This experiment was conducted within the appropriate ethical framework of the Ethiopian Institute of Agricultural Research, as confirmed by an institutional letter in Ref. No.: 23.11/660/2023 written on 06 October 2023.

The experiment was conducted at the Ambo Agricultural Research Center, Entomology greenhouse. Parthenium seeds were sown on seedling beds, and seedlings were transferred at the stage of 12 leaves to pots (18 × 20 cm dimensions) ( Figure 1A). The soil used in the pot was a combination of sand, compost, and clay loam, at a ratio of 1:1:2, respectively. Seedlings of the same stage, with 18–24 leaves, were transferred to the experimental cage and used for the experiment ( Figure 1B). The plants were watered daily to provide free moisture for adult bioagents, and the leaves were sprayed with a sodium hypochlorite (Bleach, product of the Ghion Industrial and Chemical PLC., Addis Ababa, Ethiopia) solution to prevent disease development. Because sodium hypochlorite is the most often used disinfectant, it is a broad-spectrum disinfectant that is efficient against viruses, bacteria, fungi, and mycobacterium. The sodium hypochlorite was mixed with sterilized distilled water at 2% of the solution by using a 1-liter hand sprayer.

The beetles were reared in an insect-rearing cage, which was constructed using metal and nylon mesh as a frame and cover, respectively. The size of each cage was 0.5 × 0.5*1m. Twenty-five pairs of beetles were released from the parthenium seedlings in the rearing cage. After two to three days of introduction and the plants were checked for oviposition, the adults were transferred to another rearing cage. The deposited eggs were monitored daily until they reached the adult stage. Within two days, pairs of emerged adults were transferred to the experimental cages ( Figure 1C). Beetle rearing and experiments were conducted in a greenhouse.

A completely randomized design with three replicates was used for the experiment. Six treatments, viz., two pairs, five pairs, ten pairs, 15 pairs, and 20 pairs of Z. bicolorata adults per parthenium seedling, and a control without the beetle, were used. The number of leaves per plant was counted before the introduction of the beetles and at the end of the experiment. Similarly, the number of eggs deposited, hatched larvae, and damaged leaves were counted, and plant damage was measured on a scale of 0–100% daily. ¹⁵

The collected data were checked for normal distribution prior to data analysis. Data on the number of eggs deposited, hatched larvae, and damaged leaves were analyzed by ANOVA, with the means separated by the least significant difference (LSD) using SAS version 9.4. ¹⁶ Additionally, a correlation analysis was performed between the independent variable (exposure day) and the dependent variable (plant leaf damage).

Adult beetles released on parthenium plants deposited eggs from the first days of introduction. A lower egg number (71.67) was recorded from the plants treated with two pairs, while a higher egg number (328.67) was deposited from the 20 pairs used ( Figure 2).

The larvae of Z. bicolorata began to emerge after seven days of exposure. Similar to eggs, Z. bicolorata larvae were recorded on all parthenium plants treated with beetles. The data showed that the number of larvae that hatched increased with the population of beetles applied ( Figure 3). The number of hatched larvae per plant treated with 20 pairs of beetles exceeds 75.89%, 57.53%, 35.13%, and 19.58% of the number of plants treated with fifteen, ten, five, and two pairs, respectively.

The number of leaves per plant did not vary during the initial period among the treatments used ( Figure 4). However, based on the number of released beetles, leaf defoliation varied among treatments (P < 0.001) ( Figure 5). Three days after release, the 20 pairs defoliated 53% of the leaves, which is a significant leaf defoliation, whereas no visible defoliation was observed under two pairs of beetles. After six days of release, no significant leaf defoliation was recorded under the fifteen and twenty-pairs of beetles, and complete defoliation was recorded on the twelfth day. Similarly, ten pairs recorded complete leaf defoliation after 15 days of beetle release. At the first day after release, all pairs defoliated 100% of the leaves. The higher population of beetles (20 and 15 pairs) completely defoliated within 1.75 folds shorter time compared with two and five pairs of beetles. Additionally, correlation analysis indicated that the number of beetle populations was highly positively correlated with leaf defoliation (r = 0.944) (p < 0.01) ( Table 1).

This study indicated that all released pairs of beetle adults defoliated the parthenium plant leaves. The shortest time (12 d) for complete leaf defoliation was recorded for 20 and 15 pairs, whereas the longest (21 d) was recorded for plants treated with two and five pairs. This implies that beetle populations per plant are important for the effective management of weeds, and increasing the population of beetles minimizes the number of days needed to achieve complete leaf defoliation. This result is in line with the results of Shabbir et al.’s ¹⁷ and Kanagwa et al.’s, ¹¹ who reported that beetles had defoliated leaves up to 100% and reduced weed performance within 28 days. Additionally, the defoliated leaves increased as the exposure time increased, and the beetles effectively controlled the weed and significantly reduced its vegetative and reproductive stages in Australia, India, Tanzania, and South Africa. ^{11 , 18 , 19} Parthenium leaf consumption increased rapidly from 14 to 28 days by 115%, 112%, and 100% at populations of 0.4, 0.8, and 1.2 beetles per plant, respectively, in Tanzania. ¹¹ In Tanzania, 1.2 beetles per plant (which was used as a higher population) caused 100% leaf defoliation after 28 days of exposure in the wet season, whereas in this experiment, two pairs per plant recorded 100% leaf defoliation within 21 days. This indicates that the beetle population is negatively correlated with the date of complete leaf defoliation in the plant. Similarly, Chandravanshi et al. ¹⁴ reported that a higher population (five pairs) of beetles recorded more severe damage to leaves within a shorter time than lower populations (two pairs). This result agreed with that of Hasan et al., ¹⁹ who showed that damage from weeds was positively related to the exposure time.

After larval emergence, the leaves of weed plants were effectively defoliated. This indicates that the larvae play an important role in weed control. In contrast, the collaboration between adults and larvae ensured that the leaf of the weed was quickly eaten and growth was retarded. Similarly, Mohapatra et al. ²⁰ reported that the larvae have the capacity to feed more on parthenium weed than adult females and recommended it as a better management option for the weed. Additionally, in Bangalore (India), larvae were fed the weed effectively and suppressed their growth and flower production, causing them to dry up completely. ¹⁸ This implies that the bioagent is florivorous in nature at both the larval and adult stages and is a voracious defoliator of the weed, which enhances its susceptibility to biotic factors. ^{21 , 22} It was understood that the plants that were not infested by the beetles continued and flourished in their growth stages, while others dried up ( Figure 6A-D).