Effect of Metarhizium anisopliae on the nutritional physiology of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), was recorded for the first time in Pakistan in 2019, and now it has spread in several regions, i.e., Punjab and Sindh, affecting maize production. Farmers are using widely synthetic pesticides to control the pest. Further, the resistance development in insects and the non-target effect of chemicals on the environment and humans pose serious threats of using insecticides. The use of entomopathogenic fungi (EPF) is being considered an important tool in integrated pest management program. The main objective of this study was to check the impact of different conidial concentrations of Metarhizium anisopliae fungus on the nutritional physiology of S. frugiperda. The dose-dependent effect of tested EPF was observed, and the highest concentration (1 × 109 conidia/ml) considerably affected nutritional parameters. Reduced relative consumption rate (21.7%), relative growth rate (19.5%), the efficiency of conversion of ingested food (24.2%), and approximate digestibility (16.3%) were observed in treated larvae compared to the untreated by using a higher concentration of EPF. Pupal weight was also found lower (77.9 mg and 84.2 mg, respectively), when larvae were treated by 1 × 109 and 1 × 108 conidia/ml concentrations of EPF. Further, 46.7% of larvae were found dead with this conidial concentration. This study suggests that the application of a higher concentration of M. anisopliae could be an effective option to control S. frugiperda. The EPF can enhance the integrated pest management programs and could be useful in reducing the environmental impact of synthetic insecticides.


Background
The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a migratory polyphagous insect pest and is native to the American continent, including tropical and subtropical regions (Montezano et al. 2018). It has invaded Africa, Australia, and Asia, and has a greater impact on global food security. It was first reported in West Africa (Goergen et al. 2016), and then in a short period, it was dispersed to sub-Saharan Africa (Stokstad 2017). In May 2018, S. frugiperda was observed in the Karnataka region of India (Kalleshwaraswamy et al. 2018). Occurrence of S. frugiperda has also been reported in southeastern Asian countries, including Bangladesh, Thailand, and Myanmar (Guo et al. 2018). In Pakistan, S. frugiperda species was reported in 2019 (Naeem-Ullah et al. 2019). There are two strains of this pest: the rice strain and the corn strain. The corn strain Page 2 of 5 Mubeen et al. Egyptian Journal of Biological Pest Control (2022) 32:73 is more notorious for many crops than the rice strain (Dumas et al. 2015). In Pakistan, the rice strain of S. frugiperda has also been reported recently (Yousaf et al. 2021). Its population is spreading rapidly due to its biology, ability to migrate with air, and high fecundity rate (Prasanna et al. 2018). Spodoptera frugiperda damages over 350 host plants worldwide, including major crops; corn, wheat, sugarcane, rice, cotton, cabbage, millets, and sorghum (Montezano et al. 2018). Although the use of insecticide is a quick tool to control the insect pests in the field, the insecticidal control of S. frugiperda is very difficult due to its feeding behavior, as the inner leaves of the plants with larval excrement protect them from toxicants (Paredes-Sánchez et al. 2021). Resistance development in insects is also another concern. The resistance in S. frugiperda against various insecticides has already been reported (Mota-Sanchez and Wise 2019). Thus, it is necessary to minimize the application of insecticides and find out some sustainable IPM technologies against this pest.
Microbial control is being considered a safer and ecofriendly approach to manage the population of insect pests. About 750 species of entomopathogenic microorganisms from about 85 genera have been identified (Norjmaa et al. 2019). The entomopathogenic fungi (EPFs) use enzymes to penetrate the cuticle of insects (Rajula et al. 2020). Various genera of fungi, Beauveria, Isaria, Lecanicillium, Cordyceps, Metarhizium, and Nomuraea have been utilized in the effective management of various aboveground and soil insect pests (Thaochan and Sausa-Ard, 2017). Metarhizium spp. have been studied against many insect pests due to its vast host range, environmental friendliness, and ease to produce (Greenfield et al. 2015). Metarhizium anisopliae species is being widely commercialized by companies and has the highest number of product registration (Mascarin et al. 2019). Although the mortality rate of insect pests by using EPFs have been studied, knowledge about their effect on consumption and growth rate of insect pests is limited. Keeping in view the increasing interest and successful use of entomopathogens to control insect pests, the present study aimed to determine the effect of different concentrations of M. anisopliae on the nutritional physiology of S. frugiperda.

Spodoptera frugiperda culture
Larvae and egg batches of S. frugiperda were collected from corn fields (32° 07′ 53.8″ N 72° 41′ 34.4″ E) in the Sargodha region, Pakistan. The collected population of larvae was brought to the laboratory of Entomology for rearing. Larvae of S. frugiperda were kept individually in small plastic Petri plates (90 × 15 mm). Fresh leaves of corn were provided to the larvae on daily basis. The diet was replaced daily, and the feces were removed from the Petri plates regularly. After the pupation, the pupae were placed in small plastic cages for adult emergence. The male and female adults were introduced in jars for mating and egg-laying. Honey solution (10%) as soaked cotton plugs was provided to adults. The stripes of muslin clothes were hung in jars for egg-laying. The culture was maintained under controlled conditions of 26 ± 2 °C and 65 ± 5% R.H. The culture was maintained up to F 3 generations to be used in the further experiment.

Nutritional physiology parameters
Thirty 3rd instar larvae were dipped in solution of each EPF concentration for 10 s. After application, larvae were placed in Petri plates. Fresh corn leaves were provided to larvae. Before and after 24 h of feeding, diet weight was recorded using weight balance. The larval length was recorded daily before and after the feeding of 24 h using a measuring scale. Similarly, the weight of each larva and feces was also recorded daily using digital weight balance. When the larva developed to a pupa, the pupal weight was recorded. The mortality data of larvae were also recorded daily. The parameters related to nutritional physiology such as RGR, relative growth rate; RCR, relative consumption rate; ECI, the efficiency of conversion of ingested food; and AD, approximate digestibility were calculated by the method suggested by Waldbauer (1968).

Data analysis
Data of all the nutritional physiology parameters including RCR, RGR, ECI, AD, and pupal weight, and larval mortality were analyzed by one-way analysis of variance (ANOVA). Means were separated by the least significant difference LSD test at a probability level of 5%. The analyses were performed using SPSS 20.0 software.

Discussion
Use of entomopathogens as biocontrol agents is a promising and alternative tool in the integrated management of various insect pests (Deka and Baruah 2021). The impact of various concentrations of M. anisopliae was studied on S. frugiperda. During the penetration, M. anisopliae secretes the proteins such as trypsins, subtilisins, and chymotrypsins that digest the protein-rich procuticle of arthropods (Wang et al. 2008). Due to the penetration of EPF, the consumption of food biomass of larvae was reduced in terms of ECI and RCR. Obtained findings are supported by previous studies that reported a reduction in ECI and RCR values of treated insects than in untreated with the application of entomopathogens (Moorthi et al. 2015). The AD values were also reduced, and it could be due to indiscriminate food consumption or not retaining food to the intestine of larvae to get energy and nitrogen (Hussain et al. 2009). Obtained results are similar to the findings of Moorthi et al. (2015) who reported a reduction in feeding parameters including RCR, RGR, ECI, and AD of Spodoptera litura (Fabricius) after application of EPF, Isaria fumosorosea. Overall, the reduction in food consumption of insects is due to toxic substances secreted by EPF inside the host's body that ultimately disrupt the structural integrity (Tefera and Pringle 2003).
All the feeding indices parameters were reduced by increasing the concentration of EPF. Thus the effect of EPF is proportionally dependent on the conidial concentration (El Husseini 2019). Using higher concentrations is not challenging as the EPFs are safer for humans and the environment (Yasin et al. 2019). Lower values of RGR, RCR, and ECI possibly after exposure to EPF led to the formation of smaller pupa (Khosravi et al. 2010

Conclusions
The impact of M. anisopliae with various conidial concentrations was studied on the feeding indices parameters of S. frugiperda. The treated larvae with higher concentrations showed lower consumption and growth rate. Further, higher larval mortality (antifeedant effects) was observed using a higher concentration of EPF. With the benefits of environmental safety of microbial insecticides, M. anisopliae can be used in the IPM programs concerned with S. frugiperda. However, to better understand the biopesticidal effect of this EPF under field conditions, a more detailed study is needed before adding EPF into integrated management of S. frugiperda.