In this study, mites were intentionally fed on various plants to ensure a generalizable mortality response across different pest populations from a range of host plants. The positive control (Sunfire® at a test concentration of 0.06% w/v) exhibited significantly higher mortality rates (54.6-89.7%) compared to the relative control (0.42-7.6%) from 48 to 96 h (P<0.05) (Table 1). This result aligns with expectations and indicates that the variability in female diet, due to different host plant species, did not significantly alter the response to the evaluated treatments. Unfractionated botanical extracts were prepared using 96% ethanol as the primary extractant due to its availability, ease of recovery, and ability to solubilize and extract a broad range of bioactive specialized metabolites. The bioassay demonstrated that the ethanolic extracts induced mortality and affected oviposition in T. urticae females at varying levels at the selected concentration (0.06% w/v). This dose was chosen as a representative value within the concentration range commonly used for biopesticides, such as those derived from Azadirachta indica (0.02-0.10% w/v)(Biswas 2013).

Table 1

The corrected mortality percentages of T. urticae females exposed via direct contact with various crude ethanol extracts under laboratory conditions over 24 to 96 h are presented in Table 1. Among the tested extracts, N. amazonum exhibited the highest corrected mortality (14.4%) at 96 h, with significant differences from the relative control (P<0.01). Solanum nigrum-EC2 showed significantly different mortality rates at 24, 48, and 72 h (P<0.05), whereas U. europaeus exhibited its highest mortality at 96 h. Conversely, S. nigrum-EC1, T. diversifolia, and V. elongata displayed low corrected mortality rates, with S. nigrum-EC2 reaching a maximum of 8.9% after 96 h. These findings indicate that the test extracts had weak lethal effects (<15%). For T. diversifolia, its low impact on T. urticae agrees with Pavela et al. (2018), who reported a maximum mortality of 50% at a dose of 150 µg cm^-³ of a polar extract in acute toxicity assays. The observed low mortality may be attributed to the characteristics and traits of the tested mite population. This observation was evident from the results obtained for a reference active extract derived from S. nigrum-EC2 (collected in Casanare, Colombia) (Numa et al. 2016). This extract was previously tested on a homogeneous T. urticae population composed of susceptible individuals, yielding high corrected mortality (85.0±4.3% after 72 h) (Numa et al. 2016). However, when tested on the current heterogeneous population under identical exposure conditions, mortality was markedly lower (5.9±1.6%). To rule out chemical composition differences as a cause of these discrepancies, LC-MS analyses were performed on the botanical extracts, confirming identical chemical profiles. Thus, the differential mortality between assays can be attributed to variations in T. urticae populations. In this study, S. nigrum ecotype 1 (EC1) was included among the tested botanicals to compare the effects of extracts from the same species growing in different locations. The extract from S. nigrum-EC1 (collected in Cundinamarca, Colombia) exhibited lower mortality compared to that of S. nigrum-EC2. This difference is likely due to variations in chemical composition, with ecotype 2 containing higher levels of certain metabolites, particularly alkaloidal triterpenes.

Despite the low impact on T. urticae mortality, the main effect of test botanicals was observed on reproductive alterations. Females from positive control (Sunfire®) increased oviposition at 72 and 96 h of exposure (Table 2). A similar effect was observed for the N. amazonum-derived extract, which was the most active botanical in terms of mite mortality. This response can be attributed to physiological stress, leading to hormonal changes that accelerate reproduction. A similar phenomenon has recently been reported in the brown planthooper Nilaparvata lugens, where exposure to certain insecticides has been associated with increased juvenile hormone levels, responsible for stimulating egg development and oviposition (Gao et al. 2025).

Table 2

Significantly lower oviposition rates were recorded for S. nigrum (ecotype-2) at 96 h (P<0.05) and T. diversifolia, V elongata, and V. peruviana at 24 h. Additionally, U. europaeus induced an oviposition reduction below one egg per day per female at 96 h (Table 2). These extracts exhibited a sublethal effect by reducing T. urticae oviposition, consistent with findings for other plant extracts. Pavela et al. (2018) reported that polar extracts from T. diversifolia, despite their low direct toxicity, strongly inhibited oviposition. This effect was attributed to the presence of tagitinin C and A, which act as repellents and antifeedants. In the present study, tagitinin C and A were not detected in the T. diversifolia extract; however, a structurally related compound, tagitinin D, was identified. This finding suggests that sesquiterpene lactones such as tagitinins may play a role as bioactive metabolites in the genus Tithonia, which has been previously associated with plant defense mechanisms (Chagas-Paula et al. 2012).

Several studies have investigated the oviposition-deterring effects of plant by-products on T. urticae, demonstrating that phenolic- and terpene-rich botanicals significantly impact mite reproduction (Rincón et al. 2019). In this context, flavonoids and phenolics (from V. elongata, V. peruviana, and U. europaeus), alkaloidal triterpenes (from S. nigrum-EC2), and sesquiterpenes (from T. diversifolia) may be the active phytochemicals responsible for the observed reductions in oviposition. Similar effects have been reported by Chacón-Hernández et al. (2020b), who found that exposure to Magnolia tamaulipana powder extract led to a concentration-dependent reduction in T. urticae oviposition, with the effect increasing between 5 and 1,000 μg mL-1. Additionally, ethanolic extracts of Lippia origanoides and Gliricidia sepium at a concentration of 5% v/v inhibited oviposition in Tetranychus cinnabarinus by 43.7-57.0% after 72 h of exposure (Sivira et al. 2011). This oviposition-inhibitory effect may have two possible explanations: 1) specialized metabolites produced by plants could directly impair ovarian function (Dimetry et al. 2003), or 2) these compounds may induce incomplete or temporary sterility, leading to reduced oviposition (Hosny et al. 2010). Further research is needed to elucidate the physiological mechanisms underlying these sublethal effects.

Although preference tests were not conducted in this study, previous experiments suggest that botanicals can modify oviposition behavior, as females often avoid locating and ovipositing on treated surfaces (Keskin et al. 2020). This response could be mediated by specific phytoconstituents, such as volatile compounds, present in botanicals (Keskin et al. 2020). In multiple-choice tests, Hata et al. (2020) demonstrated that aromatic plants with low acceptance by T. urticae females also induced significantly lower oviposition rates per day. Similarly, Zhang et al. (2008) reported that an extract derived from Artemisia annua leaves significantly reduced T. cinnabarinus oviposition. Kumral et al. (2010) further demonstrated the effectiveness of Datura stramonium L. seed- and leaf-derived extracts against T. urticae, not only as an acaricide but also as a repellent, oviposition deterrent, and hatch rate reducer after 24 h of exposure. These findings highlight the potential efficacy of compounds in the tested extracts for disrupting T. urticae reproduction.

LC/MS-based chemical characterization of the unfractionated extracts used in the bioassay detected 42 main compounds (annotated at level three based on their mass spectral data, Table 3), comprising phenolics, flavonoids, alkaloids, and terpene-related metabolites. Some of these compounds shared retention times in certain extracts, while most were detected only in a specific extract, reflecting the taxonomic variability of the tested botanicals (Figure 1, Table 3). Thus, this comparative LC/MS-based analysis provides an overview of the number and semiquantitative levels of metabolites present in a single extract, particularly those derived from leaves, which exhibit highly varied phytoconstituent profiles. These plant-derived metabolites can act as toxic agents or oviposition disruptors for many pests (Numa et al. 2015), causing various single or concomitant effects on mites (Numa et al. 2018). This complexity presents a challenge in identifying the specific compounds responsible for the observed acaricidal activity. However, existing records on the toxicity of certain metabolites can guide further investigation. For instance, by-products from Eugenia langsdorffii and Ocotea spp. contain high concentrations of sesquiterpenes, primarily found in leaves, which exhibit strong toxic effects on T. urticae, attributed to these terpene-related compounds (de Moraes et al. 2017).

Figure 1

Table 3

Table 3

Table 3

The findings of this study serve as a foundation for developing alternative control strategies targeting T. urticae reproduction using test plant extracts, particularly U. europaeus, an invasive species in South America. In this context, quinolizidine alkaloids (e.g., rhombifoline) and prenylated isoflavones (e.g., isowighteone) were identified in the U. europaeus extract and may contribute to oviposition inhibition (Isman 2020). Indeed, prenylated isoflavones, precursors of rotenoids, have demonstrated acaricidal activity and completely inhibited the oviposition of the female tick Rhipicephalus appendiculatus (Van Puyvelde et al. 1987), while matrine-type quinolizidine alkaloids have been shown to affect T. urticae oviposition (Marčić and Međo 2014). Additionally, nitrogenated triterpenes (e.g., abutiloside J), lignans (e.g., grandisin), and sesquiterpene lactones (e.g., tagitinin D) were detected in extracts with the highest oviposition inhibitory effects, such as those from S. nigrum, V. elongata, and T. diversifolia. These findings warrant further bio-guided fractionation studies to isolate and evaluate the active compounds responsible for anti-mite effects, defining the active principles and their specific roles in T. urticae oviposition alteration.

T. urticae susceptibility to acaricidal agents has been primarily assessed through acute contact toxicity assays. However, resistance development is well-documented and is often linked to genetic traits enabling adaptation to acaricide pressure via transposable elements in its genome (Rincón et al. 2019). Resistance mechanisms include reduced target site sensitivity, metabolic detoxification, and decreased penetration, all classified as physiological resistance, while behavioral adaptations remain poorly studied (Adesanya et al. 2019). Developing behavioral manipulation techniques is a promising approach to mitigating resistance and optimizing synthetic insecticide use (de França et al. 2013). From this perspective, botanical extracts from S. nigrum, T. diversifolia, V. elongata, and U. europaeus show potential for use as oviposition inhibitors or disruptors. Information on the extraction yield and crop productivity of these plants is essential for assessing the feasibility of incorporating oviposition inhibitors into integrated pest management (IPM) programs for controlling T. urticae. Additionally, further studies should evaluate the dose-dependent effectiveness of the most promising extracts under laboratory conditions to validate their potential in T. urticae management through targeted field trials. Once high effectiveness is confirmed, standardization processes must be established, including plant selection, cultivation management, and formulation development for field application. A common challenge with botanical products is the inconsistency in efficacy and lack of reproducibility, often due to variations in agronomic practices and extract stability (Ivase et al. 2021). Therefore, caution is advised in generalizing these findings, as physiological and behavioral aspects may influence the magnitude of the observed effects (Thompson and Reddy 2016).

This study evaluated the capacity of 10 leaf-derived unfractionated ethanol extracts to alter T. urticae survival and oviposition behavior, identifying promising candidates for disrupting its reproduction. Among the tested extracts, S. nigrum EC2 and U. europaeus significantly inhibited oviposition after 96 h, while N. amazonum, S. nigrum EC1, and T. diversifolia were most effective in inducing mite mortality. These findings suggest that specific phytochemicals, such as alkaloidal triterpenes, phenolics, and sesquiterpenes, may play crucial roles in these biological activities. The results highlight the importance of understanding phytochemical-pest interactions and emphasize the need for further research to isolate and characterize the active compounds responsible for these effects. Additionally, the observed reduction in T. urticae oviposition may be attributed to the sublethal effects of the extracts, potentially leading to decreased population growth and reduced dependence on synthetic pesticides. This research underscores the potential of botanical extracts as sustainable IPM tools, offering an environmentally friendly alternative to conventional chemical controls and contributing to the reduction of pesticide use in commercial agriculture.

The authors have no conflict of interest.

The authors declare they have not violated or omitted ethical or legal norms when conducting this study.