The Potential of Two Entomopathogenic Fungi and Enhanced Diatomaceous Earth Mixed with Abamectin: A Comprehensive Study on Mortality, Progeny Production, Application Method, and Surface Application against Tribolium castaneum

This study determined the efficacy of Beauveria bassiana (Bals. -Criv.) Vuill., Metarhizium anisopliae (Metchnikoff) Sorokin, and diatomaceous earth mixed with abamectin (DEA) alone and in their combinations for the integrated management of larvae and adults of Tribolium castaneum (Herbst) from three field populations of Pakistan (Multan, Rawalpindi, and Rahim Yar Khan) and one laboratory population (Faisalabad). Treatments were applied on three surfaces, namely, viz. steel, concrete, and jute bags, implementing two application methods, dusting and spraying. The combined treatments were more effective in comparison with single treatments for both larvae and adults. Overall, the highest mortality rates were recorded in the Faisalabad population, followed by the Rehaim Yar Khan, Rawalpindi, and Multan populations. Progeny production was suspended 21 days after exposure to the combined treatment of DEA and both fungi in all populations except Rawalpindi. Larvae were found to be more susceptible than adults in all treatments and intervals. Dusting was more efficient than spraying for both larvae and adults and for all the populations studied. The present study provides a wholistic understanding of the impact of different factors on the success of the combined treatments using DEA and entomopathogenic fungi, supporting their use as surface treatments.


Introduction
The presence of extraneous matter in stored commodities, such as insect pests and their fragments, filth, and insect excreta, indicate unhygienic practices during the process, production, and storage of products [1], compromising their quality and rendering them unfit for human consumption [2]. Approximately 5-10% of the world's grain production is lost each year due to insect damage [3]. It is estimated that developing countries such as Pakistan experience storage losses of 20% or higher [4], which translates to a yearly monetary loss of USD 500 million to USD 1 billion [5]. In contrast, developed countries experience storage losses of about 9% [6]. Containing over 250,000 described species, Coleoptera is the largest order of insects [7], which includes 600 notorious stored grain species, causing substantial grain loss [8].
of resistance, rendering the fungi less effective over time [57]. Insect infection with EPF increases enzyme activity, resulting in their sensitivity to insecticides and, consequently, to insect mortality [60]. This opens possibilities for developing effective combined biological products, implementing EPF [60]. Padín et al. [61] investigated the impact of B. bassiana on T. castaneum, observing that B. bassiana did not exhibit significant control over the grain loss caused by T. castaneum. However, the combination of the desiccant pesticide B. bassiana and diatomaceous earth (DE) has been exhibited to have an additive effect on the adults of T. castaneum [53,62].
Diatomaceous earths (DEs), which are comprised of amorphous silicon dioxide [63], are the fossilized remains of diatoms, a major group of algae [64]. DEs are considered an advantageous alternative to pesticides due to their non-toxic nature to non-target organisms [65,66], and their convenient substraction from the grains prior to the milling proccess [67]. Their mode of action involves the absorption of lipid contents (wax) from the cuticle of the insect, ultimately leading to the insect's demise [68,69]. A disadvantage of their use is linked with the high rate of application, as this issue can have a notable impact on the bulk density of the grains and the presence of dusty residues [70]. To overpower this limitation, DEs can be combined with other control methods, such as insecticides [71], EPF [32,72,73], and oils [74]. Commercially available DEs are effective against various insect species on different stored grains, but the effectiveness of DEs frequently differs depending on various factors (e.g., formulation, treatment approach, stored product) [75,76]. Employing DEs and EPF in conjunction as grain protectants could reduce the necessary application doses due to their distinct mechanisms of action and impact on the insect cuticle [51]. Furthermore, a blend of EPF and DE could be advantageous as a long-lasting protectant, given that both EPF and DE are persistent on grains [51].
There are several reports on the additive effects of DEs combined with either B. bassiana [77,78] or M. anisopliae [55,79] against T. castaneum. Nevertheless, the combination of DEs with both B. bassiana and M. anisopliae has not yet been studied. Therefore, the objective of this study is to explore the effects of the combined formulations of DE and EPF, B. bassiana and M. anisopliae, the application method, and the treated surface on the mortality and offspring production of T. castaneum populations under laboratory conditions, with the aim of simulating the real-world application scenarios employed for the preservation of hard wheat [80]. This study aims to provide useful insights into the potential use of this combination as a combined biological/natural product for the sustainable management of T. castaneum.

Insect Culture
Field populations of T. castaneum were collected from three sites of Pakistan, i.e., Rahim Yar Khan, Multan, and Rawalpindi. One laboratory population from Faisalabad (Pakistan) was reared for >10 years in the Microbial Control Laboratory, University of Agriculture, Faisalabad, Pakistan. This population had not been subjected to any chemicals, including phosphine. The culture has been conducted on wheat flour +5% brewer's yeast by weight at 30 • C and 65% RH in 0:100 (Light: Dark) [81].

Grains
Clean, non-infested, and contamination-free wheat, Triticum aestivum L. (var. Faisalabad 2008) without dockage, was used. Before the trials, the moisture content of the grains was 12.0%, as calculated by a moisture meter (Dickey-John Multigrain CAC II; Dickey-John Co., Lawrence, KS, USA).

DE Formulation
The DE formulation (DEA) consisting of freshwater DE (90%) + 0.25% abamectin as an active ingredient (a.i.) was utilized at 35 ppm or 25 ppm for bioassays related to grains, and 2 gr/m 2 for surface treatment, against larval and adult individuals of T. castaneum [77].

Fungal Formulation
Two EPF isolates, namely, B. bassiana (WG-13) and M. anisopliae (WG-03) sourced from of the Microbial Control Laboratory, were used in the experimental assays. The isolates were preserved on Sabouraud Dextrose Agar (SDA) slopes in test tubes at 4 • C. Mass cultivation of the cultures was performed on Petri dishes (10 cm diameter) with Potato Dextrose Agar (PDA), incubated for 10 days at 25 • C and 14 h Light:10 h dark. Subsequently, using a sterile scalpel, the conidia were collected from the dishes and placed into a falcon tube (50 mL) containing 30 mL of a sterile solution of Tween 80 (0.05%) (Merck, Kenilworth, NJ, USA). Following this, the conidia suspension was subjected to agitation using a Vortex (Velp Scientifca Srl, Usmate Velate, Italy) for 5 min, together with eight sterile beads made of glass to facilitate the process. The concentration of both B. bassiana and M. anisopliae was standardized to 1.2 × 10 7 , 1.2 × 10 6 , and 1.2 × 10 5 conidia/mL for each species of fungi, using a Neubauer-improved hemocytometer (Marienfeld, Lauda-Königshofen, Germany) and a microscope (BB.1152-PLi, Euromex Microscopen bv, Arnhem, The Netherlands). To estimate the germination of the conidia, two dishes (6 cm diameter) were prepared with a mixture of yeast (1%) and SDA. Each dish was inoculated with 0.1 mL of a solution that contained 1 × 10 6 conidia/mL. The dishes were then sealed with parafilm and transferred into an incubator for 16 h at 25 • C under a 14:10 h (Light: Dark) cycle. Following this period, a non-contaminated cover slip was set on top of the dishes, and a total of 200 conidia were counted for each dish. Prior to each assay, the conidia germination was assessed under 400× magnification. It was found that >91% of both isolates had germinated.

Treatment of Wheat with a Single Method
A total of six treatments, plus the control, were executed for each developmental stage of T. castaneum (larvae and adults). A single dose rate of B. bassiana or M. anisopliae alone or combined with DEA were applied against larvae and adults of T. castaneum. Specifically, the treatments were B. bassiana alone at 1.2 × 10 5 conidia/kg wheat, M. anisopliae alone at 1.2 × 10 5 conidia/kg wheat, DEA at 35 ppm (35 mg DEA/kg wheat) alone, a combination of B. bassiana + DEA, a combination of M. anisopliae + DEA, and a combination of B. bassiana + M. anisopliae + DEA. Two additional kilograms of grains were left untreated to serve as control groups. One control group was used for larvae, and the other one was used for adults of T. castaneum. The treatment for the control was water that contained Tween 80 (0.05%) [77]. For each treatment, 1 kg lots were laid in slim layers on individual trays. The EPF were applied as liquids, while DEA was applied as dust. Spraying was conducted using a different airbrush for each treatment (Master Multipurpose Airbrush, San Diego, CA, USA). One milliliter of each conidial suspension or control was treated on 1 kg wheat. The treated grains were then conveyed into individual 3 L glass jars and shaken manually for 10 min to accomplish an equal distribution of conidia inside the mass of the grain. Considering the application of DEA, 1 kg of treated wheat was conveyed into a 3 L glass jar and shaken as aforementioned. For combined treatments, the aqueous conidial suspensions were applied first, followed by the DEA, conveyed to 3 L glass jars, and shaken as described above. Subsequently, three 60 g samples from each treated lot and the control were weighted using a balance (ELB 300 Shimadzu, Kyoto, Japan), conveyed into separate glass vials (diameter = 7 cm, height = 12 cm), and labeled appropriately. Thereafter, 60 individuals of T. castaneum were liberated in each vial and preserved at 30 • C and 65% RH. The lid of the vial carried a central hole 15 mm in diameter, which was cladded with gauze to facilitate adequate aeration within the vial. To prevent the escape of insects, the upper interior surfaces of the vials were smoothed using polytetrafluoroethylene (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). New vials were prepared per exposure. Following the completion experimental period, the insects subjected to testing were removed from the wheat. Subsequently, the wheat was reintroduced into vials and repositioned in the incubators under the previously mentioned conditions for the documentation of offspring production. The mortality rate was estimated after 7, 14, and 21 days of exposure of the individuals to treated wheat samples, while the progeny was determined after 62 days [78]. The aforementioned protocol was conducted independently for each developmental stage of T. castaneum (adults of mixed sex, <14 days old, and larvae between third and fourth instar) and repeated three times, using a new series of vials for each replication (3 × 3 = 9 replications per treatment for each of the 6 treatments). The same procedure was replicated for each of the four populations studied in this investigation (Multan, Rawalpindi, Rahim Yar Khan, and Faisalabad). Progeny were adults and immatures of T. castaneum.

Treatment of Wheat with Different Methods
The treatments of fungi and DEA were applied to wheat (var. Faisalabad 2008) by two different application methods: dusting and spraying. Single and combined treatments are as mentioned above. The spraying of combined DEA and fungal conidia was carried out by preparing aqueous suspensions of DEA and each fungus at 1.2 × 10 7 conidia/kg wheat. For the treatment of DEA alone, DEA at 25 mg was made up with water (distilled) to 1 mL. Spraying of the fungal suspensions was carried out as above. Spraying of DEA suspension was performed using a different airbrush (Master Multipurpose Airbrush, US Art Supply, San Diego, CA, USA). One milliliter of DEA suspension was applied on 1 kg wheat. The treated grains were then conveyed into individual 3 L glass jars and shaken manually for 10 min to accomplish an equal distribution of DEA particles into the mass of the grains. Concerning dusting, single and combined treatments were conducted as described above, with concentrations of the aqueous conidial suspensions at 1.2 × 10 7 conidia/kg wheat and the DEA at 25 ppm. Fungal treatments were applied as liquids, while DEA was applied as dust, as described above. In addition, two lots containing 1 kg wheat were left untreated to serve as the control, one for each application method, as previously mentioned. The mortality rates were documented in the manner explained earlier, with the mortality rate assessed 10 days after the exposure of the insects to the sprayed or dusted wheat. The entire bioassay was replicated three times in total per population and per method, with each iteration involving the use of fresh insect individuals and wheat. This process was conducted independently for larvae and adults of T. castaneum.

Surface Treatment
For the surface treatment bioassays, three distinct surfaces were employed, namely, 1 mm thick galvanized steel (Pakistan Steel Mills Corporation, Karachi, Pakistan), approx. 40 mm thick concrete (D.G. Khan Cement, Lahore, Pakistan), and 250 GSM (g/m 2 ) jute bags (Punjab Food Department, Faisalabad, Pakistan). The steel and jute bags were meticulously crafted to match the dimensions of the Petri dishes (diameter = 8 cm, height = 1.5 cm high, surface = 50.27 cm 2 ). To prepare the concrete surface, a mixture of water and concrete (f = 0.4-0.6 w/c) was made into a slurry, which was subsequently poured into the dishes and allowed to dry for 24 h. Prior to the commencement of the experiment, surfaces were thoroughly cleaned to remove any debris and subsequently placed under experimental conditions at 30 • C and 65% RH. To impede insects from escaping from treated surfaces, sides of dishes were smoothed using polytetrafluoroethylene (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).
For surface treatment, three dishes were prepared for each type of surface. Single and combined treatments were as mentioned above. The B. bassiana and M. anisopliae solutions were applied as liquids, while DEA was applied as dust. A total of 1 mL of each conidial suspension (B. bassiana and M. anisopliae: 1.2 × 10 6 conidia/mL) was sprayed, using different airbrushes for each treatment, to three dishes for each type of surface. DEA (2 gr/m 2 i.e., 0.0100 g/dish) [84] was sprinkled evenly over each substrate by sieving through a U.S. standard mesh sieve No 60 (0.250 mm mesh openings) (Advantech Manufacturing, Inc., New Berlin, WI, USA). For combined treatments, the spraying of conidia preceded the DEA application. Seven dishes (corresponding to six treatments and the control) for each type of surface were used. Thereafter, 10 larvae between the third and fourth instar obtained from the Faisalabad population were introduced to each dish and incubated at 30 • C and 65% RH. Mortality was assessed after 10 days of initiation. The above procedure was replicated three times, involving the use of fresh insect individuals and wheat. The bioassay was replicated three times in total (3 × 3). The above procedure was repeated for adults of mixed sex, <14 days old.

Data Analysis
Mortality was adjusted according to the formula of Abbott [85]. To normalize variance, the data were subjected to log(x + 1) transformation before analysis [86]. For the treatment of wheat with a single method, data were analyzed using a three-way ANOVA for each developmental stage of T. castaneum, with the population, concentration, and exposure as the main effects and mortality as the response variable. For the treatment of wheat with different methods, a three-way ANOVA was performed. The treatment, population, and method of application were the main effects, and mortality was the response variable. Concerning the surface treatment, a two-way ANOVA was conducted, with the dose and treatment as the main effects and mortality as the response variable. The data on the production of progeny were subjected to a two-way ANOVA, where the main effects were the treatment and species, and the response variable was the number of offspring produced. Data were analyzed separately for each developmental stage using the statistical software Minitab 13.2 (Minitab, 2002 Software Inc., Northampton, MA, USA). The test Tukey-Kramer (HSD) was utilized to separate means for mortality and offspring counts (p = 0.05) [87].

Treatment of Wheat with a Single Method
The main effects and interactions affected larval and adult mortality significantly ( Table 1). The mortality of both larvae and adults differed among treatments (p < 0.05) for all four tested populations. After a 7-day period of exposure, mortality was lower in single treatments (application of DEA and fungal isolates alone) compared to combined treatments. After exposure to B. bassiana + M. anisopliae + DEA, larval mortality ranged from 51.35% to 60.73%, and adult mortality ranged from 37.42% to 57.63% among the four populations (Table 2). At 14 days post-exposure, mortality was higher in combined treatments compared to single treatments. Concerning the combined treatment of B. bassiana + M. anisopliae + DEA, the Faisalabad and R.Y. Khan populations exhibited 100% larval mortality, followed by the Multan and Rawalpindi populations. Adult mortality ranged from 78.72% to 92.40% (Table 3) (Table 4).
Concerning progeny, the main effects and interactions significantly affected the progeny emergence of T. castaneum in all four populations (Table 1). Significant differences were noted between all treatments applied compared to the control and all tested populations. Overall, the application of the combination of DEA and EPF resulted in lower progeny emergence compared to the application of either treatment alone ( Table 5). The treatment of B. bassiana + M. anisopliae + DEA suppressed progeny emergence in all populations except for the Rawalpindi population, while progeny implementing B. bassiana + DEA was suppressed only in the Faisalabad and R.Y. Khan populations (Table 5).

Treatment of Wheat with Different Methods
Regarding the application method, the main effects and interactions for the mortality levels of larvae and adults were significant, apart from treatment × application methods for adults (Table 6). Upon the application of the treatments in wheat, there was a significant rise in the mortalities of both larvae and adults (following 10 days of exposure) through dusting compared to spraying. In the case of larvae, 100% larval mortality was observed for the Faisalabad and R.Y. Khan populations implementing B. bassiana + M. anisopliae + DEA with the dusting application method. The same combined method exhibited 100% larval mortality with the spraying application method only for the Faisalabad population. However, in the case of adult mortality, only the Faisalabad laboratory population exhibited 100% mortality for both dusting and spraying application methods implementing B. bassiana + M. anisopliae + DEA (Table 7).

Surface Treatment
Concerning surface treatment, the main effects and interactions on the mortality of larvae and adults were significantly affected by exposure (Table 8). A significant difference was observed in the mortality rates across all surfaces and treatments applied. Overall, all treatments were effective against larvae and adults of T. castaneum compared to the control. The combined treatments were more effective in comparison to a single application of DEA or EPF alone on all surfaces employed (Table 9). In particular, 100% larval mortality was observed for combined treatments of DEA and EPF on steel. Combined treatments of B. bassiana + DEA and B. bassiana + M. anisopliae + DEA exhibited 100% larval mortality on concrete. However, only the B. bassiana + M. anisopliae + DEA treatment was equally effective on jute bags (Table 9). Concerning adult mortality, only the B. bassiana + M. anisopliae + DEA treatment was 100% effective on all surfaces employed, apart from Jute bags ( Table 9).

Discussion
Our findings indicate that longer exposures led to higher mortalities, particularly higher in T. castaneum larvae compared to adults. Additionally, the highest mortality rates were recorded in the The effectiveness of IPM programs depends on understanding the compatibility of EPF with pesticides used to protect crops [88,89]. The maximum mortality incurred due to additive effects between DEs and EPF is poorly understood. However, Dal Bello et al. [13] exhibited that DE and B. bassiana work jointly to combat various pests of stored grains, with no adverse effects on the EPF's germination. Furthermore, previous research highlighted that DE increases the effectiveness of B. bassiana against larvae of T. castaneum, particularly due to the abrasive action of DE on the insect's cuticle [51,53]. This leads to cuticle desiccation, thus facilitating the conidial adhesion, germination, and conidial penetration of the fungus inside the insect [53]. Concerning the combination of B. bassiana + M. anisopliae, it has been documented that these two EPF species have been more effective against Amblyomma variegatum (F.) (Ixodida: Ixodidae) as a combination rather than alone [90]. The EPF B. bassiana and M. anisopliae can exhibit an enhanced effect against pests, as demonstrated in this investigation and previous research. This effect appears to be further elevated when applied in combination with DE, due to its ability to damage the insect's cuticle. However, further research is required to fully understand the mechanism behind this combined effect and to optimize the application of this combination treatment for sustainable and effective pest management.
Concerning progeny production, a significant effect on the progeny of T. castaneum was observed in all treatments compared to the control, with the combined treatments demonstrating greater efficacy than single treatments. Furthermore, the combined treatment of DEA and both EPF species suppressed the progeny production during a period of 21 days in all populations, apart from the Rawalpindi population. Rizwan et al. [51] observed reduced progeny production in T. castaneum with the combined application of high concentrations of B. bassiana and DE formulation Dafil 610. According to our findings, the combination of enhanced DE with abamectin and single or both EPF was found to be virulent against T. castaneum. The reduction in insect progeny production is a critical parameter in stored-product protection, as it serves to decrease grain damage in the absence of a parental adult population [95,96].
Previous research has indicated that the effectiveness of EPF against insect pests is influenced by various factors, including the abiotic conditions, the virulence of the specific EPF species or strain, the type of grain, and the susceptibility of the target species/instar. For example, the mortality rate of T. castaneum adults treated with B. bassiana was found to be higher at 30 • C compared to 25 • C or 20 • C [45]. Commercial formulations of B. bassiana (Naturalis-L), Verticillium lecanii (Mycotal), and M. anisopliae (Met-52), evaluated against adults and larval instars of T. castaneum, have highlighted B. bassiana and M. anisopliae as more virulent than V. lecanii against this pest [97]. Likewise, Wakil et al. [98] demonstrated significant differences in the mortalities of T. castaneum when exposed to four isolates of B. bassiana and three isolates of M. anisopliae. The observed differences were significant at both species and isolate levels. The host specificity of EPF varies substantially both among and within genera. While many EPF species are highly specific to certain insect hosts, others, such as M. anisopliae and B. bassiana, have been widely studied, having a broad range of hosts [99]. Concerning the abiotic factors affecting the fungal virulence, the grain type has been identified as a highly influential factor. In fact, the type of grain can play an equally important role as other abiotic factors in determining the virulence of EPF [100]. The susceptibility of different instars of a particular species to EPF is known to vary, as observed for T. castaneum. For instance, Baek et al. [101] demonstrated that a higher mortality rate was recorded in larvae of T. castaneum compared to adults after 72 h of exposure to a B. bassiana isolate. Likewise, our results document higher larval susceptibility to combined treatments compared to adults. In the realm of EPF, virulence is predicated upon the attachment of conidia to the body of the host, their subsequent germination, and, ultimately, their penetration into the insect [42,43]. The variable virulence of EPF may be partially explained by the fact that insect stages possess different cuticular layers, which vary in the morphology, softness, and thickness of the epicuticle [102,103], as suggested in previous research on the susceptibility of T. castaneum to EPF [77,104].
Selective application methods, such as dusting, slurries, or liquid aqueous spraying, have been employed to manage stored-grain insect pests [67]. Fields and Korunić [105] reported that dust application is more effective than aqueous spray, in line with Athanassiou et al. [106], who also found that DE applied as dust was more efficient than spraying against T. confusum and S. oryzae. It is worth noting that the efficacy of DE Perma-Guard was reduced when the moisture content increased to 14% against three stored-grain beetles [107]. Admixing dry conidia of EPF with dusts such as DE enhanced their efficacy in both laboratory and storage conditions [72,[108][109][110]. In addition, the use of liquid formulations of EPF, where conidia are suspended in oily liquid ingredients, was successful; however, little is known about the success of this combination in storage environments [110]. In our study, dusting was more efficient than spraying in larvae and adults of T. castaneum when implementing B. bassiana + M. anisopliae + DEA treatments, further confirming previous research on the efficacy of different application methods applying DE. Nevertheless, the efficacy of ten different isolates of EPF against R. dominica was higher when applied through dusting rather than spraying [111], further supporting our results.
The grain commodities are stored in different storage facilities all over the world. In Pakistan, various types of storage structures are employed to store wheat commodities, such as house-type warehouses commonly known as "godowns", bunkers, hexagonal bins, binishells, steel/concrete silos, and temporary open storage bags made of polyethylene, jute, and hessian ("ganjis") [71,112]. On the basis of the findings of this research, the combined use of EPF with DEs as structural treatments offers an alternative approach for the management of noxious insects, taking into account that their mortalities may vary among different types of surfaces. Among the three tested surfaces, higher mortality was observed when treatments were applied on steel, followed by concrete and jute bags. Previously, Arthur [113] found that T. castaneum was susceptible to deltamethrin dusts applied on concrete, tile, and wood surfaces, while in latter research, the author found that the survival rate of T. castaneum was lower on concrete than on plywood or tile surfaces when treated with the insecticidal pyrrole chlorfenapyr [114]. The efficacy of four organophosphates, which included pirimiphos-methyl, was found to persist longer on galvanized steel compared to concrete when tested against three species of psocids [115]. By studying the effectiveness of malathion on several storage bag fabrics, Paudyal et al. [116] observed higher mortality and lower progeny production of T. castaneum on polypropylene bags rather than any other absorbent fabric, including jute bags. The effectiveness of a treatment on different surfaces can be attributed to the physical nature of porosity. Porous surfaces, such as concrete, wood, and fabric, exhibit a lower residual efficacy compared to non-porous surfaces (e.g., metal, tile, glass), directly affecting the insect's insecticide uptake [114,117]. This stresses the necessity for treatments to be tested on a wide range of surfaces to account for the potential variations in efficacy that may arise due to the surface type, pest species, and insecticide formulation.

Conclusions
The present study has demonstrated that biological insecticides provide effective control in storage conditions as compared to chemical insecticides. This scenario represents a more practical option akin to real-world field conditions. Biological insecticides exhibit greater efficacy than chemical insecticides when used at doses compatible with IPM practices. Our results imply that combining M. anisopliae and B. bassiana with DEA can be an effective strategy for managing populations of T. castaneum. The study also highlights that EPF and DEA enhanced with abamectin offer long-term and enhanced management of T. castaneum, inhibiting the pest's ability to reproduce effectively.