Repellent and Larvicidal Effects of Some Indigenous Plants in Abia State, Nigeria Against Female Anopheles gambiae

 Corresponding author: Mary Oluchi Iwuagwu Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Repellent and Larvicidal Effects of Some Indigenous Plants in Abia State, Nigeria Against Female Anopheles gambiae Boniface Okon Ekpo 1, Mary Oluchi Iwuagwu 2, *, Nneka Constance Emmanuel 2 and Jude Chibuzor Igwe 3 1 Department of Medical Biochemistry, College of Medicine and Health Sciences, Abia State University, Uturu. 2 Department of Plant Science and Biotechnology, Faculty of Biological Sciences, Abia State University, Uturu. 3 Department of Pure and Industrial Chemistry, Faculty of Physical Sciences, Abia State University, Uturu. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 Publication history: Received on 15 September 2025; revised on 22 October 2025; accepted on 25 October 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.1.0920 Abstract This study investigated the repellent and larvicidal activities of ethanol leaf extracts from three plants, Azadirachta indica, Carica papaya and Annona muricata, against female Anopheles gambiae mosquitoes with the aim of identifying effective plant-based alternatives for malaria vector control. Adult mosquitoes were collected from Garki Market in Okigwe, Imo State, Nigeria using an aspirator and transported to the laboratory for identification and rearing. Female An. gambiae mosquitoes were isolated and maintained under standard insectary conditions for reproduction. Third instar larvae were used for larvicidal assays, while fourth instar larvae were reared to adulthood for repellency tests. Repellency was assessed using the WHO Arm-in-Cage protocol. The ethanol leaf extracts of the three plant species were tested at concentrations of 0%, 2.5%, 5%, 10%, 15%, and 20%. All three plant extracts demonstrated significant, dosedependent repellent and larvicidal effects. At 20% concentration, A. muricata showed the highest repellency (96.97%), followed by A. indica (95.15%) and C. papaya (89.09%). A strong negative correlation was observed between extract concentration and mosquito landings (r = -0.978, -0.949, and -0.981, respectively). Complete larval mortality (100%) was achieved at concentrations above 10% for A. indica and C. papaya, and above 5% for A. muricata. Phytochemical analysis revealed a high presence of flavonoids in all three species, suggesting a potential link to their bioactivity. These findings highlight the potential of these plant extracts as environmentally friendly and accessible alternatives for malaria vector control, particularly in resource-limited settings. Keywords: Anopheles; Repellency; Larvicidal; Malaria-Vector Control; Plant Products 1 Introduction Malaria is an acute fever illness which affects millions of people and claims millions of lives globally. It is a serious and important public health threat and accounts for 17% of the global burden of all communicable diseases put together [1]. The increasing incidence of mortality and morbidity associated with malaria significantly weakens the economies and impedes the development of rural and urban communities in endemic communities especially those in low income countries in sub-Saharan Africa [2]. Vulnerable groups, including newborns, young children, pregnant women, and nonimmune travelers, accounted for 90% of all malaria-related deaths in 2020 [1,3,4]. In Nigeria, over 70% of the population is at risk of infection annually, making it one of the countries with the highest disease burden [5,6]. The etiology of malaria is attributed to the infection caused by intracellular protozoan parasites of the genus Plasmodium, transmitted through the bite of infected female Anopheles mosquitoes [7,8]. The species most commonly infecting humans include P. falciparum, P. vivax, P. ovale, and P. knowlesi, with P. falciparum being the most virulent and deadly [9-11]. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 529 Mosquito bionomics and targeted vector control/eradication are essential in reducing mosquito populations and consequently malaria transmission and outbreaks [12]. Conventionally, the focus of mosquito eradication programmes and control interventions relies heavily on chemical insecticide use. Interventions such as insecticide-treated mosquito nets, indoor residual spraying, and larval source management under the Roll Back Malaria Initiative have yielded measurable success [4,13]. However, the repeated use of chemical insecticides has led to several challenges, including environmental degradation, harm to non-target organisms, and widespread development of insecticide resistance in mosquito populations [14-16]. These issues have contributed to the resurgence of mosquito populations and reemergence of malaria in certain regions [17,18]. Additionally, the growing resistance of Plasmodium parasites to existing antimalarial drugs has further intensified the need for alternative, sustainable, and ecologically sound vector control methods [19]. In response to these challenges, biological control particularly the use of botanicals, has gained attention as a viable and environmentally friendly alternative. Many plant-based products are known to possess insecticidal and repellent properties that are safe, biodegradable, and less likely to induce resistance in mosquito vectors [20,21]. Some plant products have been reported to possess activities which are specifically mosquitocidal making them promising candidates for use in integrated vector management systems [20,22]. Consequently, plant-derived biopesticides are rapidly gaining ground and carving a niche in the integrated strategy aimed at eradicating mosquitoes and combating malaria [23]. Investigating plant-based alternatives is vital not only for broadening the range of vector control strategies but also for promoting locally available, cost-effective solutions that align with sustainable public health practices. Many studies on biolarvicides focus on Culex and Aedes mosquitoes with fewer on Anopheles particularly on An. gambiae. This study was therefore conducted to evaluate the repellent and larvicidal effects of three indigenous plants viz Azadirachta indica, Carica papaya, and Annona muricata, against female Anopheles gambiae mosquitoes in Abia State, Nigeria. These plants were selected based on their traditional use in local communities and preliminary reports of their insecticidal potential. 2 Materials and Methods 2.1 Study Area The study area of this research was Abia State, one of the 36 states in Nigeria. It is situated in the South East geopolitical zone of the country. The main tribe inhibiting Abia State, and indeed the entire South East Zone, is the Igbo tribe. This is a rain forest zone with a wet equatorial climate, and lies within 5º25'N 7º30'E and 7.500º/5.417, with average annual rainfall of 79 inches and average annual temperature of 20℃ - 31℃. It is one of the malaria endemic regions of the country. 2.2 Plant collection and authentication Fresh and healthy leaf samples of Azadirachta indica (Neem), Carica papaya (paw-paw), and Annona muricata (soursop), were collected from Uturu, Abia State, Nigeria. These plants were identified and authenticated at the Department of Plant Science and Biotechnology. 2.3 Sample preparation and extraction The fresh leaves samples were washed with clean water to remove dust and debris and air-dried under shade at room temperature (25 - 30℃) until they were completely dry. The dried leaves were ground into fine powder using a blender and stored in properly labeled, airtight containers until extraction. Extraction was done using the soxhlet extraction procedure with ethanol (95%) as the solvent. The soxhlet apparatus was washed using 50ml of 70% ethanol and the edge of the soxhlet timble was lubricated using blue seal vaseline to avoid friction. Fifty (50) grammes of the powdered leaf samples were weighed and wrapped securely in filter paper. The filter paper was inserted into the main chamber of the Soxhlet extractor. A 500 ml round-bottom flask was filled with 95% ethanol. The Soxhlet apparatus was assembled: round-bottom flask (with ethanol) at the bottom, Soxhlet chamber in the middle, and a condenser on top. The setup was heated on a heating mantle at a temperature of 70°C. The solvent was allowed to boil, evaporate, and condense, so it continuously cycles through the leaf material, extracting phytochemicals each time. The extraction lasted for 7 hours when solvent in the siphon tube became colorless (an indication of complete extraction). After extraction, the setup was allowed to cool, then carefully dismantled. The extract was concentrated using firstly a rotary evaporator to achieve 70% extract concentrate, and secondly a water bath to achieve 100% concentrate. This procedure was done for each plant leaves sample. The crude extract was stored in amber bottles at 4°C until further use in repellency tests and phytochemicals analysis. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 530 2.4 Test organism Adult mosquitoes were collected with the help of an aspirator from the Garki Cattle market in Okigwe town - a 30 minute driving distance from the main campus of Abia State University, Uturu, Abia State, Nigeria. They were then transferred into one-foot mosquito cage and taken to the laboratory of Animal and Environmental Biology where their identity was ascertained using the prescribed protocol of WHO [6]. The ones identified as adult female Anopheles gambiae were used for the study. 2.5 Mosquito Rearing The female Anopheles gambiae mosquitoes were reared at the Insectary of the Department of Animal and Environmental Biology laboratory, Abia State University, Uturu, Nigeria under standard conditions (27 ± 2°C, 70–80% RH, 12:12 h light:dark cycle). The mosquitoes were kept in a non-insecticide netted cage and fed with the blood of live hen to aid in their reproduction. The eggs were extracted using ovi-trap (an amber coloured cup with a white ribbon) folded such that it laps on the walls of the cup and half-filled with water. The ovi-trap was recovered after three (3) days. The ribbon was removed from the cup and soaked inside water for three days. The larvae, which developed after three days were then fed with fish-feed for four days. The larvae were allowed to develop into 1st, 2nd, 3rd and 4th instar larva. The 4th instar larvae were separated from the others and placed in a netted cage (30×30×30 cm) before emerging as adult female Anopheles gambiae mosquitoes. 2.6 Bioassays Different concentrations (0, 2.5, 5, 10, 15 and 20 %) of each of the crude extract were prepared. This was done by first preparing 20% stock solution followed by serial dilutions. These concentrations were used in the following bio-assays: 2.6.1 Repellency bioassay The method adopted in this bioassay was the WHO protocol of Arm-in-Cage Test. A total of 50 adult A. gambiae were kept in each non-insecticide netted cage. The adult mosquitoes were deprived of blood meal 24 h prior to their use and were instead maintained on a 10% sucrose solution. This was to make the mosquitoes hungry and eager to bite. Eighteen (18) human volunteers were used for this study. The human volunteer’s forearm (from wrist to elbow) were washed with scentless soap, rinsed with clean water and allowed to dry. The test was simply done by applying 1 ml of the extract concentration evenly on the arm up to elbow and placing the hand inside the cage. The test proper started with control and thereafter the different concentrations. For the control, 1 ml of ethanol was applied evenly on the left forearm, allowed to dry for 1 minute and then placed into the test cage containing 50 adult female An. gambiae mosquitoes. The number of mosquitoes that landed/bite was recorded every minute for a 3-minute exposure. Each extract per sample at 2.5, 5, 10, 15 and 20 % was then applied separately, starting with the lowest concentration. The number of mosquitoes that landed/bite was also recorded every minute for a 3-minute exposure. These tests were repeated thrice. Percentage repellency was calculated using the formula reported by Yoon et al. [24]: % Repellency = 𝒂−𝒃 𝒂×100 1 Where a = Number of mosquitoes in the control, b = Number of mosquitoes in the treated 2.6.2 Larvicidal Bioassy 3rd instar larva stage of the mosquito was used for this assay. A total of 20 larvae were placed in each bioassay cup using a dropping pippette. Each extract concentration was added separately into the cup, covered with an untreated mosquito net and properly labeled. Larvae death was monitored and recorded at every 1 hour for 3 hours by probing larva with a needle. 2.7 Phytochemical analysis The extracts were screened for the presence of the following secondary metabolites: alkaloids, flavonoids, phenols, saponins, steroids, tannins and glycosides using the methods of Harbone [25] and Sofowora [26]. 2.8 Statistical Analysis Data generated was statistically analyzed using SPSS 20. Significant means were partitioned using Post Hoc test. Correlation analysis was also performed. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 531 3 Results Table 1 shows the repellency activities of the different concentrations of Azadirachta indica leaf extracts against An. gambiae. As seen in the Table, the number of mosquitoes that landed on the human volunteer’s hand was significantly lowest at the highest concentration of the extract (20%) with the percentage repellency of 95.15. This was followed by the 15% and 10% concentrations, which had statistically similar values that were significantly different from the lower concentrations of 5% and 2.5%, and the control (0%), with repellency values of 90.91 and 86.97 respectively. Similarly, the numbers of mosquitoes that landed on the human volunteer’s hand at extract concentrations of 2.5% and 5.0% (with repellencies of 74.85 and 71.82, respectively) were not different from each other significantly, but were significantly different from the control. Correlation analysis revealed a highly significant negative relationship between concentration and repellency activity of A indica extract with P-value of 0.00006, correlation coefficient, r, of -0.978, and coefficient of determination, R2 of 0.956. Table 1 Repellency activities of different concentrations of Azadirachta indica (leaf) extract against An. gambiae. Extract Concentration (%) Mean landed mosquitoes±SD % Repellency 0 33±3.13a 0.00 2.5 9.3±1.73b 71.82 5 8.3±1.69b 74.85 10 4.3±0.34c 86.97 15 3.0±1.33c 90.91 20 1.6±0.07d 95.15 Means±SD followed by different superscript letter down the column are significantly different at P<0.05. Table 2 shows the repellency activities of different concentration of the extract from the leaf of Carica papaya against An. gambiae. The result shows that the numbers of mosquitoes that landed on the human volunteers hand at higher concentrations of the extract of 15% and 20% (with repellencies of 89.09 and 88.78 respectively) were not significantly different from each other, but were significantly lower when compared to other concentrations. In the same vein, the numbers of mosquitoes that landed on the volunteer’s hand at extract concentrations of 10% and 5% were statistically similar with repellencies of 77.87% and 80.91% respectively, but significantly lower than the 5% concentration (repellency of 67.88%) as well as the control. Correlation analysis showed a significant but negative moderate relationship between concentration and the repellency activities of pawpaw extract with P-value of 0.00004, correlation coefficient, r=-0.949 and coefficient of determination, R2 of 0.901. Table 2 Repellency activities of different concentrations of the leaf extract of Carica papaya against An.gambiae. Extract Concentration (%) Mean landed mosquitoes±SD % Repellency 0 33±3.13a 0.00 2.5 10.6±3.21b 67.88 5 6.3±2.33c 80.91 10 7.3±1.34c 77.87 15 3.7±1.69d 88.78 20 3.6±0.07d 89.09 Means±SD followed by different superscript letter down the column are significantly different at P<0.05. Repellency activities of different concentrations of Annona muricata leaf extract against An. gambiae are as presented in Table 3. Significantly lowest number of mosquitoes landed on the hand of the human volunteer at the highest (20%) extract concentration with the repellency percentage of 96.97, compared to the lower concentrations. This was followed by the 15% and 10% concentrations (percentage repellencies of 87.88 and 86.97, respectively) which were statistically similar but significantly different from the repellencies of the lower concentrations of 5% (77.88%) and 2.5% (72.73%). The human volunteer’s hand that had none of the extract concentrations, had the highest number of World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 532 mosquitoes that landed (33±3.13). Correlation analysis revealed a highly significant negative relationship between concentration and repellency with P-value of 0.00000, correlation coefficient, r, of – 0.981, and coefficient of determination, R2 of 0.962. Table 3 Repellency activity of different concentrations of Annona muricata extract against An. gambiae Extract Concentration Mean landed mosquitoes±SD % Repellency 0 33±3.13a 0.00 2.5 9.0±0.67b 72.73 5 7.3±2.43c 77.88 10 4.3±0.34d 86.97 15 4.0±1.33d 87.88 20 1.0±0.00e 96.97 Means±SD followed by different superscript letter down the column are significantly different at P<0.05. The effects of the different concentrations of the leaf extract of A. indica against the larvae of An. gambiae are as recorded in Table 4. As shown in the Table, increase in concentration of the extract from 0% to 10% led to significant (P≤0.05) increase in larval death. Above the 10% extract concentration, death of all larvae was observed. There was increase in larval death as duration of exposure increased, but the recorded increase was not significant. Table 4 Larvicidal effect of the different concentrations of A. indica extract against An. gambiae at varing exposure time Concentration (%) Time of exposure (hr) 1 2 3 0 0 0 0 2.5 5±2.00d 5±1.31c 7±1.99c 5 12±1.60c 14.3±2.53b 15±2.22b 10 17±1.00b 18±1.20a 18±1.08a 15 20±0.00a 20±0.00a 20±0.00a 20 20±0.00a 20±0.00a 20±0.00a Means±SD followed by different superscript letter across the rows are significantly different at P<0.05. The larvicidal effect of the different concentrations of Carica papaya against An. gambiae at varying exposure time is as shown in Table 5. Increase in extract concentration from 0% to 5% led to significant increase in larval death. Above the 10% concentration, death of all larvae was recorded. There was no significant change in larval death throughout the concentrations as the exposure duration increased from 1.0 to 3.0 hours. The larvicidal effect of different concentrations of the leaf extract of A. muricata against An. gambiae at different exposure time is as recorded in Table 6. Increase in concentration of the leaf extract of A. muricata from 0% to 5% results in significant increase in larval death. Above the 5% concentration, no significant increase in larval death was recorded. At the concentrations of 10% to 20%, there was 100% of larval death. An erratic response of the larvae at the concentration of 2.5% in the exposure period of 1 to 3 hours was recorded. At 2.5 and 5% concentration and exposure duration of 1 to 2 hours, a slight insignificant decrease in larval death was observed. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 533 Table 5 Effect of different concentrations of C. papaya leaf extract on the larvae of An.gambiae at different exposure time. Concentration (%) Time of exposure (hr) 1 2 3 0 0 0 0 2.5 5±±1.20b 6.0±1.00c 6.0±1.50c 5 14.0±1.05b 14.0±1.50b 14.0±1.50b 10 18.0±0.50a 18.0±0.02a 18.0±0.05a 15 20±0.00a 20±0.00a 20±0.00a 20 20±0.00a 20±0.00a 20±0.00a Means±SD followed by different superscript letter down the column are significantly different at P<0.05. Table 6 Larvicidal effect of different concentrations of A. muricata extract against An. Gambiae at varing exposure time Concentration (%) Time of exposure (hr) 1 2 3 0 0 0 0 2.5 7.0±2.89c 6.0±2.61c 8.0±2.03c 5 16.0±3.06b 15.0±2.33b 15.0±3.21b 10 20±0.00a 20±0.00a 20±0.00a 15 20±0.00a 20±0.00a 20±0.00a 20 20±0.00a 20±0.00a 20±0.00a Means±SD followed by different superscript letter down the column are significantly different at P<0.05. Result on the qualitative phytochemical contents of the ethanol leaf extracts of A. indica, C. papaya and A. muricata is as presented in Table 7. The leaf extracts of the three plants contained alkaloids, phenols, tannins, saponins, glycosides and flavonoids. However, steroids were absent in A. muriata leaf extract but present in the other two plant leaf extracts. Flavonoids were very highly present in all the three plants. Phenols were very highly present in both A indica and A. muricata, while saponins and steroids were very highly present in C. papaya. Glycosides were moderately present in all three plant leaf extracts. Additionally, tannins and saponins were also moderately present in A. muricata. Table 7 Phytochemical screening of the leaf extracts of Azadirachta indica, Carica papaya and Annona muricata Phytochemical indica C. papaya A. muricata Alkaloids ++ ++ ++ Phenol +++ ++ +++ Tannins ++ ++ + Saponins ++ +++ + Glycosides + + + Steriods + +++ - Flavonoids +++ +++ +++ +++ = very highly present; ++ = highly present; + = moderately present; - absent. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 534 4 Discussion The results of this study demonstrate the repellent and larvicidal properties of ethanol leaf extracts of Azadirachta indica, Carica papaya, and Annona muricata against female Anopheles gambiae, a major vector of malaria in sub-Saharan Africa. It also showed the phytochemical groups present in the three plant extracts that may have contributed to their repellent and larvicidal efficacy. The three plant extracts exhibited dose-dependent repellency activity against female Anopheles gambiae mosquitoes, although with varying degrees of effectiveness and statistical patterns. The observed increase in repellency with higher extract concentrations corresponds with the dose-dependent responses often reported in plant-based repellents [27,28]. Azadirachta indica extract showed a clear dose-dependent repellency effect: as the concentration increases, the number of mosquito landings decreases, and percentage repellency increases. The highest concentration of 20 % achieved the strongest repellency while the least was the lowest concentration of 2.5%. The percentage repellency was in this order: 95.15>90.1>86.97>74.85>71.82 for 20, 15, 10, 5 and 2.5% A. indica extract concentration, respectively. This result agrees with the findings by Aremu et al. [29] and Aidoo et al. [30], who reported increasing repellency of neem seed oil with concentration, although the maximum repellency in their studies ranged from 85-90% at 10-20% formulations. Jahan and Arju [31] reported that silk fabrics dyed with higher concentrations of A. indica (neem) leaf extract exhibited a mosquito repellent activity up to 75%, with lower concentrations showing proportionally lower effectiveness. Aizoun et al. a and b [32,33] reported that neem extracts provided better protection at higher concentrations against both Culex and Aedes species, often outperforming comparable natural repellents. Neem contains potent bioactive compounds such as azadirachtin and nimbin, which are known for their insect-repellent and antifeedant properties [34]. The correlation metrics (r = －0.978, R² = 0.956, p = 0.00006) confirm a very strong negative linear relationship between concentration and mosquito landings, i.e. a strong positive effect of concentration on repellency. These results support the potential of neem as a plant-based alternative to synthetic repellents like DEET. The evaluation of Carica papaya (pawpaw) extracts as a botanical repellent is an area of growing scientific interest, especially as plant-based alternatives to synthetic chemicals are sought for safe and effective mosquito control. Leaf extract of Carica papaya also showed increased repellency with concentration, but the pattern was irregular and the relationship weaker. At 15% and 20% C. papaya extract concentration, repellency was relatively high (88.78% and 89.09%, respectively) while intermediate concentrations (5% and 10%) showed only modest repellency of 80.91% and 77.87%, respectively. The repellency at 5% was slightly higher than at 10%, suggesting possible variability in effect or experimental conditions. The result showed that plateau begins at 15-20% extract concentration. These findings corroborate with other studies: Aizoun et al. c [35] reported that C. papaya leaf extract has repellent effect on Culex mosquitoes at higher concentration with lower concentration less effective.; Cahyati et al. [36] also found dose-response repellency effect of Carica papaya against Aedes aegypti mosquito. They reported that papaya leaf extract lotion is effective in repelling Aedes aegypti mosquitoes at a concentration of 30% with repellent percentage of greater than 90%. The negative moderate correlation (r = -0.949, R2 = 0.901, p = 0.00004) observed between C. papaya extract concentration and number of mosquito landings confirms a strong inverse dose-response: as the concentration of C. papaya extracts increases, mosquito landing rates decrease substantially, an indication of increased repellency. This finding is consistent with previous studies on plant-derived insecticides and repellents, which show that higher concentrations tend to be more effective, although the effect may plateau at very high doses due to solubility or saturation effects [28]. A similar clear dose-response trend to A. indica was observed in A. muricata, with the highest extract concentration of 20% producing nearly complete protection (96.97% repellency), followed by 15% (87.88%) and 10% (86.97%). This result is in agreement with observations by Alhamda et al. [37], who reported best effective Aedes aegypti mosquito repellent activity of A. muricata seed extract at the highest concentration of 100%. It also corroborates with the findings made by Ubulom et al. [38] who observed decrease both in number of female An. gambiae landing and biting on Annona muricata seed oil treated arms of volunteers compared to the untreated arm. They concluded that A. muricata has a significant direct repellent effect against An. gambiae. Yanti and Sari [39] in their study reported a linear dosedependant repellency effect of soursop against Aedes aegypti mosquitoes with protection percentage range of 75.2% - 97.7% for extract concentration of 20% - 100%. More so, statistical analysis from their experiment showed a highly significant negative correlation (high r and R2 values) between extract concentration and the number of mosquito landings, which is in conformity with the result of the present study. They concluded that soursop seed extract could serve as a natural alternative to chemical repellents, reducing the reliance on DEET-containing products. In another study, Sazali [40] found that Annona muricata leaf extract exhibited significant repellency against Aedes aegypti World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 535 mosquitoes at a 60% concentration. Again, correlation analysis showed a very strong inverse relationship (r = –0.981, p < 0.00001, R² = 0.962), suggesting that the repellency of A. muricata extract is highly dependent on concentration. The larvicidal effect of the plant extracts on An. gambiae larvae was primarily dependent on the concentration of the plant extracts, with exposure duration having less influence on mortality. This concentration-dependence holds consistently across the three plant leaf extractsAzadirachta indica, Carica papaya, and Annona muricata. This result is in conformity with earlier assertions that the effectiveness of plant-based larvicides is largely dose-dependent rather than duration-dependent [41-43]. However, for Azadirachta indica, significant increases in larval mortality were observed as the concentration increased from 0 to 10%, above which complete mortality was achieved and further increases did not affect the outcome. Similar studies corroborate this finding [44,45]. Contrary to the results of this study, Atenafu and Atnaf [46] found that An. gambiae larval mortality increased with both increase in concentration and exposure time to seed oils from A. indica and Schinus molle. In the same vein, complete larvae mortality (100%) was achieved with Carica papaya extract at concentrations above 10% (15-20%). This is in line with the result of Pakan et al. [47] who reported optimum larvicidal activity of C. papaya flower extract against Aedes aegypti at 20% concentration. Strong larvicidal effect of C. papaya extract against mosquitoes have been reported. For instance, Ilham et al. [48] reported strong larvicidal effect of ethanol leaf extract of C. papaya to Aedes spp. Nasiruddin et al. [49] found Carica papaya seed extract to be the most toxic larvicide against Anopheles annularis and Culex quinquefasciatus among seed extracts tested from Sinapis alba, Momordica charantia, and Capsicum annuum. In a different study, Astuti et al. [50] revealed that C. papaya extract showed the highest potency as biolarvicide of Ae. aegypti mostquitoes compared to Salacca zalacca and Sonchus arvensis. With Annona muricata, a comparable trend was observed. Increasing extract concentration from 0 to 5% significantly enhanced larval mortality; concentrations of 10-20% led to 100% mortality. Many studies have demonstrated the larvicidal efficacy of A. muricata against different mosquitoes including Anopheles gambiae [38], Aedes aegypti [51-53], Ae. albopictus [52], Ae. stephensi [53] and Culex quinquefasciatus [53]. Among the three plants tested, A. muricata extract had the highest repellent and larvicidal activity. This agrees with earlier reports that the plants of the Annonaceae family show higher larvicidal activity in comparison with plants from other families including Myrtaceae, Rutaceae, Euphorbiaceae, Piperaceae, Asteraceae and Liliaceae [52]. The qualitative phytochemical screening of the plant extracts revealed the presence of alkaloids, phenols, tannins, saponins, glycosides, and flavonoids in all three species. Notably, steroids were detected in A. indica and C. papaya, but absent in A. muricata. These secondary metabolites are known to contribute to various biological and insecticidal activities [38,48,54]. All three plant extracts exhibited a rich presence of flavonoids. Flavonoids are well-documented for their antioxidant, antimicrobial, and insecticidal properties [54,55]. The high abundance of flavonoids across the extracts suggests a potential shared mechanism in their bioactivity, including larvicidal effects, through oxidative stress induction and disruption of metabolic pathways in insect larvae [48]. Phenols were also found in high concentrations in A. indica and A. muricata. Phenolic compounds are known to exert larvicidal activity through various mechanisms such as enzyme inhibition, disruption of cell membranes, and interference with larval development [48,56]. Saponins, which were very highly present in C. papaya are known for their surfactant properties, that can cause membrane disruption and haemolysis in insects, making them potent contributors to larvicidal activity [48,55]. Alkaloids were highly present in all three plant extracts. Alkaloids are nitrogen-containing compounds known for their broad spectrum of bioactivities, including insecticidal, antifeedant, neurotoxic, and cytotoxic effects [48,55,56]. Alkaloids can disrupt the normal physiological functions of mosquito larvae by interfering with neurotransmission, enzyme systems, and hormonal regulation. Alkaloids such as azadirachtin (from A. indica) has been linked to growth inhibition, molting disruption, and feeding deterrence in various mosquito species [57]. Similarly, alkaloids in Carica papaya such as carpaine have demonstrated insecticidal and larvicidal properties through their ability to interfere with the neuromuscular activity of insects [48]. In Annona muricata, alkaloids including coreximine and anonaine have been identified and are thought to contribute to cytotoxic and larvicidal mechanisms [54,55]. Glycosides moderately present in all three extracts, are also important. Glycosides can be hydrolyzed into toxic aglycones, which may interfere with metabolic processes in larvae [55,55]. Overall, the qualitative phytochemical profiles suggest that the combined or synergistic effects of multiple bioactive compounds contribute to the insecticidal properties of these plant extracts. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 528–539 536 5 Conclusion The three plant extracts were found to have repellent and larvicidal activity against female An. gambiae mosquito with A. muricata having the strongest activity. These findings support the potential of these indigenous plants as environmentally friendly and natural alternatives to synthetic chemicals in malaria vector control. These plants therefore, represent promising candidates for the development of sustainable, community-based malaria vector control interventions. Compliance with ethical standards Acknowledgments The authors are grateful to Tertiary Education Trust Fund (TETFund) for the grant given in conducting this research work. Disclosure of conflict of interest Authors declare that no conflict of interest exits. Statement of ethical approval The ethics governing the use and conduct of experiments on animals and humans were strictly observed. Ethical approval was gotten from Abia State University ethical committee. Statement of Informed consent A written consent was obtained from the human volunteers who were used for the repellent assay. References [1] WHO. World Malaria Report 2022. Geneva, Switzerland, WHO; 2022a [2] Dey P, Goyary D, Mandal S, Dey BK, Verna A. Vector-borne diseases: prevalene, impacts, and strategies to address disease burden and threats. Int. J. Mosq. Res. 2023; 10(1): 29-38. 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