 IU/mg and 75 Gy, 1.5 × 10 4 IU/mg and 75 Gy, 7.5 × 10 3 IU/mg and 125 Gy and 1.5 × 10 4 IU/mg and 125 Gy.

Discussion
The study aimed to develop the approach of simultaneous IST and Bta, which could potentially be applied for S. littoralis management. Given the above data and considering the desire to develop environmentally promising approach, simulations treatment of IST and Bta could be used as a novel protocol for control lepidopteran pests effectively. The noteworthy difference in the toxicity of Bta combined with gamma radiation doses comparing with individual treatment in the present study goes in line with (AMER et al. 2012) who found that the gamma radiation doses 150, 250, 350 reduced the LC 50 of Bt. Moreover, (Sayed and El-Helaly 2018) found that the low doses 40 and 60 Gy were highly effective on the  Sayed et al. Egyptian Journal of Biological Pest Control (2022) 32:102 S. littoralis F 1 larvae to be more susceptible to SpliM-NPV than un-irradiated ones. Synergistic effect has been noticed in the combination of Bt with Chlorantraniliprole as well as reduced the resistance of diamondback moth (Shabbir et al. 2021).
Results of the duration of larval stage to reach pupae revealed that the larval duration was extended in various treatments, and these data are coincident with (Carpenter et al. 2001) on Cactoblastis cactorum and (Walton and Conlong 2016) on Eldana saccharina who found an extended time of larvae to reach pupae as a result of irradiated male with sub-sterilizing doses of gamma radiation. Moreover, it has been observed prolonged of Helicoverpa armigera larvae with the low concentrations of Bt (Mohan et al. 2008).
Obtained findings revealed that the insect fitness was reduced at the low concentration of Bta which agree with Eizaguirre et al. (2005) on Sesamia nonagrioides, Spodoptera frugiperda (Sousa et al. 2016) and Plutella xylostella (Zhu et al. 2016).
Sex ratio was more declined at the favor of male at most simultaneous treatments than either control or individual treatments. These results are in line with (Bloem et al. 2003) who shifted in favor of males in the F 1 sex ratio of false codling moth, Cryptophlebia leucotreta irradiated with sub-sterilizing doses of gamma radiation. Obtained results revealed that there was a significant reduction in the F 1 population of S. littoralis derived from the combination treatment than individual one. Moreover, the results for the impact of sublethal concentrations of Bt are in line with that finding by (Fathipour et al. 2019) on Helicoverpa armigera and (Camacho-Millán et al. 2017) on Diatraea considerate. The finding indicated that sublethal concentrations of Bt caused significant fitness costs in S. littoralis which coincident with (Sedaratian et al. 2013) who observed a low rate of H. armigera development exposed to low concentrations of Bt. Synergistic effect was reported by (Magholifard et al. 2020) on S. littoralis using Bt and SpliNPV.

Conclusion
Simulations treatment of IST and Bta was assessed against S. littoralis as a novel branch of integrated lepidopteran pest management. Both treatments had a positive correlation to be considered as an effective control method. The toxicity of Bta increased twofold against irradiated larvae than un-irradiated ones. The pupation, adult emergence and survival were significantly decreased in simultaneous treatment than individual ones. Results of F 1 sterility study revealed that production of insect was significantly lowered in the simulations treatment than only IST. The durations of developmental stages were extended in various treatments. The sex ratio was more declined at the favor of male at ST as compared either with the control or the individual treatments. The findings indicated that IST based on F 1 sterility combined with Bt was common sense control methods than the use of IST individually and takes advantage of compatible control methods against lepidopteran pests.