1 Introduction

The Tigray region is largely covered by cacti (Opuntia ficus-indica). This plant is traditionally grown in rural areas of Tigray, and the fruit, locally known by the vernacular name 'Beles,' belongs to the genus Opuntia in the family Cactaceae. Cactus is the primary source of food for many farmers during the rainy season, especially from June to September, just before the harvesting of staple crops (cereals). A cactus thrives in this environment and plays a special role in food security for both humans and livestock [1, 2]. The importance of cactus is increasing over time. Research findings have indicated that cactus pear has become an integral part of the culture and economy of Tigray, serving as a source of seasonal household food, income, and employment, as well as livestock feed and providing other environmental benefits. It is also a source of income for school children and has become an occupation for adults, especially women, due to a steady increase in the price of fresh fruits. In southern Tigray, much of these fruits are harvested from wild prickly pear cactus plantations [3, 4].

Despite the benefits to humans and livestock, the cactus plant is a suitable host for diseases and pests such as the cochineal insect. Cochineal insects, particularly Dactylopius opuntiae, are known as potentially damaging pests that grow and reproduce on pear cladodes [5]. The cochineal insect, often referred to as a mealybug, is a parasitoid of the cactus plant and is a sucking insect that feeds exclusively on certain cactus species [6]. There are various species of cochineal insects, each of which feeds on specific cactus species. Dactylopius coccus, also known as 'Red Gold,' is a valuable resource for producing carminic acid (a colorant) and is a significant livelihood in Mexico, Peru, and India [7]. However, the introduction of D. opuntiae to Ethiopia's Tigray region has led to significant damage to cactus plants [4, 8].

D. coccus is known for producing a dye of superior quality and is the only commercial species reared specifically for carminic acid production [9]. Cochineal insects cover themselves with a waxy coat, appearing as white cottony tufts attached to cactus pads and stems. These white sticky mounds serve as housing for cochineal bugs. Cochineal insects exist in two forms based on their survival in the environment: wild and domestic (commercial or cultivated) cochineals [10]. Wild cochineals are smaller in size compared to domestic cochineals and produce lower-quality dye. Domestic cochineals, such as D. coccus, are delicate, sensitive to cold winters, and vulnerable to insects or natural enemies. On the other hand, wild types like D. opuntiae have no identified natural enemies and are resistant to harsh environmental and climatic conditions [11].

The introduction of wild-type D. opuntiae from South Africa to the Tigray region in April 2003, supported by a FAO-Technical Cooperation Program (TCP) project hosted by Mekelle University, aimed to establish cochineal insect nurseries for commercial purposes. However, the uncontrolled spread of cochineal insects has become a serious issue due to the failure to harvest and utilize them effectively. Cochineal insects have spread rapidly across geographical areas through wind dispersal, flood transportation, human activity, and animal interactions, resulting in extensive infestation of cactus-covered areas [12,13,14].

The insect infestation causes damage to cactus cladodes, reduces cactus pear size, and threatens cactus plantations. Many people view this invasion as a future threat to cactus cultivation. The communities that rely on cactus plants are experiencing significant destruction, and the impact of the cochineal insect has become a serious concern in the region. The absence of effective mechanisms or quarantine laws to control the spread of cochineal insects to other regions and the lack of designated safe zones for cochineal production in Tigray have exacerbated the severity of the infestation. Additionally, the low market price of fresh cochineal (less than USD 1 per kilogram) offered by Foodsafe Company, combined with the difficulty of harvesting the insect from spiny cactus cladodes, has contributed to the damage of cactus plants and the resulting food and economic insecurity in the region [15, 16].

Scientific efforts have been made by the Tigray Agricultural Research Institute (TARI), Tigray Regional Agricultural Bureau, Mekelle University, and other sectors to control cochineal infestations using chemical sprays, liquid soap solutions, mechanical removal of infested cacti, and burning. However, these attempts have been largely unsuccessful despite ongoing efforts. There is a hypothesis that repeated spraying of macerated herbal materials may prevent cochineal infestations. Herbal crude extracts are complex mixtures of chemical components that exhibit synergistic effects. Most herbal extracts function as:

(i) Repellents that drive insects away from plants due to their smell or taste [17], (ii) Anti-feedants that cause insects to reduce food intake, leading to starvation [18], (iii) Oviposition deterrents that prevent insects from laying eggs [19], (iv) Inhibitors that disrupt the development of various insect stages [20,21,22].

Therefore, this research aimed to investigate effective herbal extracts against cochineal insects. The locally available herbs included in the investigation were Solanum linnaeanum, Euphorbia tirucalli, Nerium oleander, Tephrosia vogelii, Calpurnia aurea, Argemone mexicana, Datura stramonium, and Ricinus communis (Table 1).

Table 1 Plant species used in the experiment, availability, and ecological status in the study area
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2 Materials and methods

2.1 Experiment site, study subjects, and study design

The experiment was conducted within the compound of Mekelle University in the Tigray region of Ethiopia. The university's mountainous border is covered with various varieties of cactus. The experimental plants were sourced from the local environment, and 200 wild-type female cochineal insects (Dactylopius opuntiae) were used in the experiment.

A randomized block design was implemented from August 2022 to February 2023 to assess the efficacy of herbal extracts against cochineal insect. Each plant extract constituted a block, and nine concentrations (1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%) were considered within each block. Three replicates were conducted for each plant extract.

2.2 Sampling method

Locally available plants, namely S. linnaeanum (fruits), E. tirucalli (leaf), N. oleander (leaf), T. vogelii (leaf), C. aurea (leaf), A. mexicana (leaf and stem), D. stramonium (seeds), and R. communis (leaf), were purposely selected for the study.

2.3 Study methods

2.3.1 Collection and extraction of herbal materials

The herbs were collected from the field, including leaves, fruits, seeds, and stems of the experimental plants, and transported freshly with clean and dry trays to the laboratory. The herbal materials were washed with tap water to remove dust and surface contaminants, then shade-dried and ground into a fine powder for maceration. Based on the nature and characteristics of the herbs described in the literature, ethanol (97%) and methanol (99.8%) were chosen as co-solvents, along with distilled water, for maceration.

Five hundred grams (500 g) of each experimental plant powder was macerated in flasks containing 2 L of solvent. Each plant powder was macerated with three different solvents. The flasks were agitated for 10 min, four times a day, over 24 h, and allowed to settle at room temperature. The supernatant was decanted into clean 3-L glass containers. Solvents were added to the sediment, and the process was repeated three times over 72 h to maximize extract yield.

The collected supernatant liquid was filtered through Whatman No. 1 filter paper using an electrical suction pump. The filtered liquid was then evaporated in a water bath at 42 °C for 6 h for methanol [23] and 12 h for ethanol [24] to obtain dried crude oil extracts, which were stored at 4 °C. The dried extracts of the experimental plants were collected and weighed to determine the yield of each herb using the following formula [25, 26]:

$${\text{Yield }}\% = \frac{{{\text{Weight of crude oil in grams}}}}{{{\text{The initialweight of the herbal powder in grams}}}}~~ \times 100$$

2.3.2 Establishment, inoculation, and insect count

Cochineal insect-free cactus cladodes were collected from the field and established in the laboratory in plastic containers filled with wet soil. The experiment with herbal extracts was conducted both indoors and outdoors to test different herbs at various concentrations. Engorged adult female cochineals (200 in number), along with their white waxy substance containing both adults and eggs, were collected from infested cladodes in the field. These cochineals were then placed on each of the established experimental cladodes indoors and outdoors. The inoculated experimental cladodes were left for 21 weeks (5 months) to achieve a high level of infestation similar to the natural environment. Viable cochineal insects were carefully counted by three individuals before spraying to obtain an average count as pre-treatment data (resulting in different average values for the 27 groups of cacti). Similarly, dead insects were counted and recorded after the application of the experimental spray preparations (post-treatment data), and mortality rates were calculated for each experimental extract solution.

2.3.3 Preparation, application of spray, and determination of LC50 of extracts

Dried crude oil extracts and their combinations, extracted with methanol 99.5%, were prepared at different concentrations (1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%), and then tested against adult cochineals and their eggs. The preparations were applied to established experimental infested cactus cladodes indoors and outdoors. Cochineal colonies were counted before spraying and then sprayed with different extract solutions to determine the lethal concentration 50 (LC50). At the end of week 21 (5 months), insects were carefully counted by three individuals before spraying to obtain pre-treatment data. Similarly, viable insects were counted after spraying (post-treatment data) at 24, 48, and 72 h following application. Mortality rates or percentage reduction (% R) were calculated to determine the efficiency of the extract using the formula provided by Carey [27].

$$\% {\text{ Reduction }}\left( {{\text{mortality}}} \right) = \frac{{{\text{Number of deaths }}\left( {{\text{Pre}} - {\text{treatment}}{-}{\text{Post}} - {\text{treatmentdata}}} \right)}}{{{\text{Total number of insects before spray }}\left( {{\text{Pre}} - {\text{treatmentdata}}} \right)}} \times 100$$

2.3.4 Comparison for efficiency

The effectiveness of herbal extracts was determined based on mortality percentages relative to their corresponding experimental concentrations. Equal amounts of each extract were combined and prepared at different experimental concentrations ranging from 1 to 5% at 0.5% intervals to assess their synergistic effects. Additionally, the LC50 (median lethal concentration) was determined for each herbal extract.

2.3.5 Differentiation between dead and viable cochineal insects and eggs

  1. (i)

    Adult females: Experimentally infested cactus cladodes were sprayed with different experimental spray solutions. Subsequently, the cladodes were tightly covered with a mosquito net featuring a 0.6 mm pore diameter, preventing crawlers from entering or exiting. After 72 h, the cladodes were uncovered and carefully observed for the secretion of a white waxy substance on the black surface of the adult females, indicating their viability.

  2. (ii)

    Adult males and crawling stages: Moving adult male cochineals and crawlers were directly exposed to the spray and observed for any movement indicating recovery for up to 30 min after application.

  3. (iii)

    Eggs: A cochineal-infested cladode was sprayed with distilled water to remove the white waxy materials from the surface of adult females and crawling insects. All female insects were then removed from the cladode surface to expose the tiny gray-colored eggs. The cladodes were placed in shade to protect the eggs from direct sunlight. Subsequently, experimental sprays were applied to a cladode containing only eggs, which was covered with a mosquito net. After 90 days of the insect life cycle, the cladodes were thoroughly observed for insect development. The presence of any developmental stage (refer to Fig. 1) on the cladodes indicated that the eggs had survived the spray's effects and hatched into crawlers, which eventually grew into adults.

Fig. 1
figure 1

A cladode infested with female cochineal and their eggs

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2.4 Observation for re-infestation

Two highly infested cactus cladodes were placed side by side, with one cladode sprayed using the most effective spray preparation and the other left untreated. The sprayed cladodes were observed daily for any signs of re-infestation.

2.5 Stability and persistence of extract

The stability of herbal extract products refers to their ability to maintain their physical, chemical, and insecticidal properties (efficacy) during storage and application on target plants [28, 29]. Monitoring storage period, temperature, and relative humidity is one approach used in stability testing to assess changes in chemical and physical characteristics, as well as any alteration in insecticidal effectiveness over time.

2.5.1 Effect of storage time

The most effective sprayable extract solutions were stored at room temperature, ranging from 27 °C in April to 23 °C to 24 °C in May and June. The relative humidity (RH) during storage fluctuated between 62% (from January to June) and 75% (from July to December).

2.5.2 Physical and pH changes

The extracts were stored for a duration of 24 months, and their efficacy against cochineal insects was assessed at specific intervals: day 0, 1 month, 6 months, 1 year, and 1.5 years of storage. Short-term environmental changes resulting from opening the doors of the storage compartment were acknowledged as unavoidable. Killing percentages were determined at each storage period.

2.6 Skin patch test of the extract solution

The toxicological analysis using the Patch Test Technique (PTT) to assess the acute effects of an herbal extract solution against cochineal insects was conducted following established protocols at the Ethiopian Food, Medicine, and Health Care Administration and Control Authority (FMHACA). The test involved directly applying the final sprayable extract solution onto the skin of laboratory mice as animal models. Small patches containing the test solution were secured onto the skin of the mice using hypoallergenic adhesive tape, with careful consideration to minimize stress and discomfort to the animals. The patches remained in place for 24 h to allow for skin exposure. Following patch removal, the skin sites were closely observed for signs of acute toxicity, irritation, erythema, edema, or other adverse reactions at 24, 48, and 72 h post-exposure. This procedure was conducted by ethical guidelines and animal welfare regulations to ensure the safety and integrity of the study.

2.7 Qualitative phytochemical screening of experimental herbs

Phytochemical analysis was conducted to assess the presence of various secondary metabolites, including terpenoids, phenols, tannins, flavonoids, alkaloids, and saponins, using standard methods (Table 2).

Table 2 Preliminary qualitative phytochemical screening testing methods and observations
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2.8 Data analysis

Analysis of Covariance (ANCOVA) was employed to compare the mean mortality percentages, assess the homogeneity of variance across different levels of the independent variable (experimental herbs), and determine the effect of both the main independent variable and the covariate (concentration of herbal extracts) on the dependent variable (mortality %). A significance level below 0.05 (indicating a higher F-value) was considered statistically significant, indicating a difference in mean mortality among the experimental herbs.

3 Results

3.1 Extraction yield of the experimental herbs

The yield percentages of the herbs were calculated for the different solvents. Among all the experimental herbal materials, relatively higher yield percentages were obtained with methanol (99.8%) maceration, while the lowest yields were obtained from distilled water maceration (Table 3).

Table 3 Yield percentage of the experimental herbs macerated with different solvents
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3.2 Killing efficiency and lethal concentrations of extracts

The mean mortality percentage recorded by the admixture of extracts of S. linnaeanum and N. oleander was about 81%, whereas that of S. linnaeanum alone was 71.8% and N. oleander alone was 68.8% (Table 4). The plant D. stramonium caused the lowest mean mortality at 57.8% (Table 4). The actual insect mortalities of the experimental herbs at different extract concentrations are displayed in Table 5, and the LC50 of the extracts ranges between 1 and 3% (Table 5).

Table 4 Analysis of covariance (ANCOVA) results of mean mortality of herbal extracts
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Table 5 Herbs with their corresponding mortality (%) at different extract concentrations
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The test of homogeneity of variances using Levene’s test showed that there was a statistically significant difference (F (8, 72) = 4.613, p = 0.0001) in the variances of mortality of cochineal insects across the different experimental concentrations of the extracts. The pair-wise comparison tests showed that there is a statistically significant difference (p < 0.05) in the mean mortality of insects among the different experimental concentrations of the herbal extracts except for E. tirucalli and N. oleander (p = 0.184), E. tirucalli and A. Mexicana (p = 0.236), T. vogelii and C. aurea (p = 0.120), T. vogelii and R. communis (p = 0.510), and C. aurea and R. communis (p = 0.366). Levene's Test of Equality of Error Variances table is not given here.

There was statistically significant difference in the mean mortality of insects across the levels of the main effect (experimental herbs) (F (8, 71) = 57.812, p = 0.0001, ƞ2 = 0.867) and the covariate (concentration of extracts) (F (1, 71) = 4198.3, p = 0.0001, ƞ2 = 0.983) with the effect sizes (Partial Eta Squared) of 86.7% and 98.3%, respectively. This tells that the different herbs and their corresponding experimental concentrations have a high effect on cochineal insect mortality (Table 6).

Table 6 ANCOVA tests between subjects’ effects (herbs and concentration) on insect mortality
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3.3 Physical and pH changes and effect of storage time on insect mortality

The admixture of extracts of S. linnaenum and N. oleander was kept at room temperature for 24 h, one month, 6 months, one year, and for a year and a half with mortality of 99.6%, 99.54%, 99.52%, 99.4%, and 99.21%, respectively. This showed that there was no statistically significant (F = 2.132, p = 0.053) difference in the mean mortality of insects on the cactus cladodes sprayed with the admixture. Physical and pH changes through the storage period were determined, and the initial pH values of the spray preparations fell in the acidic (pH = 4.3) and dark green, and there was no change for a year and a half period. The admixture of extracts of Solanum and Nerium plants prevented cochineal re-infestation for 93 days.

3.4 Qualitative phytochemical screening of herbal extracts

The present study carried out on the herbal crude oil extract samples revealed the presence of active metabolites. Tannins were detected in all of the extracts and saponins were found in all of the extracts except in E. tirucalli (leaf) and A. mexicana (leaf and stem) (Table 7).

Table 7 Results of phytochemical analyses of the selected eight experimental plants
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3.5 Skin patch test result

The toxicological analysis using the Patch Test Technique (PTT) assessed the acute effects of a sprayable herbal extract solution against cochineal insects. The solution was applied to laboratory mice via skin patches and monitored for 24, 48, and 72 h post-exposure. No signs of acute toxicity, irritation, or allergic reactions were observed during the observation period.

4 Discussion

The application of synthetic insecticides like organochlorine, organophosphate, and carbamates are major tools in insect pest control programs [36, 37]. Large-scale application of these chemical insecticides affects human and animal health and leaves environmental residues and these chemical insecticides contribute to biological magnification. Moreover, other non-target organisms or beneficial insects get affected and it leads to insecticide resistance [38, 39]. The biological control program is, therefore, an alternative approach and is a sustainable method of insect pest control. Plants produce numerous phytochemicals, many of which have medicinal and pesticide properties like larvicidal and repellent activities against different species of mosquitoes [40,41,42,43,44]. More than 1,200 plant species have been reported with potential insecticidal value [45, 46]. Secondary metabolites of the plants act as a defense mechanism against herbivores and other environmental factors. Insecticidal activities of some groups of phytochemicals such as saponins, alkaloids, tannins, flavonoids, terpenoids, phenols, coumarins, quinines, and steroids have been reported previously from different plant species [39, 40, 47, 48]. Farmers may lack the capacity for methanol extraction to prepare herbal insecticides, suggesting a need for institutions like universities or agricultural bureaus to establish extraction labs for ready-made solutions. This initiative can facilitate access to herbal insecticides for local farmers, aiding in pest control efforts.

The present work investigated the effect of extracts of experimental herbs on the cochineal insect of cactus. In the investigation, the mean mortality percentage recorded by the admixture of fruit extract of S. linnaenum and leaf of N. oleander was about 81%, whereas that of S. linnaenum and Nerium oleander was 71.8% and 68.8%, respectively. Despite the difference in the killing efficacy, methods of extraction and concentration of the herbal extracts used, the present study revealed a positive effect in killing the cochineal insect and studies in the same region by Weldemariam and Welderufael [49] and Fitiwy et al. [8]) who used extracts of hydro-distillation of E. globulus and simple water maceration of N. glauca, respectively, showed similar effect in reducing the population of the insect from experimental cochineal infested cladodes.

The phytochemical screening and qualitative estimation of the plants studied showed that the leaf, seed, and fruit extracts were rich in alkaloids, flavonoids, saponins, phenolics, tannins, terpenoids, and steroids. The ethanol extracts obtained from both leaf and seed in D. stramonium have insecticidal, repellent, acaricidal, and oviposition deterrent properties and this is due to the phytochemical constituents of alkaloids and tannins in it [50] and this was in agreement with the current finding that the extract of Datura to show a killing effect on cochineal insect of cactus. Reegan et al. [44], and Warikoo and Kumar [51] reported that A. Mexicana extract has an effect on insects and this supports the result of the current study that the extract has a killing effect on cochineal insects. The present study shows that the A. mexicana plant extracts have the potential for the development of new and may be safe control products for cochineals. As naturally occurring insecticides, these plant-derived materials could be useful as an alternative to synthetic insecticides.

In the present study, leaf extract of C. aurea 5% caused a mean mortality of 92.8% which is similar to the findings of Hiruy and Getu [52], and Abubeker et al. [53] that the plant extract has an insecticidal effect on agricultural insect pests. Mwine and Van-Damme [54] reported that the extract and latex of E. tirucalli have insecticidal, nematicidal, piscicidal, and molluscicidal features and this finding is in agreement with the current study showed a killing effect on the cochineal insect of cactus. The finding of Kodjo et al. [55] is similar to the current study in that the extract of R. communis has a bio-insecticidal effect. Extracts from leaves and seeds of castor bean R. communis L. (Euphorbiaceae) have been used successfully in the management of curculionids of agricultural importance [56]. They cause death by ingestion and contact (which include clogging of the respiratory spiracles) or repel insects [57]. Ethanol extracts have been shown to have biological activity against insects [58].

The present study revealed that extract of Tephrosia vogelii caused high mean mortality (91%) of cochineal insects and similarly Kerebba et al. [59] and Alao and Adebayo [60] noted that it has an insecticidal effect on different agriculture pests. Moreover, WHO [61] reported that the higher mortality of agricultural insects is because of the lipophilic contents of the plant extract that is easily taken up through the respiratory spiracles. Medicinal and or toxic herbal extracts have different phytochemical constituents among which alkaloids, flavonoids, saponin, phenolics, tannins, terpenoids, and steroids have insecticidal and antimicrobial effects [62], which is in agreement with the present study findings that the phytochemical contents of the experimental herbs had an interesting killing potential on the cochineal insect pest of the cactus plant. In the present investigation, the efficacy of the extracts doesn’t change with the storage periods, and this finding is similar to the study by Meena et al. [63] that shows no change in efficacy due to storage time and sunlight exposure of extracts. The present study showed three months of prevention of cochineal re-infestation of cactus plants by the herbal extracts, and a similar finding was presented by Montanucci [64] on cactus mealy bugs.

5 Conclusion and recommendations

The wild type of the cochineal insect, D. opuntia-ficus biotype, in the Tigray region, can be effectively controlled using environmentally friendly herbal extracts, particularly those derived from S. linnaenum fruit and N. oleander leaf, either individually or in combination with a 5% concentration. However, it is important to note that farmers may not have the capacity to undertake methanol extraction to prepare these herbal insecticides themselves. Instead, local governmental institutions such as universities or agricultural bureaus could establish extraction laboratories to provide ready-made herbal insecticides to local farmers.

Therefore, to address the issue of cactus pest management effectively, there needs to be a comprehensive approach involving education and awareness among farmers about the nature of the insect pest and the available control methods. Local institutions should take the lead in establishing extraction facilities and distributing herbal insecticides to farmers. Additionally, farmers should be encouraged to adopt sustainable pest management practices, including creating buffer zones, manually removing infested cacti, and using organic insecticides. Collaboration between farmers, governmental institutions, and agricultural stakeholders is crucial to managing the cochineal insect pest in the region. By utilizing locally available herbal resources and establishing extraction facilities, sustainable control measures can be implemented to protect cactus plantations and ensure food security for the community.