Two-Sex Life Table Analysis for Optimizing Beauveria bassiana Application against Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is a highly dispersive, polyphagous insect pest that severely defoliates crops. Excessive reliance on synthetic insecticides leads to ecological pollution and resistance development, urging scientists to probe eco-friendly biopesticides. Here, we explore the virulence of an entomopathogenic fungus, Beauveria bassiana, against S. exigua, resulting in 88% larval mortality. Using an age–stage, two-sex life table, we evaluated the lethal and sublethal effects of B. bassiana on the demographic parameters of S. exigua, including survival, development, and reproduction. Sublethal (LC20) and lethal concentrations (LC50) of B. bassiana impacted the parental generation (F0), with these effects further influencing the demographic parameters of the first filial generation (F1). The infected F1 offsprings showed a reduced intrinsic rate of increase (r), mean generation time (T), and net reproduction rate (R0). Larval developmental duration varied significantly between the control (10.98 d) and treated groups (LC20: 10.42; LC50: 9.37 d). Adults in the treated groups had significantly reduced lifespans (M: 8.22; F: 7.32 d) than the control (M: 10.00; F: 8.22 d). Reduced fecundity was observed in the B. bassiana-infected groups (LC20: 313.45; LC50: 223.92 eggs/female) compared to the control (359.55 eggs/female). A biochemical assay revealed elevated levels of detoxification enzymes (esterases, glutathione S-transferases, and acetylcholinesterase) in the F0 generation after B. bassiana infection. However, the enzymatic activity remained non-significant in the F1 generation likely due to the lack of direct fungal exposure. Our findings highlight the enduring effects of B. bassiana on the biological parameters and population dynamics of S. exigua, stressing its use in eco-friendly management programs.


Introduction
Life tables are powerful tools for studying arthropod population dynamics [1].Traditional age-specific life tables that solely consider females [2] can misrepresent the life history traits [3].However, recent advancements in age-and stage-specific life table methodologies highlight the importance of incorporating both age-stage and two-sex-based data.This integrated approach provides a more accurate representation of life history parameters for organisms with complex developmental stages, such as insects [4,5].Age-stage, twosex life table analysis employs a comprehensive (often daily) schedule to track survival rates for both male and female individuals across a life cycle.This method facilitates the estimation of crucial life history characteristics, including age-specific survival, fecundity, and generation time [6].Notably, these parameters can be assessed in response to various environmental factors, such as constant or fluctuating temperatures, pesticide resistance, and insect-pathogen interactions for biological control [7][8][9].and 75-80% RH.Isolates were passaged multiple times to prevent aging [33].Conidia were harvested into a 0.05% Tween-80 (Sigma Aldrich P1754, St. Louis, MO, USA) solution, and desired concentrations were prepared.

Bioassays
Five concentrations (3 × 10 4 , 3 × 10 5 , 3 × 10 6 , 3 × 10 7 , and 3 × 10 8 conidia/mL) for each fungus were tested against 3rd instar larvae, while 0.05% aqueous Tween-80 and distilled water alone were used as controls.To ensure assay effectiveness, germination tests were conducted by following previously described methods.Fungal suspensions were plated onto PDA plates and incubated in complete darkness at 25 • C ± 2 • C for 18 h [34].Conidium was considered germinated when its germ tube reached a length at least twice the diameter of the conidium itself [35].Moreover, 40 larvae (10/replicate × 4 replicates/assay) were exposed to each concentration by direct spraying using a fine aerosol sprayer and kept in sterilized containers containing an artificial diet.Mortality data were recorded every 24 h for 7 days and larvae with no movement were considered dead.Fungal pathogenicity was confirmed by placing the dead carcasses in a humid chamber to observe conidial growth.All mortality data were corrected via the Abbott formula.Lethal (LC 50 ) and sublethal (LC 20 ) concentrations were calculated and experimentally validated.The fungus with the highest mortality and least LC 50 was selected for subsequent studies.

Effects of B. bassiana on Parental Generation (F 0 )
The 3rd instar larvae (10/replicate × 4 replicates/assay) were exposed to LC 20 and LC 50 concentrations of B. bassiana, with aqueous Tween-80 serving as the control.Larval mortality, percent pupation, percent emergence, male/female longevity, and fecundity were evaluated.

Transgenerational Effects of B. bassiana on the First Filial Generation (F 1 )
B. bassiana-infected (LC 20 and LC 50 ) and control group eggs (n = 100) were randomly selected and examined for transgenerational effects.Developmental changes from larvae through pupae to adult emergence were recorded.The emerged adults were counted, paired into opposite sexes, and transferred to cages (1 pair/cage) for data regarding fecundity and survival.

EST
EST levels were determined by using p-nitrophenyl acetate (pNPA) and 50 mM phosphate buffer (pH 7.4) as substrate.Absorbance activity was recorded for 4 min at a 405 nm wavelength [37].

Statistical Analysis
LC 20 and LC 50 were calculated using POLO-PC (version 2.0) software [43].Mortality data underwent one-way ANOVA analysis, with means distinguished by Tukey's HSD test in Minitab (version 16) software at a 5% significance level.Enzymatic activity was analyzed using ANOVA and means were separated by Tukey's HSD test.Development, fecundity, and longevity were analyzed using the age-stage, two-sex life table (TWO-SEX MS Chart) [4,5,44].The bootstrap technique (n = 100,000) [45] was used for the mean and standard error of life table parameters [4,5,44].
R 0 (net reproductive rate) is the total offspring produced by an adult throughout its lifetime.
x indicates the survival likelihood to age x for a newly laid egg.
is the mean fecundity of individuals at age x and can be obtained from the following equation: The intrinsic rate of increase (r) was assessed using the iterative bisection method and adjusted with the age-indexed Euler-Lotka equation [46]: The finite rate was calculated using the following equation: The mean generational time (T) indicates the duration for a population to increase R 0 -fold at the stable age-stage distribution: T = (ln R 0 )|r e xj signifies that the expected lifespan of an individual at age (x) and stage (j) was calculated from the following equation [47]: The age-stage reproductive value (v xj ) (role of individuals of age x and stage j to the population) was calculated as follows [48,49]: Fungi 2024, 10, 469 5 of 18

Effects of Lethal (LC 50 ) and Sublethal (LC 20 ) Concentrations of B. bassiana on F 0
Concentration-dependent results were observed after treating LC 20 and LC 50 concentrations of B. bassiana on the F 0 generation, with larval mortality percentages of 21.92% and 52.33%, respectively.Significant differences in percent pupation were observed in B. bassiana-treated groups (47.67% in LC 50 and 78.01% in LC 20 ) and control groups (98.80%).Similarly, the maximum percentage of emergence was recorded in the control group (96.55%) compared to the treated groups (42.4% in LC 50 and 69.11% in LC 20 ).Maximum male longevity of 10.22 days was observed in the control group, which was significantly reduced to 9.71 and 7.11 days in the LC 20 and LC 50 treated groups, respectively.Similarly, adult females in the control group had longer lifespans (9.0 ± 0.22 days) compared to those in the treated groups (LC 50 : 6.22 ± 0.23; LC 20 : 8.22 ± 1.54 days) (p < 0.05).In addition, reduced fecundity (eggs/female) was observed in the treated females at LC 50 (273 ± 6.22) and LC 20 (380.18 ± 7.21) compared to the control (450.11± 7.11) (F: 84, df : 2, p < 0.05) (Table 2).Together, these findings illustrate that B. bassiana not only causes larval mortality but also disrupts developmental stages, reduces adult longevity, and impairs reproductive capacity in S. exigua, thereby influencing both the population dynamics and life history traits.(22.7 days) and LC 50 (20.99days)-treated groups.Lastly, a significant difference (per day) was observed in the mean finite rate of increase (λ) between the control (1.278 ± 0.02) and LC 50 -treated (1.217 ± 0.05) groups (F: 91, p < 0.03).These results highlight the transgenerational effects of B. bassiana on S. exigua population dynamics, revealing not only reduced reproductive capacity and accelerated generation turnover but also diminished intrinsic growth potential.The age-specific life expectancy (e x ), fecundity (m x ), reproductive value (v x ), and survival rate (l x ) are plotted in Figure 2. The age-specific life expectancy (e x ) approximates the longevity of an individual of age x.The results showed that the longevity of S. exigua in the LC50-treated individual at age zero (e 0 ) was lower compared to those in the control group (Figure 2A).The curve of age-specific fecundity (m x ) demonstrated that reproduction began at different ages in different groups (control/treated), with a significantly lower number of eggs recorded in the B. bassiana-infected group (Figure 2B).The age-specific reproductive value (v x ) computes the expected future reproductive contribution of an individual at a specific age (x) to the population.When the reproduction commenced, consistent reproductive values were observed between the control and B. bassiana-treated groups.However, over time, i.e., on the 21st day, the difference became noticeable, with the value of v x peaking at a maximum in the control group (89.10 days) and remaining lower in the LC 50 group (50.88 days).This trend continued throughout the remaining days as shown in Figure 2C.At early stages, the survival rate (l x ) curve between the control and treated groups remained non-significant; however, with time, i.e., at 25 days, the survival rate of insects in the LC 50 group reduced significantly as shown in Figure 2D.
longevity of an individual of age x.The results showed that the longevity of S. exigua in the LC50-treated individual at age zero (e0) was lower compared to those in the control group (Figure 2A).The curve of age-specific fecundity (mx) demonstrated that reproduction began at different ages in different groups (control/treated), with a significantly lower number of eggs recorded in the B. bassiana-infected group (Figure 2B).The age-specific reproductive value (vₓ) computes the expected future reproductive contribution of an individual at a specific age (x) to the population.When the reproduction commenced, consistent reproductive values were observed between the control and B. bassiana-treated groups.However, over time, i.e., on the 21st day, the difference became noticeable, with the value of vₓ peaking at a maximum in the control group (89.10 days) and remaining lower in the LC50 group (50.88 days).This trend continued throughout the remaining days as shown in Figure 2C.At early stages, the survival rate (lx) curve between the control and treated groups remained non-significant; however, with time, i.e., at 25 days, the survival rate of insects in the LC50 group reduced significantly as shown in Figure 2D.The cohort-specific egg-to-adult survival rates (sxj) were significantly reduced in B. bassiana-infected groups compared to the control group (Figure 3).The sxj values for adult females and males in the LC50 treated group were 0.29 and 0.50, respectively, compared to 0.37 and 0.53 in the control group, indicating a significant effect of B. bassiana (Figure 3A-C).The age-stage, two-sex life table also estimated the expected lifespans of different stages in the population (Figure 4).Our analysis of the life expectancy of S. exigua across different life stages (exj) revealed a higher survival probability of the first larval instar (L1) The cohort-specific egg-to-adult survival rates (s xj ) were significantly reduced in B. bassiana-infected groups compared to the control group (Figure 3).The s xj values for adult females and males in the LC 50 treated group were 0.29 and 0.50, respectively, compared to 0.37 and 0.53 in the control group, indicating a significant effect of B. bassiana (Figure 3A-C).The age-stage, two-sex life table also estimated the expected lifespans of different stages in the population (Figure 4).Our analysis of the life expectancy of S. exigua across different life stages (e xj ) revealed a higher survival probability of the first larval instar (L1) in the control group than in LC 50 -treated groups.Additionally, the control group exhibited a longer lifespan compared to the treated groups (Figure 4A-C).The age-stage reproductive value (vxj) for females was significantly lower in the LC20and LC50-treated groups than in the control (Figure 5).Peak reproduction occurred at 13-17 days in the control (Figure 5A), whereas maximum reproduction rates in the LC20 and The age-stage reproductive value (v xj ) for females was significantly lower in the LC 20and LC 50 -treated groups than in the control (Figure 5).Peak reproduction occurred at 13-17 days in the control (Figure 5A), whereas maximum reproduction rates in the LC 20 and LC 50 groups were observed at 13-15 and 14-16 days, respectively (Figure 5B,C).In addition, the LC 20 and LC 50 -treated groups exhibited reduced fecundity, demonstrating the impact of B. bassiana on the population dynamics (Figure 6).LC50 groups were observed at 13-15 and 14-16 days, respectively (Figure 5B,C).In addition, the LC20 and LC50-treated groups exhibited reduced fecundity, demonstrating the impact of B. bassiana on the population dynamics (Figure 6).

Detoxification Enzyme Activity in S. exigua following B. bassiana Infection (F0 and F1)
AChE, EST, and GST enzymes play crucial roles in the detoxification of xenobiotics in insects, influencing their response to intoxication and potentially contributing to resistance development [23,27].Figure 7 shows the detoxification enzyme activity in F0 and F1 generations of S. exigua in response to B. bassiana (LC20 and LC50) infection.AChE, EST, and GST enzymes play crucial roles in the detoxification of xenobiotics in insects, influencing their response to intoxication and potentially contributing to resistance development [23,27].Figure 7 shows the detoxification enzyme activity in F 0 and F 1 generations of S. exigua in response to B. bassiana (LC 20 and LC 50 ) infection.

Detoxification Enzyme Activity in S. exigua following B. bassiana Infection (F0 and F1)
AChE, EST, and GST enzymes play crucial roles in the detoxification of xenobiotics in insects, influencing their response to intoxication and potentially contributing to resistance development [23,27].Figure 7 shows the detoxification enzyme activity in F0 and F1 generations of S. exigua in response to B. bassiana (LC20 and LC50) infection.

AChE
In F 0 generation, the maximum AChE activity was observed at 24 h in both LC 50 (13.49µmol/min/mg protein) and LC 20 (10.59 µmol/min/mg protein)-treated groups compared to the control (2.4 µmol/min/mg protein), which gradually decreased as time progressed (Figure 7A).F 1 generation exhibited similar trends (non-significant) in the control and B. bassiana (LC 20 and LC 50 )-treated groups (Figure 7A1).

EST
Maximum EST activity in F 0 generation was observed at 12 h post-treatment, with the LC 50 (15.07µmol/min/mg protein)-treated group exhibiting the highest activity followed by LC 20 (12.24 µmol/min/mg protein) compared to the control (2.09 µmol/min/mg protein) (Figure 7B).Similar to the AChE enzyme, the EST showed non-significant trends in F 1 generation (Figure 7B1).

GST
The peak GST activity was observed at 12 h post-treatment in F 0 generation, with higher activity detected in the LC 50 (15.5 µmol/min/mg protein) and LC 20 (8.43 µmol/min/mg protein)-treated groups compared to the untreated control (1.62 µmol/min/mg protein).A noticeable decrease was observed at 36-72 h post-treatment (Figure 7C).However, no significant differences in GST activity were observed in the F 1 generation (Figure 7C1).
The stable activity of detoxification enzymes in the F 1 generation could be attributed to the lack of exposure to harmful fungal compounds.However, further studies are required to elucidate the exact mechanisms behind this observation

Discussion
B. bassiana is a potent biocontrol agent, capable of controlling several susceptible and resistant/multi-resistant insect pests [50], including S. exigua [19].Here, we investigate the virulence of B. bassiana against S. exigua and its impact on fitness parameters across the F 0 and F 1 generations using age-stage, two-sex life table analysis.This knowledge can be valuable for developing integrated pest management (IPM) programs by potentially reducing the lethal time and increasing S. exigua mortality.
B. bassiana infection disturbed the fitness parameters of S. exigua.In the F 0 generation, concentration-dependent results were observed after being treated with LC 20 and LC 50 of B. bassiana with reported larval mortality rates of 21.92% and 52.33%, respectively.In addition to lethal effects, decreases in percent emergence, adult longevity, and fecundity were also reported as potential effects of B. bassiana infection.Studies have documented similar fitness costs associated with entomopathogenic fungi [56].Consistent with our findings, sublethal exposure to B. bassiana significantly impaired the development and reproductive capacity of various insect pests, including Nilaparvata lugens [57], Eurygaster integriceps [58], Sogatella furcifera [59], and H. armigera [60].These sublethal effects are potentially linked to entomopathogenic fungi-mediated nutritional deficiency [61].Upon penetrating the insect host, fungal pathogens not only release secondary toxins but also absorb essential sugars from tracheoles, weakening the insects and ultimately impacting their biology and population dynamics [62,63].
The negative effects of B. bassiana on S. exigua extended beyond the directly exposed generation.In the F 1 generation, declines in pre-adult duration, adult longevity, and female fecundity were observed, consistent with the findings reported for B. bassiana infection in Cyclocephala lurida [64] and M. anisopliae infection in H. armigera [65].Notably, prolonged pupal duration was observed in the LC 50 (7.27days)-treated group compared to the control (6.58); similar to our study, the extended pupal period was reported in B. bassiana-exposed H. armigera [56,60].We theorize that the fungal infection imposes physiological and ecological costs on the developing organism, resulting in nutritionally deficient eggs and potentially leading to the continuation of sublethal effects into the F 1 generation [57,66].Moreover, the values of the intrinsic rate of increase (r), net reproduction rate (R 0 ), mean length of generation (T), and finite rate of increase (λ) showed significant reduction in response to lethal and sublethal treatments of B. bassiana.Supporting our findings, studies have shown reduced λ and R 0 but prolonged T values in the M. anisopliae-infected tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae) [67].Similarly, lethal and sublethal treatments of B. bassiana had detrimental effects on the life-history parameters of Bactericera cockerelli in the F 0 and F 1 generations [68].The reduced r, R 0 , and λ values in treatment groups relative to the control indicate the potential impacts of B. bassiana infection on the population growth rate and generation.Notably, r is considered a particularly sensitive measure of insect response to stressors, as it directly reflects the population's growth potential [69,70].
Cohort-specific (egg-to-adult) biological parameters (e xj , s xj , and v xj ) are vital indicators for assessing the fitness of the insect population.Similar to our findings, the overlapping age-stage survival rate (s xj ) curves between control and treated groups in M. anisopliaeinfected Oxycarenus hyalinipennis were reported [71].In addition, developmental timings were also affected by fungal infection.Similar trends were also reported in B. bassianainfected Aedes albopictus [29], supporting our findings.
AChE, EST, and GST are important physiological metabolic detoxification enzymes that help insects cleanse and resist harmful intoxicants [72].Our results indicated that the detoxification enzyme activity was significantly elevated in response to B. bassiana infection compared to the control.Significant increases in the levels of EST and GST activities in Dendrolimus tabulaeformis (Lepidoptera: Lasiocampidae) larvae were closely related to the concentration of conidia and the metabolites of Beauveria brongniartii [73], potentially due to the larvae reacting to conidial infection and fungal toxins by relieving oxidative stress.Likewise, in Diaphorina citri, higher levels of GST and EST were reported in response to I. fumosorosea and B. bassiana infections [37], indicating the activation of the antifungal immune response.Exposure to B. bassiana and its secondary metabolites has been linked to changes in the activity of AChE in the Sunn pest, E. integriceps [74].A significant increase in AChE activity was observed in the hemolymph of H. armigera in response to B. bassiana infection [75].These alterations in AChE activity can be attributed to the production of the secondary metabolite bassianolide by the Beauveria spp.Bassianolide, a cyclooligomer depsipeptide, has been detected in the cadavers of silkworm larvae infected with B. bassiana [76], demonstrating that its production coincides with infection.Bassianolide can inhibit the acetylcholine receptors of insect muscles, reducing the production of AChE [77].Furthermore, secondary toxins produced by entomopathogenic fungi are known to induce host cell apoptosis via an increase in oxidative stress and interference with hormonal and mitochondrial signaling while also affecting acetylcholine receptors [78].However, the enzymatic activity remained non-significant in the F 1 generation potentially due to the absence or lack of direct fungal exposure.These findings highlight the potential

Figure 2 .
Figure 2. The age-specific (A) life expectancy (e x ); (B) fecundity (m x ); (C) reproductive value (v x ); and (D) survival rate (l x ).The sublethal and lethal concentrations of B. bassiana-treated groups are represented as LC 20 and LC 50 , respectively.

Figure 6 .
Figure 6.Daily mean number of eggs from S. exigua after treatment with B. bassiana.The sublethal and lethal concentrations of B. bassiana are shown as LC20 and LC50, respectively.

Figure 7 .
Figure 7.The detoxification enzyme activity in S. exigua in response to sublethal (LC20) and lethal (LC50) concentrations of B. bassiana.Figure (A-C) show the enzymatic activity in the parental/filial (F0) generation, while (A1-C1) show the enzymatic activity in the first filial generation (F1).Different colors and symbols are used for different treatments.

Figure 6 .
Figure 6.Daily mean number of eggs from S. exigua after treatment with B. bassiana.The sublethal and lethal concentrations of B. bassiana are shown as LC 20 and LC 50 , respectively.

Figure 6 .
Figure 6.Daily mean number of eggs from S. exigua after treatment with B. bassiana.The sublethal and lethal concentrations of B. bassiana are shown as LC20 and LC50, respectively.

Figure 7 .
Figure 7.The detoxification enzyme activity in S. exigua in response to sublethal (LC20) and lethal (LC50) concentrations of B. bassiana.Figure (A-C) show the enzymatic activity in the parental/filial (F0) generation, while (A1-C1) show the enzymatic activity in the first filial generation (F1).Different colors and symbols are used for different treatments.

Figure 7 .
Figure 7.The detoxification enzyme activity in S. exigua in response to sublethal (LC 20 ) and lethal (LC 50 ) concentrations of B. bassiana.Figure (A-C) show the enzymatic activity in the parental/filial (F 0 ) generation, while (A1-C1) show the enzymatic activity in the first filial generation (F 1 ).Different colors and symbols are used for different treatments.

Table 2 .
Influence of B. bassiana on parental generation (F 0 ) of S. exigua.
Note: Units are days for male/female longevity.Fecundity (eggs/female).Different letters within rows mark statistically significant differences (p < 0.05) between means.Values with the same letter are not statistically significant.3.3.Effects of Lethal (LC50 ) and Sublethal (LC 20 ) Concentrations of B. bassiana on F 1 3.3.1.Biological Parameters Transgenerational effects of B. bassiana (LC 20 and LC 50 ) on S. exigua are presented in

Table 3 .
Effects of B. bassiana on the first filial generation (F 1 ) of S. exigua.

Table 4 .
Lethal (LC 50 ) and sublethal (LC 20 ) effects of B. bassiana on the population parameters of S. exigua.
r = Intrinsic rate of increase (per days); R 0 = net reproduction rate (offspring/individual); T = mean length of a generation (days); λ = finite rate of increase (per days); SASD = stable age-stage distribution.Different letters within rows mark statistically significant differences (p < 0.05) between means.Values with the same letter are not statistically significant.