Bio-Insecticide Impact of Sida Rhombifolia Fabric Treated with Aloe Vera on Wild Anopheles Gambiae

BULETINUL INSTITUTULUI POLITEHNIC DIN IAŞI Publicat de Universitatea Tehnică „Gheorghe Asachi” din Iaşi Volumul 71 (75), Numărul 3, 2025 Secţia CHIMIE şi INGINERIE CHIMICĂ DOI: 10.5281/zenodo.17357350 BIO-INSECTICIDE IMPACT OF SIDA RHOMBIFOLIA FABRIC TREATED WITH ALOE VERA ON WILD ANOPHELES GAMBIAE BY NSANGOU ABDOURAMANE1,2, ETEME ENAMA SERGE3, NKEMAJA DYDIMUS EFEZE4, BETENE FABIEN EBANDA1, DOINA SIBIESCU5,, NOAH PIERRE MARCEL ANICET1, MEWOLI ARMEL EDWIGE1, BETENE OMGBA ACHILLE DESIRE1, NGUM LESLEY NGUM6 and NDOUM ANIGUE NGOULOUMA GASPARD3 1University of Douala, Mechanical Laboratory (LME), ENSET Douala, B.P 1872 Douala, Cameroon 2University of Dschang, Department of Decorative Arts, Institute of Fine Arts 3University of Yaounde 1, Laboratory of Parasitology and Ecology, Department of Animal Biology and Physiology, Faculty of Sciences, B.P 812 Yaounde, Cameroon 4University of Bamenda, HTTTC Bambili, P.O. Box:39 Bambili, Cameroon. 5“Gheorghe Asachi” Technical University of Iaşi, “Cristofor Simionescu” Faculty of Chemical Engineering and Environmental Protection, Iaşi, Romania 6Researcher, Institute of Medical Research and Medical Plant Studies, Cameroon Received: July 4, 2025 Accepted for publication: August 25, 2025 Abstract. Malaria is transmitted to humans through the bite of infected female Anopheles mosquitoes carrying Plasmodium parasites. In Cameroon, mosquito control primarily relies on long-lasting insecticide-treated nets (LLINs), which may cause skin irritation and respiratory discomfort due to synthetic pyrethroids. This study explores a potential natural alternative: weft-knitted Sida rhombifolia fabrics treated with Aloe vera gel. Nine (9) samples were tested, including a positive control (permethrintreated net), a negative control (untreated net), and seven experimental fabrics: untreated Sida rhombifolia fabric, fabric treated with Aloe vera, the same fabric Corresponding author; e-mail: d[email protected] 72 Nsangou Abdouramane et al. washed twice, permethrin-treated Sida fabric, and Aloe vera-treated fabrics stored for 1, 2, and 3 months. Two WHO-standard bioassays were conducted: cone tests (3-minute exposure) and cylinder tests (15-minute exposure), using wild Anopheles gambiae populations from Yaoundé. Fabrics treated with Aloe vera showed promising mortality rates, reaching up to 22% in cone tests and 25% in cylinder tests, comparable to those of permethrin. Efficacy declined slightly over time, particularly after 3 months of storage or brief contact exposure. These findings suggest that Aloe vera-treated Sida rhombifolia textiles could serve as natural, biodegradable alternatives to LLINs, subject to further optimisation for long-term stability and field deployment. Keywords: Sida rhombifolia, Aloe vera, natural insecticide, mosquito control, Anopheles gambiae, bio-fabric. 1. Introduction Worldwide, over 3,500 mosquito species are classified into 41 genera. In Africa alone, 837 species have been documented (Clements, 1992). Malaria transmission to humans occurs solely through female mosquitoes of the genus Anopheles. Of approximately 430 Anopheles species globally, only 30 to 40 are known to transmit malaria. The disease primarily targets three major vectors that are widely distributed across sub-Saharan Africa: Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis (Eleazar et al., 2022; Clements, 1992 and 1999; Islam et al., 2017; Kakou, 2021; Nsangou et al., 2022, 2023a, 2023b). These three species share identical morphology and belong to the same species complex, known as Anopheles gambiae sensu lato (Clements, 1992; Nsangou et al., 2023a; Yadouleton et al., 2018). One effective method to combat mosquito bites is the use of impregnated mosquito nets. Long-lasting impregnated mosquito nets (LLINs) in Cameroon are available in three types: Olyset Net, featuring polyethylene support impregnated with permethrin; Perma Net 3.0, supported by polyethylene and impregnated with deltamethrin; and Mag Net, made of polyester with alphacypermethrin and a PBO mixture (Nkemaja et al., 2024). These nets may cause adverse effects upon contact with skin or when breathing, so it is advised to leave them in the shade for 24 hours before use (Eleazar et al., 2024). Chemicals in the nets can lead to negative health effects, such as headaches, oxidative stress, and pollution (Oyetunji et al., 2024). The fibre from the bast of SR is white, flexible, soft, shiny, and has a uniform texture. The fibres of Sida rhombifolia are suitable for textiles (Nsangou et al., 2022). The gel is the transparent mucilage found in the parenchymal cells of the fresh leaf of AV (Nsangou et al., 2022). AV gel, with a pH of 4 to 5, contains approximately 98.5% water, with the cuticle containing about 90%. The total solid content is 0.66%, with soluble solids accounting for 0.56%, considering seasonal variations (OMS, 2017; Youssouf et al., 2020). The choice of knitwear for SR treated with Bul. Inst. Polit. Iaşi, Vol. 71 (75), Nr. 3, 2025 73 Aloe vera gel stems from three prior studies (Nkemaja et al., 2024; Nsangou et al., 2021a; Nsangou et al., 2023a). These studies indicate that textiles of plant origin with antimicrobial properties (such as cotton, jute, wool, cotton-rayon blends) are often treated with chemical agents. For example, cotton can be treated with bifenthrin to repel mosquitoes. Natural and naturally treated textiles for combating parasitic arthropods are still not fully understood. Neem, garlic, eucalyptus, lemongrass, Aloe vera, and Sida rhombifolia are all known for their insecticidal properties. Extracts from these plants, including Aloe vera gel, have been used in textiles and tested for antimicrobial activity. Sida rhombifolia fibre completely inhibits Escherichia coli at 2g after six hours. It also inhibits Salmonella typhi, with coatings of Aloe vera gel accelerating this effect. It inhibits Pseudomonas aeruginosa and Vibrio cholerae at doses of 0.5g, 1g, 1.5g, or 2g. When macerated with Aloe vera gel, it fully inhibits Aeromonas hydrophila at 1.5g over six hours, Pseudomonas aeruginosa at 2g over six hours, and Vibrio cholerae at 1.5g over three hours (Nsangou, 2020; Nsangou et al., 2021a). These require further experimental investigation considering environmental challenges posed by climate change, which could also open new avenues for economic growth. Since this approach might reduce reliance on pyrethroids, which are produced through an exothermic substitution of alkane hydrogens with halogens (chlorine or bromine) or via addition reactions involving halogens, requiring catalysts for alkenes and alkynes. Consistent with Nkemaja et al. (2024b), phytochemical screening showed the presence of compounds such as flavonoids, sterols, and polyterpenes. The diversity of compounds identified through visible and UV spectroscopy at 366 nm, as well as various reagent tests, highlights the richness of bioactive substances in the extracts (Nkemaja et al., 2024b). Therefore, the plant's potential to combat mosquitoes may be linked to these chemical families or their synergistic effects. This study aims to replace conventional mosquito nets with Sida rhombifolia nets treated with Aloe vera gel. 2. Materials and Methods 2.1. Impregnation of knitting Sida rhombifolia with Aloe vera gel Nine samples were used to evaluate the activity of Sida rhombifolia knitted fabric impregnated with Aloe vera gel: a mosquito net sample impregnated with permethrin or a positive control (Olyset Net), and another nonimpregnated control sample or negative control (Care Plus) (MNTB). The other seven samples included a Sida rhombifolia knitted sample not impregnated with Aloe vera gel (TNT), a sample impregnated with Aloe vera gel (TTA), a sample impregnated with Aloe vera gel and washed twice (TTAL2), a sample impregnated with permethrin (TIP), a sample impregnated with Aloe vera gel for one month (EI1M), for two months (EI2M), and for three months (EI3M). All samples underwent cone tests. Additionally, a cylinder test was performed 74 Nsangou Abdouramane et al. considering these knits and curtains as papers impregnated with insecticide, following WHO recommendations when the impregnated dose is unknown. These tests utilised a wild field population of Anopheles gambiae from Yaoundé, chosen specifically to evaluate the real-world effectiveness of the knitting against field mosquitoes. 2.1.1. Washing Washing was done manually by ISO 3758 using water at room temperature. Azure brand soap (350g) was used. Excess water was removed through manual wringing and air drying for 2 minutes, with drying carried out in the shade at room temperature. For multiple washes, the process remained the same: simply wait for drying and repeat the cycle. This indicates that the number of washes correlates with the number of days: one wash per day, two washes over two days. 2.1.2. Samples They were used to assess the activity of Sida rhombifolia knitted fabric (the sample is displayed in Fig. 1) impregnated with Aloe vera gel: a mosquito net sample impregnated with permethrin or positive control (Olyset Net), and another non-impregnated control sample or negative control (Care plus) (MNTB) and the other 7 samples (a Sida rhombifolia knitted sample not impregnated with Aloe vera gel (TNT), a Sida rhombifolia knitted sample impregnated with Aloe vera gel (TTA), a Sida rhombifolia knitted sample impregnated with Aloe vera gel and washed twice (TTAL2), a Sida rhombifolia knitted sample impregnated with permethrin (TIP), a sample impregnated with Aloe vera gel for one month (EI1M), an impregnated sample impregnated with Aloe vera gel for 2 months (EI2M) and a sample impregnated with Aloe vera gel for 3 months (EI3M). Fig. 1 ‒ Samples to be tested: the type of knitting of Sida rhombifolia. These samples were subjected to cone tests. In addition to this test, the cylinder test was carried out by considering these knits and curtains as papers impregnated with insecticide. Bul. Inst. Polit. Iaşi, Vol. 71 (75), Nr. 3, 2025 75 This is a WHO recommendation when the dose of the impregnated material is not known. Said tests were carried out with a wild field population (Yaoundé) of Anopheles gambiae. The choice of the wild field population of Yaoundé was made with the sole aim of controlling the reality of the activity of the knit against the field mosquito. 2.2. Collection of immature stages and laboratory rearing Anopheles gambiae (suspected resistance) populations for insecticide sensitivity testing were collected in the larval stage from natural (puddles, ponds, lakes) and artificial (water reservoirs, pits, tyre tracks, and tin cans) breeding sites around the University of Yaoundé 1 during the period from 17 to 28 August 2024 (Chonoibon et al., 2008). Anopheles gambiae larvae and nymphs were collected using the dipping method (Yadouleton et al., 2018) with handled ladles (Fig. 2a). These larvae and nymphs were collected beneath plants bordering watercourses. A breeding site is deemed positive if at least one Anopheles larva is found; it is considered negative if no larvae are collected after 20 to 30 minutes of sampling (Merdya et al., 2022). The collected larvae and nymphs were transferred into 1litre plastic bottles filled with water from the breeding site and transported to the Laboratory of Parasitology and Ecology, Department of Animal Biology and Physiology, Faculty of Sciences, University of Yaoundé 1, for their rearing (Merdya et al., 2022). These larvae were raised in water from the breeding sites and were fed daily (Yadouleton et al., 2018) (Fig. 2c). Adult mosquitoes were fed in 10-litre basins open to the surroundings, using honey on cotton. Anopheles gambiae are identifiable by the horizontal position of their larvae in the water, and when mature, they have a pair of long, segmented antennae. Rearing took place at a room temperature of 22°C and a pressure of 1024 hPa. Fig. 2 ‒ Collection (a), transport (b) and rearing (c) of immature stages of Anopheles gambiae. 2.3. Cone test The cone test was conducted per the WHO protocol (WHO, 1998). In each cone, eight young female mosquitoes (2-5 days old) were introduced and 76 Nsangou Abdouramane et al. left in contact with the knitted fabrics for 3 minutes. They were then removed from the cones and transferred to sterile cups covered with a veil, placed on cotton discs soaked in natural honey, used for feeding the mosquitoes. After 24 hours of observation, mortality rates were recorded for each batch of mosquitoes. Mortality for the test and control samples is calculated by adding the number of dead mosquitoes and expressing the result as a percentage of the total number of mosquitoes exposed (equation 1). Fig. 3 ‒ a) Mosquito culture tank, b) Mosquitoes kept in a cone, c) mosquitoes stay in cups supplied with natural honey. 2.4. Cylinder tests This test assesses the presence of insecticide on impregnated substrates. It is used with the cone test to provide additional information on the efficacy of knitted fabrics impregnated with Sida rhombifolia. The tests were carried out using fragments of impregnated curtains and knitwear cut into 5cm-by-5cm fragments and used as insecticide-impregnated papers. The tests followed the WHO protocol in a cylindrical tube (WHO, 1998). The mosquitoes' exposure time to the impregnated curtains was 15 minutes, and the observation time before reading the results was 24 hours. After the tests, the dead and live mosquitoes were separated. Considering the WHO scale, any mosquito population was considered sensitive to the insecticide when the mortality in the test was greater than or equal to 97%. The population was described as resistant when the mortality was less than 80%. Resistance was suspected between the two values. The same apparatus as in Fig. 3 was used, with the cone replaced by the cylindrical exposure chamber (see Fig. 4) Observed mortality=𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑚𝑜𝑠𝑞𝑢𝑖𝑡𝑜𝑒𝑠 𝑇𝑜𝑡𝑎𝑙 𝑒𝑥𝑝𝑜𝑠𝑒𝑑 x100 (1) Bul. Inst. Polit. Iaşi, Vol. 71 (75), Nr. 3, 2025 77 Fig. 4 ‒ Mosquitoes stay in the cylinder. To find the replicate mean, it adds up replicates 1, 2, 3 and 4 and divides by 4. The standard deviation is equal to the sum of the replicates squared divided by 4 minus the mean squared. 3. Results 3.1. Cone test 320 mosquitoes were used for testing. The nine (9) samples to be tested, namely: one sample of net impregnated with permerine or positive control (Olyset Net), and another sample of unimpregnated control or negative control (Care plus) (MNTB) and the other eight samples (one sample of Sida rhombifolia knitted fabric not impregnated with Aloe vera gel (TNT), one sample of Sida rhombifolia knitted fabric impregnated with Aloe vera gel (TTA), a sample of Sida rhombifolia knitted fabric impregnated with Aloe vera gel and washed twice (TTAL2), a sample of Sida rhombifolia knitted fabric impregnated with permethrin (TIP), a sample impregnated with Aloe vera gel for one month (EI1M), a sample impregnated for 2 months (EI2M) and a sample impregnated for 3 months (EI3M). Bioassay results after four repetitions of the Cone tests showed that the average induced mortality rate after a 3-minute contact was 63.4% (Table 1). 78 Nsangou Abdouramane et al. Table 1 Mortality of Anopheles gambiae populations from the cone test observed after 3 minutes of contact with treated and untreated knitwear, as well as controls Morbidity (%) after 3 minutes of contact Replicate MBT MNTB TNT TTA TIP TTAL2 EI1M EI2M EI3M 1 25 12.5 16.75 25 22 18.75 22 12.5 18.75 2 25 10.75 17.75 22 22 25 12.5 8.25 12.5 3 22 8.25 12.5 22 18.75 18.75 22 18.75 3 4 12.5 3 15.75 15.75 18.75 18.75 18.75 12.5 12.5 Average 21 8.75 15.75 22 20.25 22 18.75 13 11.75 SD 02 01.75 02.00 01 01.75 01 01.75 01.75 02.75 SD: Standard deviation MBT: Net impregnated with permerine or positive control (Olyset Net), MNTB: Net unimpregnated control or negative control TNT: Sida rhombifolia knitted fabric not impregnated with Aloe vera gel (Care plus), one sample of Sida rhombifolia knitted fabric impregnated with Aloe vera gel, TTA: Sida rhombifolia knitted fabric impregnated with Aloe vera gel, TTAL2: Sida rhombifolia knitted fabric impregnated with Aloe vera gel washed twice, TIP: Sida rhombifolia knitted fabric impregnated with Permetrin EI1M: Sida rhombifolia knitted fabric impregnated with Aloe vera gel for one month. EI2M: Sida rhombifolia knitted fabric impregnated for 2 months, EI3M: Sida rhombifolia knitted fabric impregnated for 3 months. Average values facilitate comparison of the different tested conditions. MBT (positive control): high mortality (21%), as expected. MNTB (negative control): low mortality (8.75%), confirming it has no effect. TNT (knitted fabric not treated with Aloe vera gel): moderate mortality (15.75%), indicating a slight insecticidal effect of Sida rhombifolia knitted fabric without Aloe vera gel. TTA (Sida rhombifolia knitted fabric impregnated with Aloe vera gel) and TIP (Sida rhombifolia knitted fabric impregnated with permethrin) exhibited the highest mortality (22%), demonstrating a significant protective effect. EI 1 M, EI 2 M and EI 3 M (gel impregnated for 1, 2 and 3 months): mortality decreased over time (18.75%, 13%, 11.75%), suggesting a gradual decline in efficacy. Standard deviations are generally low (1 to 2.75%), indicating consistent and reliable data. For EI 3 M, the slightly higher standard deviation (2.75%) may reflect increased variability related to the gel's age. The impregnated knitted fabrics (TTA and TIP) show high mortality rates (22%), similar to the positive control (MBT), confirming their effectiveness in a short timeframe. Aloe vera gels lose effectiveness over time (EI 1 M> EI 2 M> EI 3 M). MNTB (negative control) exhibited minimal mortality (8.75%), reaffirming the absence of an insecticidal effect. TNT (knitted fabric impregnated with Sida rhombifolia) resulted in slightly higher mortality (15.75%), possibly due to mechanical effects or other Bul. Inst. Polit. Iaşi, Vol. 71 (75), Nr. 3, 2025 79 properties of Sida rhombifolia. Mortality rates are lower than in the cylinder test, reflecting a limited effect due to the short contact time (3 minutes). Maximum efficacy reached 22% with the treatment of Aloe vera gel and permethrin. Comparing this to Yadouleton's (2018) work on quality control of bifenthrinimpregnated curtains for use and dissemination in Benin, which showed that in cone bioassays, the mortality after 3 minutes of exposure over 2 hours was 48.85% for the susceptible strain and 6.96% for the wild strain. 3.2. Cylinder test As with the cone test, 320 mosquitoes were used to perform this test on the nine (9) samples to be tested. The purpose of the results of this cylinder test is to confirm the cone test. As with the cone test, 320 mosquitoes were used to perform this test on the nine (9) samples to be tested. The bioassay results after four replicates of the cylinder tests are shown in Table 2 below. Table 2 Mortality rate of Anopheles gambiae populations following the cylinder test observed after 15 minutes of contact with treated knitwear Morbidity (%) after 3 minutes of contact Replicate MBT MNTB TNT TTA TIP TTAL2 EI1M EI2M EI3M 1 25 18.75 22 25 25 25 25 25 21 2 25 12.5 25 25 25 25 25 25 25 3 25 15 22 25 25 25 25 25 25 4 25 12.5 22 25 25 25 25 25 25 Average 25 14.75 23.5 25 25 25 25 25 23.25 SD 00 01 00.5 00 00 00 00 00 00.25 SD: Standard deviation MBT: Net impregnated with permethrin or positive control (Olyset Net), MNTB: Net unimpregnated control or negative control TNT: Sida rhombifolia knitted fabric not impregnated with Aloe vera gel, one sample of Sida rhombifolia knitted fabric impregnated with Aloe vera gel, TTA: Sida rhombifolia knitted fabric impregnated with Aloe vera gel, TTAL2: Sida rhombifolia knitted fabric impregnated with Aloe vera gel washed twice, TIP: Sida rhombifolia knitted fabric impregnated with permetrin EI1M: Sida rhombifolia knitted fabric impregnated with Aloe vera gel for one month. EI2M: Sida rhombifolia knitted fabric impregnated for 2 months, EI3M: Sida rhombifolia knitted fabric impregnated for 3 months.