This study utilized a completely randomized design (CRD) with five treatments and three replications to evaluate the effect of B. bassiana concentration on the mortality of N. rufipes adults on copra. A quantitative experimental approach was employed to investigate the cause-and-effect relationship between independent (fungal concentration) and dependent (mortality and copra weight loss) variables. Two experiments were conducted: one assessing mortality and another evaluating copra weight loss.

For the mortality experiment, five concentrations of B. bassiana culture were tested: A = 10% (10 g/100 mL distilled water), B = 15% (15 g/100 mL), C = 20% (20 g/100 mL), D = 25% (25 g/100 mL), E = 30% (30 g/100 mL), and F = control (0 g/100 mL). Each treatment used 25 adult N. rufipes individuals placed with 150 g of copra as a feeding substrate. For the weight loss experiment, five pest population sizes were tested: G = 8 adults, H = 12 adults, I = 16 adults, J = 20 adults, and K = 24 adults, each with 150 g of copra. Mortality was recorded weekly for one month, starting one week post-treatment, while copra weight loss was measured after one month.

Sampling of the test insects did not differentiate between males and females; instead, individuals of the same imago age were selected, specifically 1–3 days post-emergence to ensure consistency in physiological condition. Furthermore, mortality observations did not account for potential differences in resistance between male and female insects to pathogen infection.

Data were analyzed using analysis of variance (ANOVA). Significant differences [Sig (0.000) < α (0.05)] were further assessed with the Least Significant Difference (LSD) test. The effective lethal concentration (LC₅₀) of B. bassiana for controlling N. rufipes was determined using probit analysis, focusing on treatments C (20 g), D (25 g), and E (30 g), which approached 50% mortality.

Preparation

1. Rearing of test insects (Necrobia rufipes)

• N. rufipes was propagated by collecting infested copra showing signs of pest attack from storage warehouses and transferring it to the Plant Pests and Diseases Laboratory for rearing.
• One kilogram of infested copra was placed in plastic jars, covered with white azahi cloth, and maintained for 1.5–2 months until the N. rufipes population multiplied sufficiently to serve as test insects.

1. Preparation of B. bassiana culture on rice medium

• Rice was steamed until half-cooked, cooled in a sterile cabinet, and packed into transparent plastic bags (20 g per bag).
• Pure B. bassiana cultures (derived from Paraeucosmetus spp., North Sulawesi) were inoculated into each 20-g rice package and incubated for one week until ready for application.

Experimental application

1. Application of B. bassiana to N. rufipes adults

• The pathogen was applied to test insects in jars containing 150 g of sterilized copra and 25 N. rufipes adults per treatment.
• After application, jars were sealed with white Asahi cloth (Japan).
• Observations began one-week post-application and continued for one month, with weekly intervals. Dead N. rufipes was collected to calculate mortality per treatment, assessed in week two, while copra weight loss was measured in week four.

1. Copra weight loss assessment

• Weight loss was determined as the reduction in copra mass due to N. rufipes feeding during the study period.
• Copra (150 g per treatment) were weighed at the start of the experiment using an electronic balance with 0.01 g precision, placed individually in jars, and then infested with varying N. rufipes adult populations according to the treatment levels. Jars were covered with cloth to allow for ventilation.
• After one month, the copra was removed from the jars and weighed again. The difference between the initial and final weights represented the copra weight loss.

Mortality of adult N. rufipes due to B. bassiana application was calculated using the following formula:

$\text{Mortality}(\%)=\left(\frac{\text{Number of dead insects}}{\text{Total number of observed test insects}}\right)\times 100$

Copra weight loss resulting from N. rufipes infestation was determined as:

Weight loss = Initial sample weight − Final sample weight

Data on mortality and weight loss were analyzed using analysis of variance (ANOVA). All statistical analyses were performed using SPSS 21.0 software.

The concentration of B. bassiana significantly influenced the mortality of adult N. rufipes on copra. Mortality, measured as cumulative mortality after one month of exposure, was directly proportional to fungal concentration, with higher doses resulting in greater mortality rates. Mean mortality rates are presented in Table 1.

The highest mortality (69.33% ± 3.79%) was observed at a 30% concentration, while the lowest (14.67% ± 2.31%) occurred at 10%. This trend likely results from increased toxin levels—such as beauvericin, beauverolide, bassianolide, and acids—in higher B. bassiana concentrations, which impair insect physiology and reproduction (Wang et al. 2018).

Regression analysis revealed a positive coefficient (2.263), indicating that a 1% increase in B. bassiana concentration raised mortality by 2.263%. The relationship between concentration (X) and mortality (Y) is depicted in Figure 1, with a coefficient of determination (R² = 0.953), suggesting that 95.3% of mortality variation is explained by concentration.

Previous studies support these findings, showing that N. rufipes mortality increases with higher concentrations of entomopathogenic fungi like M. anisopliae and B. bassiana (Fathy et al. 2025). This infection process culminates in the death of the insect (Turrà and Di Pietro 2015), which directly influences the subsequent impact of N. rufipes population density on copra weight loss. Impact of N. rufipes Infestation on Copra Weight Loss.

Figure 1. 

Relationship Between B. bassiana Concentration (X) and Adult N. rufipes Mortality (Y).

The study on copra weight loss due to N. rufipes infestation revealed that pest population density significantly influenced weight reduction. Results from laboratory experiments are presented in Table 2.

The highest weight loss (6.87 ± 0.42 g) occurred at a population density of 24 N. rufipes adults, while the lowest (1.13 ± 0.12 g) was observed with 8 adults. Copra weight loss increased with population density, as larger populations require more food, leading to greater copra consumption. Storage pests like N. rufipes depend entirely on stored commodities for survival, and higher densities exacerbate damage (Manueke et al. 2023). Arifin et al. (2022) define copra weight loss as the reduction of material due to pest consumption, while Nuraini et al. (2022) note that storage pests cause quantitative losses, quality degradation, and reduced seed viability. These pests vary in morphology, preferred food sources, and environmental requirements for survival and reproduction.

Regression analysis showed a positive coefficient of 0.316, indicating a direct relationship between N. rufipes population density (X) and copra weight loss (Y). The regression equation, Y = 0.316X − 0.672, implies: (1) a constant of -0.672 as the baseline treatment value; (2) a 1% increase in population density increases weight loss by 0.316 g; and (3) a coefficient of determination (R² = 0.9076) suggests that 90.76% of weight loss variation is explained by population density. This relationship is depicted in Figure 2.

Storage pests cause both quantitative (reduced quantity) and qualitative (decreased quality) damage to stored products. They can infest commodities pre-harvest, during transport, and in storage, with warehouses providing ideal conditions—abundant food, favorable environments, and low natural enemy presence—allowing rapid population growth and significant damage in a short time (Hill 1990; Tarore et al. 2023).

Figure 2. 

Relationship Between N. rufipes Population Density (X) and Copra Weight Loss (Y).

The effective lethal concentration (LC₅₀) of B. bassiana culture required to achieve 50% mortality of N. rufipes adults on copra was determined using probit analysis. The results are presented in Table 3.

The probit analysis results in Table 3 show that the effective concentration dose of the B. bassiana pathogen culture on the mortality of the pest N. rufipes on copra lies between treatment C, which is a concentration of the B. bassiana pathogen culture of 20 g/100 cc of distilled water, causing a mortality of N. rufipes adults of 36.00%, and treatment D, which is a concentration of the B. bassiana pathogen culture of 20 g/100 cc of distilled water, causing a mortality of N. rufipes adults of 52.00%. The effective concentration dose of the B. bassiana pathogen culture in controlling N. rufipes adults on copra is 23.611. This effective dose is still classified as a low active ingredient content, namely 23.611% = 23.611 g of the B. bassiana pathogen culture in 100 cc of distilled water. Determining this LC₅₀ is crucial for optimizing B. bassiana application strategies. Using the LC₅₀ as a target concentration can help minimize the amount of fungus needed, reducing costs and potential non-target effects, while still achieving effective pest control.

While this study focused on B. bassiana efficacy, the observed mortality is consistent with its known mode of action, which involves cuticle penetration and multiplication within the insect host (Kaur and Padmaja 2008; Indriyati 2009).

The toxicity of insecticides to organisms is usually expressed in terms of LD50 (lethal dose for 50% of insects). In some cases, LC₅₀ is used to express the concentration of insecticide that will kill half of the test insect population (Paramasivam and Selvi 2017). According to Hasyim et al. (2016) and Hasyim et al. (2019), the LC₅₀ value is the concentration that can cause the death of 50% of the pest insects tested in a specific observation.

While more frequent mortality assessments (daily) could have provided a more detailed time course of N. rufipes mortality, we opted for weekly intervals due to the labor-intensive nature of the counts, the desire to minimize disturbance to the experimental setup, preliminary observations that showed minimal changes in mortality at shorter intervals.