Vegetables are considered a crucial component of human nutrition due to their high nutritional value. However, they are susceptible to insect attacks, attributed to their attractive colors and soft structure, leading to approximately 40% of vegetable damage. Among the vegetable groups, bitter melon (Momordica charantia L.) stands out as a widely cultivated and valuable species (Joseph and Jini 2013). The fruits and leaves of M. charantia are rich in phytochemicals and are commonly utilized in traditional medicine for treating ailments such as joint pain, chronic fever, liver diseases, and digestive disorders (Bortolotti et al. 2019). The Bactrocera cucurbitae fly poses a significant threat to bitter melon plants. Capable of causing damage ranging from 30% to 100%, depending on environmental conditions, and this fly thrives in environments with temperatures below 32°C and relative humidity between 60% and 70%. B. cucurbitae targets non-ripe, green, and soft-skinned fruits, it particularly inflicts severe damage when the fruits are in the developmental stage. For vegetables like bitter melon, damage caused by the fruit fly is a primary factor limiting quality and productivity (Dhillon et al. 2005). Various control measures have been implemented over the years to manage harmful insects; however, heavy reliance on chemical insecticides has not yielded satisfactory results. Blind reliance on chemical insecticides without proper selection has led to the development of insecticide resistance in most harmful insect species. Another serious issue is the accumulation of pesticide residues in food due to the uncontrolled use of chemical insecticides (Safdar et al. 2020). B. cucurbitae has also demonstrated resistance to several common chemical insecticides. Currently, the most environmentally friendly and alternative solution to synthetic chemical insecticides is the use of biological insecticides. Derived from plants, these insecticides are non-toxic and can undergo safe biological degradation, offering an effective and sustainable choice in managing harmful insects (Jaleel et al. 2020).

Melia azedarach L., a widely used medicinal plant in the Meliaceae family, originates from Africa, Asia, and North Australia, and is globally distributed. The leaves of M. azedarach contain abundant limonoid compounds, along with phenolic acids, cardiac glycosides, and flavonoids, providing diverse benefits such as antibacterial, antioxidant, anticancer, antiparasitic, antifungal properties, and free radical scavenging activity (Mckenna et al. 2013). Numerous studies have substantiated the insecticidal efficacy of M. azedarach against various harmful insect species. These effects include reducing reproductive capability, inhibiting egg hatching, decreasing the lifespan of mature offspring, preventing egg laying, and exerting antifreeze and growth-regulating effects on insects during molting (Jabamo et al. 2023). Extracts from M. azedarach fruit also exhibit efficacy against the leaf miner Phyllocnistis citrella (Mckenna et al. 2013) and larvae of Xanthogaleruca luteola (Chiffelle et al. 2019). In the context of plant protection strategy, minimizing the use of synthetic compounds is crucial, with a shift towards natural alternatives. Natural insecticides often pose minimal harm to non-target organisms. In agricultural pest management, botanical insecticides prove to be the most suitable choice for organic food production in developed industrialized countries and can play a significant role in post-harvest food production and protection in developing nations (Mckenna et al. 2013). The availability of M. azedarach provides a natural resource for farmers to control economically significant pest species. The objective of this study is to elucidate the efficacy of extracts from M. azedarach fruit and seeds in managing the population of Bactrocera cucurbitae, a pest causing damage to Momordica charantia, under laboratory and greenhouse conditions.

Collection plant material

The fruits and seeds of Melia azedarach were systematically collected in May 2023 from Hung Dinh commune, Thuan An District, Binh Duong province, Vietnam. A reference sample (code MA150523VST) has been meticulously preserved at the Laboratory of Plant Biotechnology, Institute of Biotechnology and Food Technology, Ho Chi Minh City University of Industry, serving as a valuable resource for future identification and reference purposes. Careful selection criteria were applied to choose fresh fruits, ensuring the exclusion of damaged specimens, followed by thorough washing and air-drying. Subsequently, the fruits were precision-cut into small pieces and subjected to drying within the Memmert UN 110 drying cabinet (manufactured in Germany) at a temperature of 60°C. Post-drying, the fruits were promptly hermetically sealed in plastic packaging for optimal preservation.

Preparation of the extract

The extraction process was carried out using the cold extraction method according to the study conducted by Hemdan et al. (2023). Dry fruit samples from M. azedarach (500 mg) were specifically immersed in 70% ethanol solvent for 72 hours (static, 2 cycles of 5 times at room temperature). Subsequently, the extraction solution was filtered through a filter, and the solvent used was completely evaporated using a slow rotary evaporator. The obtained extracts were further concentrated using a rotary evaporator at a temperature below 40°C. These concentrated extracts were referred to as MAFS and stored in desiccators until utilized in subsequent research studies.

Phytochemical screening and quantitative analysis in the ethanol extract of M. azedarach fruits and seeds

Phytochemical screening: The botanical chemical analysis study was conducted qualitatively to identify bioactive compounds in the ethanol extract obtained from the fruits and seeds of the M. azedarach tree. The methods employed in this research adhere to the standards set by Tran et al. (2023), as presented in Table 1.

Table 1 . Phytochemical analyses of ethanol extracts derived from fruits and seeds of M. azedarach.

Phytoconstituents	Test	Observation
Tannins	2 mL extract + 2 mL H2O + 2-3 drops FeCl3 (5%)	Green precipitate
Flavonoids	1 mL extract + 1 mL Pb(OAc)4 (10%)	Yellow coloration
Terpenoids	2 mL extract + 2 mL (CH3CO)2O + 2-3 drops conc. H2SO4	Deep red coloration
Saponins	5 mL extract + 5 mL H2O + heat	Froth appears
Steroids	2 mL extract + 2 mL CHCl3 + 2 mL H2SO4 (conc.)	Reddish brown ring at the junction
Cardiac glycosides	2 mL extract + 2 mL CHCl3 + 2 mL CH3COOH	Violet to Blue to Green coloration
Alkaloids	2 mL extract + few drops of Hager’s reagent	Yellow precipitate
Phenolic compound	2 mL extract + 2 mL FeCl3	Bluish-green appearance

Phytochemical quantification: Polyphenols, flavonoids, and tannins play a crucial role in plant physiology, serving as potent antioxidants. These compounds fortify the plant’s defense mechanisms against the invasion of B. cucurbitae. Precise quantification of these constituents in plant extracts is essential and is conducted through well-established methodologies as elucidated by Nhung and Quoc (2024). The quantification of total polyphenol content was conducted using the Folin-Ciocalteu method, expressed in milligrams of gallic acid equivalent per gram of dried plant material (mg GAE/g) following the gallic acid standard. Total flavonoid content was measured using the aluminum chloride (AlCl₃) method, with results represented in milligrams of quercetin equivalent per gram of dried plant material (mg QE/g) based on the quercetin standard. The reliable determination of tannin content was achieved through the vanillin spectrophotometric method, and the total condensed tannin content was calculated in milligrams of catechin equivalent (mg CE/g) using a standard curve. These established methods yield robust data for quantifying the levels of polyphenols, flavonoids, and tannins in plant extracts. They provide valuable insights into the potential roles of these compounds in enhancing the health of M. charantia and reinforcing defense mechanisms against the damage caused by B. cucurbitae.

Formulations for treating

The concentration of the extract from the fruits and seeds of M. azedarach was achieved through solvent evaporation, forming a basic concentrated solution. This solution was then diluted into five different concentrations (1, 5, 25, 125, and 625 ppm), denoted as MAFS1, MAFS5, MAFS25, MAFS125, and MAFS625, respectively. In this study, the commercial product Sofri protein 10 DD (1.2 liters/ha) served as a positive control (Sofri protein treatment). Water was employed as the reference toxicity treatment (Water treatment). All treatment methods were implemented to manage B. cucurbitae in laboratory and greenhouse experiments.

Experimental investigations in the laboratory environment

Cultivating B. cucurbitae larvae: This investigation was conducted at the Animal Biotechnology Laboratory, Department of Biotechnology, Ho Chi Minh City University of Industry, following established protocols by Jaleel et al. (2020) with minor modifications. To procure first, second, and third instar larvae, bitter melon samples were placed in steel mesh cages containing over 100 gravid females. After 24 hours, the bitter melon samples were removed from the cages and placed in vials lined with moist sand (D10 × L15 cm). Bitter melon samples were excised using fine forceps to harvest larvae. Subsequently, the harvested larvae were rinsed with distilled water and transferred into experimental vials (D25 × L100 mm) containing an artificial diet (comprising a mixture of Dextrose-L and protein hydrolysate Protinex, Pfizer Ltd.) in a 1:1 ratio, with varying concentrations of MAFS (1, 5, 25, 125, and 625 ppm), along with corresponding control groups (Sofri protein and water). Each concentration was replicated three times, with each vial containing 15 larvae. These experimental vials were stored in a Biological Oxygen Demand (B.O.D) chamber to maintain stable temperature, humidity, and lighting conditions, thus creating an environment conducive to larval survival (27 ± 1.5°C, 60 ± 5.5% humidity, and 10-hour light/14-hour dark cycles). Daily observations were conducted on developmental parameters such as pupation time and the number of pupae formed, time and number of emerging flies emergence, and oviposition in B. cucurbitae.

Pupation time and the number of pupae formed in B. cucurbitae: The evaluation of pupation time and the number of pupae formed in B. cucurbitae was conducted following the method outlined by Thakur and Gupta (2013) with minor modifications. Collecting larvae of B. cucurbitae from both the test sample and the control group, the time taken for each larva to pupate is monitored, noting the emergence time of pupae. After pupation, the quantity of pupae formed from each group is recorded. The percentage of pupation (PP) in B. cucurbitae is calculated using the formula: $PP(\%)=\frac{NPE}{TNL}\times 100$. Where, Percentage of pupation (PP), Number of pupae formed (NPF), and Total number of larvae (TNL).

The time and number of emerging B. cucurbitae: Initiate the monitoring process by recording the starting time and collecting initial data on the number of pupae present. Monitor the duration it takes for each pupa to develop into an adult fly, making notes on the emergence time of each fly. Following fly emergence, conduct a count of the number of flies and record both the quantity and time of appearance for each fly. The emergence percentage rate (EPR) of B. cucurbitae is calculated using the formula: $EPR(\%)=\frac{NEF}{TNP}\times 100$. In there, Emergence percentage rate (EPR), Number of emerging flies (NEF), Total number of pupae (TNP).

Oviposition in B. cucurbitae: The evaluation of oviposition deterrent effects was conducted through the fruit dipping method at varying concentrations of MAFS, following the protocol outlined by Cheseto et al. (2022) with minor modifications. MAFS was tested at 1, 5, 25, 125, and 625 ppm. Bitter melons served as the substrate for B. cucurbitae and were sliced into pieces (5-6 cm), with each piece featuring thin crosswise slits from one side, preserving the fruit’s intact skin (for easy egg retrieval). The substrates were immersed in the test concentrations of MAFS for 30 seconds and, after air-drying in the shade, placed into Petri dishes (5 cm in diameter) within cages sized 45 cm × 37.5 cm × 30 cm (one cage per fruit). Ten-day-old adult flies (both male and female) of B. cucurbitae were selected and introduced into the cages (one pair per cage) provided with the treated substrate for oviposition. Three replicates were performed for each treatment. Observations on the number of eggs laid per female per fruit were recorded. Bitter melons were replenished daily during the observation period. The ovulation inhibition rate (OIR) was calculated using the formula: $OIR(\%)=\frac{NEC-NET}{NEC}\times 100$. With, ovulation inhibition rate (OIR), Number of eggs laid in control (NEC), Number of eggs laid in treatment (NET).

Experimental studies in a greenhouse environment

Experimental design: Following the growth of 2-3 true leaves, each bitter melon plant is transplanted into a pot with a volume of 17.67 mL (diameter 30 cm, height 25 cm). The soil mixture consists of topsoil supplemented with a substrate such as sawdust and organic fertilizer such as cow dung or vermicompost, in a ratio of ¼ soil, ½ substrate, and ¼ organic fertilizer. The pH level is maintained between 6 and 7.1, creating optimal conditions for bitter melon plants. The first round of fertilization (after 3-5 true leaves) involves applying liquid organic leaf fertilizer, such as Panga TC fish dung (Hoang Lien Son Agriculture Co. Ltd), and the second round (when the plant is preparing to flower) utilizes inorganic fertilizer such as NPK Phu My 15-5-20 + TE (PetroVietnam Fertilizer and Chemicals Corporation). When the plant reaches a height of 25-30 cm, stakes or a trellis are inserted to support its climbing. Before infestation with B. cucurbitae flies, the plants undergo a 5- week cultivation period at a temperature of 25°C and relative humidity (RH) of 70-80% within the greenhouse. Each pot cultivating bitter melon plants is placed in a designated area within the greenhouses. B. cucurbitae flies are collected from the Bitter melon breeding environment in the laboratory, quantified, and monitored for survival parameters. The transmission of B. cucurbitae flies to the bitter melon cultivation area is carried out by opening cages containing flies (50 flies both male and female/cage/bitter melon plant). The experiment is designed with 5 treatment groups, utilizing MAFS at corresponding concentrations (1, 5, 25, 125, and 625 ppm), and Sofri protein 10 DD (1.2 liters/ha) as the standard fly-killing agent, with distilled water serving as the negative control. MAFS extract and Sofri protein solution are applied at intervals of 40 mL per plant on days 0, 5, and 10 after fly release. The plants are maintained for 8 weeks at a temperature of approximately 25°C and a relative humidity of 70-80% within the greenhouse.

Quantification of the number of eggs in bitter melon plants: Oviposition of B. cucurbitae commonly occurs on various parts of bitter melon plants such as leaves, flower stems, and fruits. The assessment of the number of eggs laid by B. cucurbitae is conducted by counting and recording the number of eggs deposited by female flies on bitter melon plants.

Larval and pupal quantification in bitter melon plants: Regularly monitor bitter melon plants to detect the presence of larvae and pupae effectively. Conduct thorough inspections of leaves, stems, and flowers to ascertain the location and extent of infestation. Employ a sampling tool to collect specimens from various parts of the bitter melon plant, with a focus on regions displaying signs of larvae or pupae, such as nibbled leaves, darkened markings, or areas where they commonly appear. Utilize a magnifying glass for a detailed examination of the samples to accurately determine the number of larvae and pupae. Systematically record the quantity of larvae and pupae on the bitter melon plant. Survivorship of B. cucurbitae from pupae to adult (SPA) is calculated according to the formula: $SPA(\%)=\frac{NDP}{TNP}\times 100$. With, SPA - Survivorship of B. Cucurbitae from pupae to adult, NDP - Number of dead pupae, TNP - Total number of pupae on bitter melon plants.

Level of damage to bitter melon fruit: The methodology is based on Deguine et al. (2021) description with adjustments. Bitter melon fruit samples are selected from regions exhibiting signs of corrosion, such as cracks and dark spots. Direct observation is performed to determine the extent of damage on each fruit, evaluating the degree of cracks, dark spots, and other signs on the surface of each fruit. A scoring system is implemented to assess the level of damage on each bitter melon, ranging from 0 to 5, where: 0 = Normal fruit, 1 = Slight damage, only a few small blemishes, with minimal impact on the overall condition of the fruit, 2 = Moderate damage, with some punctures or dark spots, yet the fruit still maintains its shape and overall quality, 3 = Increased punctures and dark spots, affecting a small area, but the fruit remains usable, 4 = Punctures and dark spots increase, covering a significant portion of the fruit, resulting in the loss of a substantial part of the fruit; however, some areas remain undamaged, 5 = Severe damage, with most of the entire surface of the fruit punctured and darkened, leading to the loss of utility and market value of the fruit.

Statistical analysis

Statistical analyses, including ANOVA (One-way analysis of variance) and Tukey’s test for mean comparison (p ≤ 0.05), were conducted for laboratory bioassays utilizing the Statgraphics Centurion XIX software. Additionally, data obtained from greenhouse experiments were subjected to analysis through the application of the student ‘t’ test using the Statgraphics Centurion XIX software.

Screening and quantitative analysis of phytochemicals in the ethanol extract of fruits and seeds of M. azedarach

The screening process for identifying phytochemical compounds in the ethanol extract from the fruits and seeds of M. azedarach (MAFS) revealed the presence of various phytochemicals, including alkaloids, tannins, saponins, polyphenols, steroids terpenoids, and flavonoids. Conversely, cardiac glycosides were not detected in the studied sample (Table 2). Furthermore, quantifying the levels of flavonoid, alkaloid, and tannin provided detailed insights into their concentrations within MAFS, with a total flavonoid content of 41.83 ± 3.38 mg QE/g, total tannin content of 69.97 ± 3.74 mg CE/g, and total polyphenol content of 70.77 ± 4.46 mg GAE/g (Table 3). This information serves as a foundation for investigating the potential activity of M. azedarach against the B. cucurbitae pest, which poses a threat to bitter melon plants.

Table 2 . Qualitative screening of phytochemicals in the ethanol extracts derived from fruits and seeds of M. azedarach.

Phytochemicals	Present in MAFS	Phytochemicals	Present in MAFS
Alkaloids	+	Cardiac glycosides	-
Tannins	+	Steroids	+
Saponins	+	Terpenoids	+
Polyphenols	+	Flavonoids	+

Note: Presence of phytochemicals in MAFS: (+) present and (-) absent.

Table 3 . Quantification of flavonoid, alkaloid, and tannin contents in the ethanol extract from fruits and seeds of M. azedarach.

Sample	Total flavonoid content (mg QE/g)	Total tannin content (mg CE/g)	Total polyphenol content (mg GAE/g)
MAFS	41.83 ± 3.38	69.97 ± 3.74	70.77 ± 4.46

Note: GAE: Gallic acid equivalents, QE: Quercetin equivalents, CE: Catechin equivalents..

Experimental investigations in the laboratory environment

The suppressive impact of MAFS on pupation time and pupal count in B. cucurbitae

The inhibitory effects of ethanol extract from the fruit and seeds of M. azedarach (MAFS) on the pupation period, the number of appearing pupae, and the pupation percentage (PP) of B. cucurbitae were investigated, and the results are presented in Table 4. The findings indicate a significant prolongation of the pupation period in groups treated with MAFS (p < 0.05). Notably, the MSFS625 group exhibited a pupation period equivalent to the Sofri protein-treated group, an organic insecticide (p > 0.05). Conversely, the water-treated group had a significantly shorter pupation period compared to the treatment groups (p < 0.05). Furthermore, there was a significant increase in the number of pupae in the water-treated group compared to the treatment groups (p < 0.05). In contrast, the number of appearing pupae and the pupation percentage in the MAFS125 and MAFS625 groups were approximately similar to the Sofri protein-treated group (p > 0.05). These results demonstrate the capability of MAFS to efficiently regulate the pupation period, reduce the number of pupae, and influence the pupation percentage of B. cucurbitae. Remarkably, the MSFS625 group even exhibited efficacy comparable to Sofri protein, a highly effective insect control method in agriculture. This underscores the potential applicability of MAFS in the management and control of harmful insects in agricultural environments.

Table 4 . The suppressive impact of ethanol extracts from the fruits and seeds of M. azedarach on pupation time and pupal count in B. cucurbitae.

Parameters	Water group	Sofri protein group	MAFS1 group	MAFS5 group	MAFS25 group	MAFS125 group	MAFS625 group
Pupation time (day)	11.33 ± 0.58a	30.00 ± 0.00f	17.67 ± 0.58b	20.33 ± 0.58c	24.33 ± 0.58d	29.00 ± 0.00e	30.00 ± 0.00f
No. of pupae	14.33 ± 0.58e	6.67 ± 0.58a	9.33 ± 0.58d	8.33 ± 0.58c	7.67 ± 0.58bc	7.33 ± 0.58ab	7.00 ± 0.00ab
PP %	95.56 ± 3.85e	44.44 ± 3.85a	62.22 ± 3.85d	55.56 ± 3.85c	51.11 ± 3.85bc	48.89 ± 3.85ab	46.67 ± 0.00ab

The values are expressed as Mean ± SD, where the letters (a, b, c, d, e, and f) indicate differences between treatments (p < 0.05)..

The inhibitory effect of MAFS on the time emergence and the number of individuals of adult B. cucurbitae

The outcomes presented in Table 5 delineate the inhibitory effects of an ethanol extract derived from the fruit and seeds of M. azedarach (MAFS) on the emergence time of adult individuals (germination time), the number of adult individuals, and the emergence proportion (EPR) of B. cucurbitae. The germination time of B. cucurbitae in the water group significantly decreased compared to the treated groups (p < 0.05). Conversely, following treatment with MAFS, the germination time significantly increased (p < 0.05). This increase exhibited a positive correlation with the concentration of MAFS (p < 0.05). The number and emergence proportion (EPR) of adult B. cucurbitae substantially decreased in the groups treated with MAFS (p < 0.05), and this outcome significantly differed from the water group (p < 0.05). Notably, in the MAFS125 and MAFS625 groups, the quantity and emergence proportion of adult B. cucurbitae were comparable to the group treated with sofri protein (p > 0.05). These results indicate that MAFS can inhibit germination time, reduce the quantity, and decrease the emergence proportion of adult B. cucurbitae, particularly in groups with high MAFS concentrations. The efficacy of MAFS, especially in the MAFS125 and MAFS625 groups, even rivals that of sofri protein, an effective insecticide. This enhances the applicability of MAFS in the management and control of insect pests in agricultural environments.

Table 5 . The inhibitory effect of ethanol extract from the fruit and seeds of M. azedarach on the emergence time and the number of individuals of adult B. cucurbitae.

Parameters	Water group	Sofri protein group	MAFS1 group	MAFS5 group	MAFS25 group	MAFS125 group	MAFS625 group
Germination time (day)	25.33 ± 0.58a	67.67 ± 0.58g	38.67 ± 0.58b	46.67 ± 0.58c	54.33 ± 0.58d	63.33 ± 0.58e	65.33 ± 0.58f
No. of flies	10.67 ± 0.58c	2.00 ± 0.00a	5.33 ± 0.58c	4.33 ± 0.58b	3.67 ± 0.58a	2.67 ± 0.58a	2.33 ± 0.58a
EPR (%)	74.44 ± 3.70d	30.16 ± 2.75a	57.41 ± 8.49c	52.31 ± 9.25c	48.21 ± 9.94bc	36.31 ± 7.22ab	33.33 ± 8.25a

The values are expressed as Mean ± SD, where the letters (a, b, c, d, e, f, and g) indicate differences between treatments (p < 0.05)..

The suppressive impact of MAFS on oviposition of B. cucurbitae

Fig. 1 illustrates the inhibitory effects of ethanol extract from the fruit and seeds of M. azedarach (MAFS) on the oviposition of B. cucurbitae. The oviposition inhibition ratio (OIR) progressively increases with the rising concentration of MAFS treatment (p < 0.05), highlighting the potential of MAFS to inhibit the oviposition process of the insect. In the negative control group (water group), the OIR is 0%, indicating no inhibitory effect on oviposition. However, at concentrations of 125 and 625 ppm MAFS, the OIR has significantly increased and is comparable to sofri protein (p > 0.05). This suggests that, at these concentrations, MAFS can inhibit the oviposition of B. cucurbitae similarly to sofri protein, an effective organic insecticide. These results imply that MAFS may influence the reproductive capacity of B. cucurbitae by inhibiting oviposition, providing a basis for further research and development of MAFS applications in the management and control of harmful insects in agricultural environments.

Figure 1. The suppressive impact of ethanol extracts from the fruits and seeds of M. azedarach on oviposition of B. cucurbitae. The results are presented as Mean ± SD, with letters (a, b, c, d, and e) denoting significant differences between treatments (p < 0.05)

Experimental studies in a greenhouse environment

The impact of MAFS on the egg number of B. cucurbitae in bitter melon plants

The impact of ethanol extract from the fruit and seeds of M. azedarach (MAFS) on the egg quantity of B. cucurbitae on bitter melon plants is illustrated in Fig. 2. The egg quantity laid by B. cucurbitae on bitter melon plants significantly increased in the control group (water group) (p < 0.05). Conversely, in groups treated with MAFS, the egg quantity of B. cucurbitae sharply decreased after treatment (p < 0.05), and the reduction was inversely proportional to the concentration of MAFS. Particularly at concentrations of 125 and 625 ppm of MAFS, the inhibitory effect on the egg-laying of B. cucurbitae was comparable to the standard insecticide, sofri protein (p > 0.05). This suggests that MAFS has the potential to effectively inhibit the egg-laying behavior of B. cucurbitae, making it a promising candidate for pest management and control in agricultural settings.

Figure 2. The impact of ethanol extracts from the fruits and seeds of M. azedarach on the egg numbers of B. cucurbitae in bitter melon plants. The results are presented as Mean ± SD, with letters (a, b, c, d, and e) denoting significant differences between treatments (p < 0.05)

The suppressive impact of MAFS on larval and pupal quantification in bitter melon plants

The inhibitory effects of ethanol extracts from the fruits and seeds of M. azedarach (MAFS) on the larval quantity, pupal quantity, and survival ability of B. cucurbitae from larval to adult stages (SPA) in bitter melon plants are illustrated in Table 6. The larval and pupal quantities on bitter melon plants significantly decreased in the treatments with MAFS (p < 0.05). This result significantly differed from the water-treated control (p < 0.05). The survival ability of B. cucurbitae from larval to adult stages (SPA) also showed similar trends to the changes in larval and pupal quantities. The rate of pupae developing into adults sharply declined after treatment with MAFS (p < 0.05). Meanwhile, SPA in the water group increased significantly (p < 0.05). Particularly, at the treatment concentrations of 125 and 625 ppm MAFS, the inhibitory results on larval quantity, pupal quantity, and survival ability from larval to adult stages of B. cucurbitae on bitter melon plants were equivalent to the inhibitory efficacy of sofri protein. The findings suggest that MAFS has a substantial impact on the developmental stages and survival of B. cucurbitae on bitter melon plants, highlighting its potential as an effective method for controlling this insect pest in agricultural settings.

Table 6 . The inhibitory effect of ethanol extracts from the fruits and seeds of M. azedarach on the oviposition events in B. cucurbitae.

Parameters	Water group	Sofri protein group	MAFS1 group	MAFS5 group	MAFS25 group	MAFS125 group	MAFS625 group
No. of larvae	254.33 ± 7.64e	95.67 ± 5.51a	98.33 ± 7.02d	136.33 ± 4.04c	119.67 ± 5.51b	98.33 ± 7.02a	97.33 ± 4.16a
No. of pupae	56.67 ± 3.06d	21.33 ± 2.52a	37.33 ± 4.04c	30.33 ± 3.06b	26.67 ± 2.52ab	21.67 ± 5.51a	20.33 ± 3.51a
SPA (%)	70.62 ± 3.80d	26.27 ± 2.40a	46.32 ± 2.16c	37.30 ± 1.97b	33.48 ± 4.38b	27.30 ± 2.31a	26.47 ± 2.55a

The values are expressed as Mean ± SD, where the letters (a, b, c, d, and e) indicate differences between treatments (p < 0.05)..

The inhibitory effect of MAFS on the level of damage to bitter melon fruit

Fig. 3 illustrates the inhibitory effects of ethanol extracts from the fruits and seeds of M. azedarach (MAFS) on the severity of damage to bitter melon fruits. The highest level of damage to bitter melon fruits was observed in the negative control group (water group) (p < 0.05), significantly different from the groups treated with MAFS and sofri protein (p < 0.05). The severity of damage markedly decreased in bitter melon plants treated with MAFS (p < 0.05), inversely proportional to the concentration of MAFS. Particularly, at concentrations of 125 and 625 ppm MAFS, the effectiveness showed comparable impact to sofri protein (p > 0.05). MAFS have a significant inhibitory effect on the severity of damage to bitter melon fruits. The effectiveness of MAFS in reducing damage was comparable to that of sofri protein, emphasizing its potential as an alternative in controlling damage to bitter melon crops.

Figure 3. The inhibitory effect of ethanol extracts from the fruits and seeds of M. azedarach on the level of damage to bitter melon fruit. The results are presented as Mean ± SD, with letters (a, b, c, and d) denoting significant differences between treatments (p < 0.05)

Extracts from the fruits of M. azedarach have undergone extensive prior research, yielding diverse effects on insect species. Specifically, Melia fruit extracts have been shown to reduce the population of leafminer Agromyzid liriomyza huidobrensis on crops such as kale and cucumber in both field and greenhouse conditions (Hammad et al. 2000). Similarly, extracts from unripe M. azedarach fruit have demonstrated a reduction in the larvae of leaf-mining fly Liriomyza sativae on bean plants (Hammad and McAuslane 2010). Additional studies have indicated the toxic and repellent effects of Melia extracts on both adult and larvae stages of Bemisia species across various plant types Hammad and McAuslane (2006). Moreover, according to Mckenna et al.’s report in 2013, the presence of bioactive plant compounds in Melia fruit extracts effectively combated the leaf-boring pest Phyllocnistis citrella under field conditions.

In this current study, ethanol extracts from the fruits and seeds of M. azedarach (MAFS) were analyzed, revealing the presence of compounds such as alkaloids, tannins, saponins, polyphenols, steroids terpenoids, and flavonoids. These components demonstrate potential as a selective biological pesticide against B. cucurbitae. Specifically, alkaloids exhibit toxic effects or influence the nervous system of insects, saponins create an anti-insect effect through foam formation or impact on cell membranes, and steroid terpenoids are harmful to the endocrine system of insects Rattan (2010). Flavonoids, tannins, and polyphenols, major components in MAFS, are known for their antioxidant properties, reducing damage from free radicals, and antibacterial effects. Polyphenols contribute to antioxidant activity and cellular damage to insect structures, tannins create hindrance and affect digestive functions, while flavonoids inhibit biological processes in insect bodies, disrupting energy metabolism or reproductive processes Muhammed et al. (2022). These results highlight the potential of MAFS to provide antioxidant defenses and inhibit the development of B. cucurbitae, contributing to the protection of bitter melon plants. The combination of compounds in the extract from M. azedarach creates a multi-faceted effect, enhancing the ability to combat B. cucurbitae and minimize damage to bitter melon plants.

The pupation period, representing the time it takes for the larvae of an insect species to transition from the larval stage to the pupal stage, is a critical phase in the insect’s developmental cycle. During this stage, the insect transforms and matures into a pupa before emerging as an adult. In the case of B. cucurbitae, the pupation period measures the duration from the last larval stage of this species to when it becomes an adult. Prolonged pupation periods may impact the development and reproduction of the insect, potentially reducing its harmful effects on crops Puri et al. (2021). Pupation is an intermediate stage between the larval and adult stages in the developmental cycle of an insect. The number of pupae of B. cucurbitae reflects the total number of pupae in the population or the study sample, influencing the reproductive strength and synthesis capability of the insect. It serves as a crucial index when assessing the potential harm caused by the insect to crops or the agricultural environment. Typically, a reduction in the number of pupae is considered positive, as it may decrease the insect’s reproductive capacity and, consequently, minimize its harmful impact on crops or the surrounding environment Samiksha et al. (2019). In this study, the pupation period significantly increased in groups treated with MAFS, especially in the MSFS625 group, indicating that MAFS can slow down and inhibit the pupation process of B. cucurbitae under laboratory conditions. The number of appearing pupae significantly decreased in groups treated with MAFS, particularly in the MAFS125 and MAFS625 groups. Thus, MAFS has the potential to reduce the number of pupae of B. cucurbitae, diminishing the impact of this harmful insect under experimental conditions. As the pupation period increases and the number of pupae decreases, especially in groups treated with MAFS, the ability to inhibit the transition from larvae to pupae and reduce the reproductive capacity of B. cucurbitae is enhanced. This increases the efficiency of insect control in the experimental environment. Survey results suggest that MAFS could be used as an insecticidal agent in pest management, particularly against B. cucurbitae. Reducing the pupation period and the number of pupae can minimize the harmful effects of the insect on crops. The study results provide a basis to further explore the practical application of MAFS in insect management under real agricultural conditions.

The emergence time of adult B. cucurbitae (germination time) represents the period during which the larvae of this insect species transition from the larval stage to the adult stage. This is a crucial stage in the insect’s developmental cycle, where they undergo metamorphosis and mature into adult flies. The adult count of B. cucurbitae refers to the total number of mature individuals of this fly species. The Emergence rate (EPR) of adult B. cucurbitae is the ratio between the number of emerging mature individuals and the total number of mature individuals in a population or study sample (Subedi et al. 2021). Germination time can be influenced by various factors, including environmental conditions, food availability, and the effects of chemicals such as the ethanol extract from M. azedarach seeds and fruits (MAFS). The study results reveal a significant increase in the emergence time of adult B. cucurbitae after being treated with ethanol extract from M. azedarach seeds and fruits (MAFS), particularly in the group with a high MAFS concentration. This indicates the ability of MAFS to slow down the development and emergence of the insect. The current research has also quantified the impact of MAFS on the population scale of B. cucurbitae. The reduction in the number of emerging adults may signify the effectiveness of MAFS in controlling the development and reproduction of this insect, a positive sign of insect control. Additionally, the emergence proportion (EPR) is used to measure the occurrence of adult B. cucurbitae in groups treated with MAFS compared to the water-treated group. The decrease in EPR suggests a significant inhibitory effect of MAFS on the emergence of adult flies, indicating a positive sign in insect control against B. cucurbitae. Thus, these results indicate that MAFS has a significant negative impact on germination time, the number of emerging adults, and the emergence rate of B. cucurbitae under laboratory conditions, highlighting the potential application of MAFS in the management and control of harmful insects in agricultural environments.

The oviposition (egg-laying) capability of B. cucurbitae is not only a crucial factor in the reproduction of this insect species but also plays a pivotal role in the scale and stability of the insect population. The ability to lay eggs can regulate the number of eggs each fly produces, influencing the population scale, especially when facing constraints such as insecticides. A reduction in oviposition capability may lead to an increase in the number of eggs laid, enhancing reproductive potential and consequently amplifying the population scale. Conversely, if oviposition is inhibited, it can contribute to population control, preventing a sudden surge in insect numbers Chou et al. (2012). The examination of the oviposition inhibition rate (OIR) in this study holds significance in understanding the effectiveness of ethanol extracts from M. azedarach fruit and seeds (MAFS) on the reproduction of B. cucurbitae. The OIR reflects the degree of MAFS inhibition on the egg-laying process of the insect. As the OIR increases, it indicates that MAFS extract has the potential to reduce the egg-laying of B. cucurbitae, thus influencing the insect’s reproductive process by inhibiting oviposition. Diminishing egg-laying could lead to a decrease in the number of newly hatched insects, impacting the population scale. The results obtained from the OIR survey support the hypothesis that MAFS has the potential to reduce the reproduction of B. cucurbitae, providing essential insights into the ability to control harmful insects in agricultural environments.

B. cucurbitae exhibits a preference for specific oviposition sites on bitter gourd plants, such as leaves, young branches, flowers, and fruits, as these locations provide optimal conditions for the development and protection of larvae. On bitter gourd fruits, B. cucurbitae commonly deposits eggs on the surface, particularly in wrinkled or creased areas. Eggs may also be laid underneath the leaves, especially near the leaf stem or in leaf crevices. The selection of specific sites on bitter gourd plants ensures the safety and success of larval development, contributing to the effective reproduction of the fly population Dhillon et al. (2005). The assessment of the number of eggs laid by B. cucurbitae on bitter gourd plants is crucial for evaluating the insect’s impact on crops and provides fundamental information for insect management strategies. The closely related number of eggs to the damage caused by B. cucurbitae implies that an increase in egg count predicts an escalation in crop losses. When implementing insect control measures, monitoring the egg count on plants aids in assessing the effectiveness of these measures. A reduction in egg count signifies the efficacy of control measures Kubar et al. (2021). In the current study, the ethanol extract from seeds and fruits of M. azedarach (MAFS) significantly inhibits the number of eggs laid by B. cucurbitae on bitter gourd plants. This reduction in egg count translates to diminished reproductive capabilities and decreased damage to crops. MAFS, as a natural extract and biological insecticide, not only proves effective in reducing crop damage but also contributes to minimizing the use of harmful chemical agents. The notable decrease in egg count at higher MAFS concentrations (125 and 625 ppm) underscores the potential efficacy of MAFS in insect control, providing a basis for its application as an effective method in managing B. cucurbitae infestations.

The investigation into the number of larvae, pupae, and survival capability of B. cucurbitae from the larval to the adult stage (SPA) aims to provide detailed and comprehensive information on the impact of insecticides throughout the insect’s life cycle and population dynamics. The SPA survey assists in identifying specific factors influencing the developmental process from the larval to the adult stage of B. cucurbitae. Monitoring the quantity of larvae and pupae provides insights into the mechanism of action of insecticides, slowing or inhibiting the insect’s development. The larval and pupal stages are typically sensitive and crucial phases in the insect’s life cycle. Effective control during these stages can lead to a reduction in the number of mature individuals, minimizing the level of damage to crops. Evaluating the survival capability of B. cucurbitae during its development from larvae to adulthood helps determine the inhibitory effect of the insecticide on their reproduction Dhillon et al. (2005). The effectiveness of the ethanol extract from seeds and fruits of M. azedarach (MAFS) in inhibiting the larval quantity, pupal quantity, and survival capability of Bactrocera cucurbitae from the larval to the adult stage (SPA) is a crucial aspect of this research, providing valuable insights into insect control in agricultural environments. MAFS has demonstrated its inhibitory potential on the development of B. cucurbitae larvae and pupae on bitter melon plants. The significant reduction in larval and pupal quantities results from the impact of compounds within MAFS, diminishing reproductive capabilities, and the emergence of new insects. MAFS also influences the survival and development of B. cucurbitae from the larval to the adult stage. The substantial decrease in the rate of pupae maturing into adults after MAFS treatment raises questions about their ability to progress through the pupal stage and develop into adults. Particularly, at concentrations of 125 and 625 ppm MAFS, the inhibitory effects on larval and pupal quantities and the survival capability of B. cucurbitae are comparable to the efficacy of sofri protein. This outcome enhances the practical value of MAFS, especially considering its capacity to deliver comparable performance without the need for chemical insecticides. These findings support the proposition that MAFS holds potential for use in the management and control of B. cucurbitae insects on bitter melon plants.

The extent of damage on bitter melon fruits is a crucial metric for evaluating the effectiveness of insect control methods and serves as a basis for deciding on the implementation of crop management and protection measures against harmful insect infestations. Investigating the extent of damage aids in assessing the efficacy of insect control methods, providing information on the potential to reduce insect infestations, and determining the level of crop protection. The extent of damage can offer insights into the biological characteristics of insects, including their feeding habits, development, and interactions with crops, and identify key periods for implementing control measures Saeed et al. (2022). The ethanol extract from seeds and fruits of M. azedarach (MAFS) demonstrates significant efficacy in controlling the extent of damage to bitter melon fruits. MAFS has a notable impact in reducing the extent of damage to bitter melon fruits, which linked to its inhibitory effects on the growth and development of insects, diminishing their harmful impact on crops. The current study compares the effectiveness of MAFS with sofri protein, a standard organic insecticide. The equivalence between MAFS and sofri protein at specific concentrations (125 and 625 ppm) illustrates the potential of MAFS in reducing the extent of damage to bitter melon fruits. This result supports the hypothesis that MAFS holds promise as an effective method in insect management strategies. The efficacy of MAFS not only aids in minimizing crop damage but also presents an opportunity to reduce reliance on harmful chemical agents.

A comprehensive understanding of how MAFS influences the developmental, reproductive, and survival cycles of insects provide crucial information for refining management strategies. The utilization of the ethanol extract from seeds and fruits of M. azedarach emerges as an environmentally friendly solution, reducing the dependence on harmful chemical substances in agriculture and safeguarding ecological balance.

The current study reveals an inhibitory effect of the ethanol extract from seeds and fruits of M. azedarach (MAFS) on B. cucurbitae. In the laboratory environment, MAFS efficiently prolonged the pupation period, and germination time, and reduced the number of pupae and adult individuals. Moreover, MAFS demonstrated the ability to impede the egg-laying process of B. cucurbitae. In the greenhouse experiment, MAFS significantly decreased the number of eggs laid by B. cucurbitae on bitter melon plants, leading to a substantial reduction in the number of larvae and pupae. Additionally, MAFS markedly minimized the severity of damage caused by B. cucurbitae to bitter melon fruits. At concentrations of 125 and 625 ppm, MAFS exhibited efficacy equivalent to sofri protein in both experimental models. These findings collectively support the notion that MAFS holds promise as an effective and environmentally friendly solution for managing B. cucurbitae infestations.