Efficacy of Two Entomopathogenic Fungi in Combination with Diatomaceous Earth Against Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

The current investigation was conducted to study the efficacy of natural diatomaceous earth (DE) alone and combined with two fungal pathogens: Metarhizium anisopliae and Beauveria bassiana , as a new formulation against Spodoptera littoralis third instar larvae under laboratory conditions. When M . anisopliae , B . bassiana , and DE were applied alone, the mortalities of larvae were concentration-dependent. The LC 50 values were 299.2, 391.5


INTRODUCTION
Spodoptera littoralis (Boisd.) larvae attack different valuable cultivated plants in Egypt, including cotton, peanut, maize, wheat, rice, and vegetables (Pineda et al., 2007). Because S. littoralis larvae have developed resistance to various synthetic chemical pesticides, suitable alternatives are required. The widespread use of insecticides to control S. littoralis has had negative consequences for humans, beneficial insects, and the ecosystem (Chaudhuri et al., 1999). The challenges and hazards associated with the use of traditional insecticides have prompted the urgent demand for substitute insecticides. Microbial control agents are one of these, and they include bacteria, fungi, and viruses (Dent, 2000). A crucial component of a long-term pest management strategy is using entomopathogenic fungi in biological plant protection. When compared to traditional insecticides, entomopathogens have a number of advantages as bioagents. These include being affordable, highly effective, and safe for non-target organisms, minimizing environmental residues, and boosting biodiversity in human-managed settings (Lacey et al., 2001). Fungal biocontrol agents invade in a distinctive way. They may directly infect their host through the cuticle. Because of this, the entomopathogenic fungus can infect insects' non-feeding stages, such as their eggs and pupae, and due to its complex mode of action, it's very difficult for an insect to grow resistance against these pathogens (Nguyen et al., 2007).
The most prevalent entomopathogenic fungus species employed as biological control agents against insect pests is thought to be Metarhizium anisopliae (Barra et al., 2013). It is extensively distributed in nature and frequently isolated from soil or diseased insects (Razinger et al., 2014).
Beauveria bassiana is an extremely virulent entomopathogenic fungus that infects a broad spectrum of insect hosts (Tanada and Kaya, 1993). It lives in soil and is found all over the world (Klingen et al., 2002). The results of previous research revealed that this fungus is one of the most effective microbial control agents for battling various insect pests (Lacey and Goettel, 1995). B. bassiana has the capability to invade the insect through its cuticle and reach the hemocoel, inhibiting the host's immunological response (Götz, 1991). Diatomaceous earth (DE) is a natural substance that poses no hazard to the environment. It is shown to do no harm to beneficial insects and wildlife. It is simple to use and leaves no residue in the food or in the ecosystem. Because of these characteristics, DE is considered a perfect option for use as an insecticide (Korunic, 1998). Carlson and Ball (1962) reported insecticidal impacts of diatomaceous earth when applied to eight different species of stored grain insect pests. An insect dies when DE is absorbed into the cuticle's wax layer, leading to water loss and dryness (Ebeling, 1971;Quarles, 1992). The Environmental Protection Agency in the United States announced DE as a safe substance.
The combined effects of DE with fungi pathogens, Beauveria bassiana and Metarhizium anisopliae, have been observed in many studies (Athanassiou and Steenberg, 2007;Lord, 2007;Athanassiou et al., 2008;Riasat et al., 2011;Wakil et al., 2011;Wakil et al., 2012;Luz et al., 2012;Hanif et al., 2022). These researchers found that the rough attributes of DE improved the effectiveness of entomopathogenic fungi as insecticides. It enhanced the pathogenicity of both fungi. It has not yet been investigated how DE interacts with Egyptian isolates of M. anisopliae and B. bassiana and how this affects Spodoptera littoralis.
The immune system of insects is relatively effective. Both humoral and cellular defense responses make up an insect's immunity. Nodule production, encapsulation, and phagocytosis are all aspects of cellular immunity (Lavine and Strand, 2002). Antimicrobial peptides and the phenoloxidase system play significant roles in humoral defense. An essential component of the insect immune system, phenoloxidase plays a crucial role in the coagulation, melanization, and wound healing processes. In addition to the activation via the prophenoloxidase cascade, prophenoloxidase is the inactive form of phenoloxidase. It is carried out in the cuticle or in the hemolymph of the insect in response to injury. The enzyme that cleaves prophenoloxidase into phenoloxidase is believed to be a clip-domain serine protease (Ashida, 1990). To create melanin, these quinones polymerize. Fungi are affected by quinones and melanin. In the hemolymph, melanin deposition darkens parasites, and foreign bodies are encapsulated in nodules caused by hemocyte melanization (Söderhall and Cerenius, 1998). Phenoloxidase, a major enzyme in the production of melanin, is located in the hemolymph, midgut, and cuticle. Melanin and its precursors are used directly in the immune system. It is believed to play a significant part in the encapsulation of bigger organisms (Cotter et al., 2004). The chemical nature of melanin itself may also hinder the growth of fungi (St. Leger et al., 1988).
This work aims to explore the efficacy of two fungal pathogens, Metarhizium anisopliae and Beauveria bassiana, alone and combined with a natural control agent DE, against Spodoptera littoralis larvae and their effects on some biochemical elements.

Rearing Technique of The Experimental Insect:
A laboratory strain of cotton leafworm, S. littoralis (Lepidoptera: Noctuidae), was reared on fresh leaves of the castor oil plant. The culture was kept under constant conditions of 27±2°C, photoperiod of 14 hours light and 10 hours dark, and 65±5% R.H. Egg-masses obtained from the Plant Protection Research Institute (PPRI) Dokki, Egypt. The larvae were daily provided with fresh castor leaves. The emerged adults were fed on a 10% sugar solution in clean jars to allow mating. Clean filter papers were used for egg laying.

The Tested Compounds:
Diatomaceous earth (DE) was purchased from Natural Source LLC, FL, USA as a 100% pure powder.
The two entomopathogenic fungi treatments were obtained from the commercial formulation produced by the Insect Pathogen Unit, Plant Protection Research Institute, Ministry of Agriculture, Egypt. The Beauveria bassiana was tested as Biover® 10% W. P. (32 million spore /g) and the Metarhizium anisopliae was tested as Bioranza® 10% W. P. (32 million spore /g) and 200 g per 100 liters of water/Feddan was the recommended application rate. A stock solution (1000 ppm) was prepared by dissolving ten grams of each formulated fungus in one liter of distilled water. A series of different concentrations (100, 200, 300, 400, and 500 ppm) were prepared for the DE, Biover®, and Bioranza® to facilitate the comparison between the three treatments.

Bioassay Tests:
Newly molted third instar larvae were treated with the same series (100, 200, 300, 400, and 500 ppm) of the three different materials: Diatomaceous earth (DE), Beauveria bassiana (Biover®), and Metarhizium anisopliae (Bioranza®). All the tested larvae were starved for about four hours before the treatment. For each treatment, five replicates of twenty larvae in each plastic cup were used. Three different treatments were done as M. anisopliae, B. bassiana, and diatomaceous earth, independently. Two combination treatments were done as DE + M. anisopliae and DE + B. bassiana. The mixed ratio was one-to-one for each concentration. In each treatment, discs of castor leaves were dipped in the corresponding concentration of independent or combined treatments. The different treated discs were introduced to the tested larvae. In the control replicates, discs of castor leaves were dipped in distilled water. After the larvae finished feeding on the treated leaves (24 h), fresh castor leaves were provided to the tested and control larvae. The mortalities for the five treatments were recorded after ten days.
The bioassay results were handled by PC programming LdP to draw the regression line and for the identification of the concentration which kills twenty-five percent of the larvae (LC25), the concentration which kills fifty percent of the larvae (LC50), and the concentration which kills ninety-five percent of the larvae (LC95) values as previously described (Finney, 1952).

Evaluation of the Joint Toxicity:
The joint toxicity of combinations of diatomaceous earth with M. anisopliae and diatomaceous earth with B. bassiana was detected by using the LC25 obtained from each treatment independently. The LC25 of DE was mixed with the LC25 of M. anisopliae for the first combination test. The LC25 of DE was mixed with the LC25 of B. bassiana for the second combination test. The experiment was conducted as previously mentioned in the bioassay test. The observed mortalities for these two combinations were used to find the relationship between the tested compounds when applied together. The co-toxicity factor was calculated by using the following equation according to Mansour et al. (1966).
The co-toxicity factor indicated the results. Potentiation is defined as a factor of 20 or greater; a negative factor of 20 or more is considered to be antagonism, and values that are between -20 and +20 imply additive effects.

The Biochemical Analyses:
The LC50 values of the different five treatments were used in the biochemical study as previously described. The hemolymph was collected from the treated and control late fifth instar larvae. The thoracic legs were pulled out from the larvae by a fine forceps. The drained hemolymph was collected by a micro syringe and placed in Eppendorf tubes. The inhibitory effect of tyrosinase was prevented as recommended by Abou El-Ghar et al. (1996) by adding traces of phenylthiourea.
The activity of the phenoloxidase enzyme in the diluted hemolymph was measured by the colorimetric test described by Laughton and Siva-Jothy (2011) in the presence of Lβ-3,4-dihydroxyphenylalanine (L-Dopa) as a substrate. The absorption was measured by a UV spectrophotometer at 490 nm.
The activity of the prophenoloxidase enzyme in the samples was measured as described for the phenoloxidase enzyme with an extra step. The diluted hemolymph was incubated with chymotrypsin as an activator in sodium cacodylate buffer for five minutes before adding the L-Dopa. The difference between these two activity readings directly indicates the activity of the prophenoloxidase enzyme.
The lysozyme activity was quantified by using dried bacterial cell walls (Micrococcus lysodeikticus) in sodium phosphate buffer (10 mM -pH 5.5) according to the methodology described by Powning and Davidson (1973). The turbidity was measured by a UV spectrophotometer at 450 nm after adding sodium hydroxide. When measured against 10 mM phosphate buffer, the initial absorbance readings were between 1.5 and 1.6 under these conditions. The amount of enzyme that reduces the absorbance by 0.001 units per minute is considered to be one unit of lysozyme. Units of activity per mg of protein are used to measure the lysozyme's specific activity in the samples.
The original method of Gornal et al. (1949) was used to determine the total protein amounts in the hemolymph of treated and untreated samples. The change in the reaction color was measured by a UV spectrophotometer at 545 nm. The final protein concentrations were calculated as gram per deciliter.

Statistical Analyses:
The means of the mortality percentages of the larvae in the various experimental treatments were separated and statistically analyzed using ANOVA (LSD). After calculating the mean mortality percentage, the standard error of the mean (SEM) was added. A significant correlation ship was recorded when P<0.05, and a non-significant correlation ship was recorded when P≥0.05.

Toxic Effects of Metarhizium anisopliae, Beauveria bassiana, and Diatomaceous Earth on Spodoptera littoralis Larvae:
Data in Table (

The Combined Toxic Effects of Metarhizium anisopliae, Beauveria bassiana, and Diatomaceous Earth on Spodoptera littoralis larvae:
In order to evaluate the combined toxic effect of DE with M. anisopliae or with B. bassiana, several concentrations (100, 200, 300, 400, and 500 ppm from each compound) were mixed equally for each combination in a ratio of 1:1 and were applied to 3 rd instar S. littoralis larvae. The corresponding average percentage of mortalities was recorded after ten days of exposure in Table (2). Table (2) indicates that the percentage of larvae mortality was significantly increased with the increase of the combined concentration of DE with M. anisopliae, or with B. bassiana.

Data displayed in
The lethal concentration values (LC25, LC50, and LC95) in Table (2) are obtained from the regression line in Figure (2). The combination of DE and M. anisopliae is more efficient than the combination of DE and B. bassiana where the mortalities percentage at 300 ppm concentration from each compound were 93.33 ± 1.67 and 71.67 ± 6.01 at the mentioned combinations above, respectively. All larvae died at 400 and 500 ppm concentrations in the two combinations. The LC50 values were increased from 133.77 to 189 ppm in binary combinations of DE with M. anisopliae and DE with B. bassiana, respectively. This increase in the LC50 value indicates the improvement of the combination between DE and M. anisopliae over the other combination.

Joint Toxicity Actions of Diatomaceous Earth with Metarhizium anisopliae and Diatomaceous Earth with Beauveria bassiana against 3 rd instar Spodoptera littoralis Larvae:
Two combinations of the three tested compounds were applied to the larvae to evaluate the joint toxicity. The first combination was DE with M. anisopliae and the second combination was DE with B. bassiana. In these combinations, the LC25 for each compound was used.
The observed and expected mortalities were used to calculate the joint toxic action of the two combinations, which was then presented as a Co-toxicity factor for each combination (Table 3). Data in Table 3 revealed that the obtained mortality percent was increased when the mixture of DE with M. anisopliae or the mixture of DE with B. bassiana was applied. This indicates that using the two compounds together is more efficient. As a result, the larval mortality was enhanced by the combination rather than using each one independently. The two binary mixtures showed potentiating effects.

Changes of Some Biochemical Parameters in Treated Spodoptera littoralis Larvae:
The enzymatic activity of phenoloxidase, prophenoloxidase, and lysozyme (as an immune response in insects) in addition to the total protein level in the hemolymph was quantitively detected in the treated larvae with the three tested compounds independently and in two combinations. The changes in these biochemical elements were compared to their levels in the control larvae.
In Figure (3-A), the level of phenoloxidase was significantly (P<0.05) reduced in treated larvae with M. anisopliae, and with B. bassiana by -55.94% and -63.67%, respectively. When DE was added to the fungi, the erosion in the cuticle facilitated the entrance of the fungi with a high level of secreted toxins. As a result, there were significant (P<0.05) reductions in the phenoloxidase in treated larvae with M. anisopliae mixed with DE, and with B. bassiana mixed with DE by -75.51% and -77.96%, respectively. Data in Figure (3 Figure (1-D).
It was noticed from Figure (3) that larvae treated with DE did not show any significant reduction (P>0.05) in the enzymatic activity of phenoloxidase, prophenoloxidase, and lysozyme or in the total protein level in the hemolymph.

DISCUSSION
According to the findings of our study, there is a link between S. littoralis larvae mortality and a higher diatomaceous earth concentration. Mortality was reported in a significant ascending manner with the increase of the DE concentration. This death occurred as a result of DE's ability to adhere to insect cuticles and absorb their waxy, oily, outer layer, which causes water loss and promotes dryness and abrasiveness. When higher dosages are used than lower ones, the harm is more severe, causing an insect to die quickly from dehydration due to the abrasion in the cuticle layers (Athanassiou et al., 2005). The killing action of DE is a mechanical one, not a chemical one, as it has microscopically razor-sharp edges, which act like rough sandpaper. There are now a lot of commercially available DE formulations, and various studies show that they work well against a variety of pest species (Subramanyam and Roesli, 2000). Mewis and Ulrichs (2001) observed high mortality in Tribolium confusum, Plodia interpunctella, and Sitophilus granaries treated with low dosages of DE and found that it required a long exposure period to cause higher mortality. The results stand in the same line as El-Sayed et al. (2010), who claimed that the treated wheat with diatomaceous earth highly reduced the population of Sitophilus oryzae, Tribolium castaneum, and Rhyzopertha dominica. Because of its low mammalian toxicity, worker safety, the low danger of food residues, and the formation of resistant insect populations linked to the use of chemical pesticides, diatomaceous earth usage has increased during the past decades.
The present study illustrates the ability of the tested formulated entomopathogenic fungus, M. anisopliae, to show positive influences on S. littoralis larval mortalities. Our results were in agreement with El Husseini (2019), who tested the local isolate of the entomopathogenic fungus M. anisopliae against 3 rd and 5 th larval instars of the cotton leafworm S. littoralis and recorded the deaths of larvae that reached high mortality four days after treatment. Şahin and Yanar (2021) observed a high mortality rate of M. anisopliae when applied to S. littoralis larvae under laboratory conditions.
For controlling a broad range of insects with natural agents by direct penetration of the host cuticle, M. anisopliae is the most effective one (Santi et al., 2010). Lotfy et al. (2016) observed positive influences on the 2 nd and 4 th instar larvae of S. littoralis treated with spores of M. anisopliae at different concentrations in laboratory conditions. In other studies, it was found that Metarhizium brunneum was very effective against S. littoralis and can be considered as a potential biological control agent against S. littoralis larvae (Funda and Yusuf, 2021;Eleawa et al., 2022).
The mode of action of B. bassiana is through adhering to the insect cuticle, germinating, and penetrating its body. It has no effect on non-targeted insects (Goettel and Hajek, 2001). In this research, B. bassiana showed significant mortality effects. The correlation between the concentrations of B. bassiana and the mortalities was exponential. Ahmed and El-Katatny (2007) studied the effects of B. bassiana on both larval and pupal stages of S. littoralis within 5 days post-treatment. The result showed relatively high dosedependent larval and pupal mortalities. Ramos et al. (2020) hypothesized that B. bassiana began to kill Spodoptera frugiperda larvae on the 4 th day after treatment, with an eleven percent mortality rate. After that, mortality increased, reaching its highest mortality rate on the 9 th day after treatment. Meanwhile, M. anisopliae started its mortality on the 3 rd day after treatment with a nineteen percent larval mortality.
Because of the fungus' mode of action, it's expected that the fungus' efficacy would be enhanced in the presence of other surface-active agents. DE has an enormous surface area with sharp edges. Due to the physical structure of DE, it can induce cracks in the cuticle of the target pest. This action facilitates the entrance of the fungus into the bodies of the tested insects. In our results, a potentiation effect in the mortality rate was noticed after mixing DE with B. bassiana or with M. anisopliae. That was confirmed when the sublethal concentration of each in the mixture was used. The addition of DE appears to have different types of influence on the potency of fungus. According to Lord (2001), DE has a synergistic interaction with B. bassiana against some stored grain pests, while it has no negative effects on the fungus's germination. According to Akbar et al. (2004), the combination of B. bassiana and diatomaceous earth had good performance when used to control T. castaneum larvae. To decrease the amount of DE, it can be mixed with other compounds to improve their efficacy (Korunic and Fields, 1995;Subramanyam and Roesli, 2000). The use of DE in combination with other compounds is one of the potential remedies for the effects brought on by the use of DE in excessive doses. These techniques comprise a combination of entomopathogenic fungi (Michalaki et al., 2007). Experimenting with these combined compounds frequently demonstrated a synergistic and improved efficacy effect (Korunic, 2007;Athanassiou and Korunic, 2007). The effectiveness of M. anisopliae against S. oryzae, R. dominica, and T. castaneum was enhanced by the addition of inert dust, such as DE (Batta, 2008).
In the present study, B. bassiana and M. anisopliae severely deplete the protein concentration with an average of -62.15% either when used alone or when used in combination with DE. House (1963) reached the conclusion that a great depletion in protein content of a treated insect may be the leading factor in their mortality rates as it causes hindrance to ovulation and egg development as proteins are the key factor in these physiological processes. The finding that the fungus is accountable for the disturbance in the endocrine balance of the host and the lack of protein makes it a direct reason for its death. Highnam and Hill (1969) stated that the disturbance in the endocrine balance of insects by fungi may be responsible for the depletion in protein concentrations of the hemolymph of insects. The decrease in the protein content may be one of the main causes of death in treated insects. Spores connect to and germinate on the cuticle of an appropriate host, triggering a series of recognition and enzyme activation processes in both the host and the fungal parasite.
In the current work, the levels of phenoloxidase, prophenoloxidase, and lysozyme were significantly decreased in treated larvae with B. bassiana or M. anisopliae. These enzymes are involved in the immune response in insects, especially during the invasion of microorganisms. The recorded reductions could be due to the inhibition of these enzymes by the toxins produced by the entomopathogenic fungi (Huxham et al., 1989;Cerenius et al., 1990).
The phenoloxidase and other downstream enzymes are activated as a result of the defensive reactions to the fungal infection. Changes in the levels of phenoloxidase may assist in reducing microbial infection, preserving the nutritional value of the paralyzed larva as a future food supply for the parasitoid (Hagstrum, 1983).
According to Trudeau et al. (2001), once the fungus has penetrated the cuticle of the insect, it invades the insect body and hemolymph and alters the enzyme activity and protein concentrations, where insects use their cuticle, fat body, and protein as defenses against attack by fungi. The death of Schistocerca gregaria and Locusta migratoria due to infection with M. anisopliae may cause hemolymph protein, phenoloxidase, and lysosome enzyme titers to decline (Gillespie et al., 2000;Mullen and Goldsworthy, 2006). Proteolytic enzymes that are secreted into the insect's hemocoel and hydrolyze the host protein during parasitism may account for the decrease in total protein and loss of soluble protein from the host's hemolymph during parasitism (Gillespie et al., 2000). Total protein concentrations in desert locusts infected with M. anisopliae drastically decreased (El-banna et al., 2012). Our results were also in congruence with Gabarty et al. (2013), who investigated the efficacy of entomopathogenic fungi B. bassiana and M. anisopliae on the immune enzyme response of S. littoralis larvae. They recorded a significant decrease in the enzyme activities of phenoloxidase, prophenoloxidase, lysozyme, and protein concentration.

Conclusion
These results suggest that the use of inert powders, DE, in combination with entomopathogenic fungi, M. anisopliae, or with B. bassiana, may provide protection to agricultural crops while negatively affecting larvae of S. littoralis. These combinations have a great potentiation effect on S. littoralis. The most effective treatment was M. anisopliae mixed with DE, followed by B. bassiana mixed with DE, M. anisopliae, B. bassiana, and then DE alone. More studies in the field are needed to confirm the results and define the strategies and possible interactions of these management tools with other control methods for these species. Ethical Approval: This research paper was approved by the research ethics committee from Faculty of Science, Ain Shams University (ASU-SCI/ENTO/2022/11/3).