Effect of abamectin and profenofos insecticide on stingless bee, Tetragonula laeviceps Smith (Hymenoptera: Apidae: Meliponini)

Pollinators such as the bee Tetragonula laeviceps Smith are essential components of ecosystems that provide services to plants. However, the massive use of pesticides in crops can affect the existence of pollinating bees and even cause a decrease in bee populations in the field. This study aimed to determine the effect of abamectin and profenofos insecticides on T. laeviceps. The methods used in the test were bee attractiveness bioassay, toxicity test of dermal application (topical and residual), and oral application with the abamectin and profenofos. The results of the bioassay showed that bees were more attracted to the aroma of the insecticide abamectin 180 ppm than profenofos 1500 ppm. The recommended concentrations of abamectin and profenofos in the field were 180 and 1500 ppm, respectively, so this topical and oral application showed a high level of danger to bees because it can kill >80% of bees. The LD50 values of the insecticides abamectin (0.0064 ppm) and profenofos (0.0345 ppm) were classified as highly toxic based on EPA classification. Therefore, insecticides with active ingredients, particularly abamectin and profenofos, are harmful to T. laeviceps.


Introduction
Pollinators are an essential component of biodiversity, namely as providers of ecosystem services for plants [1].Plants and insect pollinators have a mutually beneficial relationship (mutualism); insects obtain food (nectar and pollen), used as a food source in their colony, while plants receive assistance from insects in pollination.One of the pollinating insects in Indonesia that has the potential to be used as a pollinator is the stingless bee, Tetragonula laeviceps (Hymenoptera: Apidae: Meliponinae) [2].This bee has a relatively small size (3.5-4.0 mm), which allows it to enter the flower.
Open pollination produces the highest percentage of fruit set compared to wind and self-pollination.Open pollination by T. laeviceps can increase crop yields 2 to 3 times compared to wind pollination and 4 to 5 times compared to self-pollination [3].Cucumber plants with bee pollination can increase fruit set success by 53.93%, human-assisted pollination produces 46.79% of fruit, and wind pollination produces 11.43% of fruit from all flowers.This shows that pollination with the help of bees can increase the quality and quantity of fruit [4].
Agricultural crop production requires the role of pollinator.Unfortunately, in the last few decades, the use of pesticides has been suspected of causing mass deaths of pollinating bees, also known as the colony collapse phenomenon [1].Neuman and Carreck [5] stated that honey bees (Apis mellifera) 1346 (2024) 012027 IOP Publishing doi:10.1088/1755-1315/1346/1/012027 2 experienced an average colony mortality of 30% in the United States and 85% in the Middle East.Insecticides containing the active ingredient abamectin (avermectin, group 6) impact Scaptotrigona bees, causing the bees' foraging activity to be disrupted [6].Apart from that, the insecticide profenofos (organophosphate group) is also toxic to Apis mellifera bees and causes their death within a short time after exposure to the insecticide [7].The impact of using the pesticides abamectin and profenofos on stingless bees, such as T. laeviceps, is unknown.Therefore, this study aimed to determine the effect of insecticides containing the active ingredients abamectin and profenofos on T. laeviceps.

Experimental materials
The T. laeviceps were obtained from Banten, West Java-Indonesia beekeepers.Worker bees (foragers) were used for experimental studies.Olfactory and toxicity bioassays were carried out using commercial insecticide products of abamectin 18 g/l and profenofos 500 g/l.

Olfactory bioassay
The bioassays can be done in several ways, such as testing with live plants, flowers, prey, or other aroma.Olfactory bioassay was used to evaluate the attractiveness of bees to insecticides, water, and honey.The olfactometric response test method for two insecticides used a Y-tube olfactometer (left and right Y arms 10 cm, bottom side 9 cm, diameter 2 cm).The odor source is an insecticide containing the active ingredients abamectin 180 ppm (ABA) and profenofos 1500 ppm (PRO).The test solution was then dropped 200 µl into foam measuring 2 cm x 2 cm x 2 cm, which was put into a glass tube connected to the end of the Y-tube olfactometer arm.The glass tube (diameter 5 cm, height 10 cm) was covered with gauze, and then the Y-olfactometer arm's base was connected to the pump's hose (Dyna Pump Model 3).A flowmeter (Gilmont GF 6541-1215) was installed between the olfactometer and the pump to check the air flow rate.The bee was placed at the end of the olfactometer arm, and the flowmeter was run at a 40 ml/min flow rate.Tests were carried out at room temperature (± 26 •C).The test comprises ten treatments divided into three subtests (Table 1).The bees in the previous test were treated for 2 hours without food.Each treatment used 15 bees (as replication), which were acclimatized for 2 hours in a gauze cage (30 cm x 30 cm x 30 cm) before treatment.
The bees of T. laeviceps were acclimatized before treatment by placing them inside a test tube (25 mm in diameter; 150 mm in height) for one hour and anesthetized in a refrigerator at -18℃ for 3 minutes.Each concentration of insecticide (1 µl) was applied to the dorsal-thorax of the bees using a micro syringe applicator [8].Each treatment was replicated three times with ten bees per replication.After treatment, the test bees were placed in a glass tube (d: 100 mm, h: 150 mm) and fed with 10% honey solution.Mortality of bees was observed at 48 hours after treatment (HAT).

Residual application
The concentrations of abamectin and profenofos tested were similar to those in the topical application test.Acetone was used as a solvent.The bioassay method used refers to Syahputra [9].The test solution (0.5 ml) was poured into a glass tube (d: 25 mm; h: 150 mm), and then the inner surface of the glass tube was evenly coated with the test solution by rolling the glass tube until it was even.After that, the tube was dried until the solvent evaporated.A total of 10 bees were exposed to each treated glass tube for 5 minutes, transferred into a new rearing tube, and fed with a 10% aqueous honey solution absorbed in cotton wool.Each treatment was repeated three times.Bee mortality was observed at 48 HAT.

Oral application
The concentrations of abamectin and profenofos tested were similar to those in the topical application test.The insecticide was diluted with a 50% aqueous honey solution.Ten bees (T.laeviceps) were fasted for 1 hour before treatment in the tube (d: 25 mm, h: 150 mm).Then, the bees were treated with each treatment concentration by placing a cotton swab containing each insecticide concentration at the top of the tube.An aqueous honey solution (50%) was used as a control.The bees were allowed to feed for 5 minutes.After that, all cotton containing insecticide was discarded and replaced with untreated cotton.Bees that have been treated were kept and fed a 10% honey solution absorbed by cotton.Each treatment was repeated three times.Mortality observations were carried out in 48 HAT.

Data analysis
Test result data is tabulated using the Microsoft Excel 2016.The chi-square test at the 5% level was used to analyze significant differences in odor source selection in the repellency test.Data on bee mortality in topical application tests, oral exposure, and residual effects were analyzed using probit to obtain LC50 and LD50 values using the POLOplus application.The LD50 value is used to classify the level of insecticide toxicity to bees based on US EPA [8] (Table 2).

Attractiveness of abamectin and profenofos to T. laeviceps
The bees (T.laeviceps) rely on their sense of smell (olfactory) when looking for food.Bees' food sources are nectar and pollen [10].Nectar is a sweet liquid containing sugar produced by flowers.Nectar is used as a source of carbohydrates, while flower pollen is used as the primary source of protein, lipids, IOP Publishing doi:10.1088/1755-1315/1346/1/0120274 minerals, and vitamins bees need.The nectar will later be processed into honey, which the honey bee colony uses as a food reserve [11].The results of the olfactory bioassay of the insecticides abamectin 180 ppm and profenofos 1500 ppm on honey showed that T. laeviceps were more attracted to honey than insecticides (Figure 1).Bioassay of insecticide mixtures with honey, both abamectin 180 ppm and profenofos 1500 ppm, showed that T. laeviceps responded more strongly to the insecticide added with honey (Figure 1-P1).Testing the insecticide abamectin 180 ppm against aquades showed that the response of T. laeviceps was more robust to aquades.Meanwhile, testing profenofos 1500 ppm, abamectin 180 ppm in 10% honey solution, and profenofos 1500 ppm in 10% honey solution against aquades showed no significant results (Figure 1-P2).Testing of the insecticide abamectin 180 ppm against profenofos 1500 ppm showed no significant results, with the percentage of interest of T. laeviceps to abamectin 180 ppm at 53.33%, while the percentage of interest of T. laeviceps to profenofos 1500 ppm was 46.67%.However, an olfactory bioassay of the insecticide mixture with honey showed that adding honey to the insecticide abamectin 180 ppm could increase bee attraction by up to 66.67% (Figure 1-P3).
In general, the test results showed that adding honey to the test insecticide increased the attractiveness of bees to the insecticide.Honey has an aroma and composition similar to nectar in plants.If the nectar is exposed to insecticide, it will threaten bees because bees will continue to visit to take nectar that has been contaminated with insecticide.Honey is the result of collecting plant nectar by bees, then processed enzymatically by bees.Honey contains carbohydrates in the form of fructose sugar (around 38.5%), glucose (around 31.0%),maltose, sucrose, and other sugars [12].Nectar contains sucrose, which will be destroyed by the invertase enzyme and broken down into simple sugars, namely fructose and glucose [13].

Mortality of T. laeviceps to abamectin and profenofos
The insecticides abamectin and profenofos are contact and gastric poisons, so they can kill bees through physical contact or by inserting the insecticide into the digestive tract.Abamectin (group 6) is a contact and gastric poison insecticide neurotoxin [14].Bees that eat flowers contaminated with insecticides can be exposed to the insecticide and die.Bees will bring food (nectar and pollen) into the hive to feed the colony so the entire bee colony can be exposed to insecticide.Bees have trophallaxis behavior, namely giving each other liquid food orally between colony members [15].So, if the food given to the colony (nest) is contaminated with insecticide, it can spread to all bee colony members.

Figure 2. Toxicity of abamectin (ABA) and profenofos (PRO) to Tetragonula laeviceps in topical (A), residual (B), and oral application (C).
Insecticides with the active ingredient abamectin showed high mortality rates at lower concentrations compared to insecticides with the active ingredient profenofos.In topical application, the insecticide containing the active ingredient abamectin was more deadly than the insecticide profenofos.The mortality of bees to profenofos and abamectin caused 90% at 150 and 13.5 ppm, respectively (Figure 2A).Meanwhile, the results of testing the insecticides abamectin and profenofos against T. laeviceps bees using the residue test method showed different results.The profenofos insecticide using the residual method caused higher mortality compared to abamectin (Figure 2B).The recommended concentration of abamectin (180 ppm) using the residue test method was 13.33% lethal to bees.When the concentration of abamectin insecticide was increased to 1080 ppm, the mortality of bees was 100%.The results of this test indicate that abamectin insecticide residues in the field tend to be less harmful to bees.
According to Djojosumarto [16], insecticides with the active ingredient abamectin quickly degrade phytochemically in the environment, reducing insecticide toxicity.
The results of the oral application test were similar to those of the topical test.Bee mortality in the oral test showed a higher bee mortality rate with the insecticide abamectin than with the insecticide profenofos (Figure 2C).The profenofos insecticide caused more than 80% of bee deaths at 600 ppm, while the abamectin insecticide caused death at 1.8 ppm.In oral tests, insecticides containing the active ingredient profenofos showed low mortality values of up to 75 ppm.They increased bee mortality against the insecticide profenofos at 150-600 ppm concentrations.The recommended concentrations of abamectin and profenofos in the field are 180 and 1500 ppm, respectively, so this oral test shows a high level of danger to bees because it can kill >80% of bees.Topical (dermal) bioassay occurs on bees when the insecticide directly touches the bee's body (Figure 3A).In the field, bees can be exposed to insecticides through contact when farmers spray insecticides on bee-infested plants.T. laeviceps lands on flowers longer in the morning than in the afternoon and evening [17].In addition, spraying insecticides in the field can contaminate nectar and pollen in plant flowers, as illustrated in the oral test (Figure 3B).Nectar contaminated by previous spraying will be taken by bees to be used as a food source for the bee colony.Insecticides do not directly contaminate bees, but bees are exposed to insecticides from nectar collected by bees from flowers in plantations.One of the advantages of synthetic insecticides is that they have high persistence properties.Insecticide persistence is the period required for a pesticide to reduce its working capacity in the soil to 0%.Spraying insecticides on plantations can leave insecticide residues, which are then ingested or stuck to bees' body parts, as shown in the residue effect test (Figure 3C).

Toxicity of abamectin and profenofos to T. laeviceps
The insecticide abamectin is an allosteric modulator of glutamate (GluCl)-gated chloride channels [14].Abamectin can disrupt the nervous system and work by stimulating amino acid receptors, which can increase the entry of chloride ions into nerve cells, causing insects to become paralyzed, stop eating, and then die [18].The insecticide profenofos works as an inhibitor of the enzyme acetylcholinesterase (AChe) [14].The AChe enzyme is an enzyme that is located in many places in the central nervous system.The nervous system regulates nerve transmission by catalyzing the hydrolysis process of the neurotransmitter acetylcholine into acetic acid and choline [19].
Abamectin had high toxicity in tests with the topical and oral application of 6.37 and 1.12 ppm, respectively.LC50 values in topical and oral tests are equivalent to 28 and 160 times more toxic than the recommended concentration (180 ppm).However, based on the residue test results, the LC50 value is deficient (522.65 ppm).According to Mahafarisoa [20], the insecticide abamectin can cause sublethal effects in the form of digestive disorders that affect the health and vitality of the colony.The residual effect has a trim level of toxicity because the insecticide content is volatile, so the insecticide tends to be less dangerous.High temperatures can cause spray droplets applied to plants to evaporate quickly, resulting in reduced residue on plants [21].
Meanwhile, the profenofos were very toxic in all three test methods compared to the recommended concentrations (Table 3).Sequentially, the LC50 values of profenofos against T. laeviceps were 39.29, 289.94, and 210.51 ppm (topical, residual, and oral application).The LC50 value in the topical test was 39.29 ppm, or the equivalent of 38 times lower than the recommended concentration (1500 ppm).The insecticide solution that is dripped on the dorso-thorax when applied causes the bees' motor nerves to be disturbed.Organophosphate (sub-group 1B) inhibits AChE (Acetylcholinesterase inhibitor), an enzyme that catalyzes the hydrolysis process of the transmitter acetylcholine.Acetylcholinesterase (AChE) reacts chemically with insecticides, resulting in AChE being unable to fulfill its function, resulting in the gradual accumulation of neurotransmitters at nerve synapses or muscle connections and continuous stimulation of nerve or muscle fibers [22].Abamectin and profenofos, based on the EPA classification (2014), have LD50 values , which are classified as very toxic insecticides (Table 4).Insecticides with high toxicity value are dangerous for bees and can cause high mortality.Profenofos has an LD 50 value against honey bees (Apis mellifera) of 24, 48, and 72 JSP, respectively, 1.25, 0.93, and 0.61 µg/bee [10].In addition, the LD50 value of the insecticide abamectin is 0.70 mg/ml [6].Meanwhile, in the testing of Mahefarisoa et al. [20], abamectin has an LD50 of 0.011 µg/bee in the oral test and 7.8 µg/bee in the contact test The use of insecticides does not only impact the existence of bees.Al-Zaidi et al. [23] stated that the use of pesticides can be a source of air, water, and soil pollution, which can harm human health and living organisms in the environment.In the long term, insecticides harm ecological systems and endanger human health.Pesticides can pollute rivers, lakes, and oceans, polluting the natural environment and food chain [24].
The profenofos as an organophosphate group does not cause environmental pollution in the long term.Insecticides with the active ingredient profenofos are more toxic to vertebrates.The persistence of profenofos residues does not last long, so chronic environmental poisoning tends not to occur because environmental factors quickly degrade profenofos compounds into non-toxic components [25].Meanwhile, the degradation period for organophosphates in the environment is around two weeks [26].

Conclusion
Insecticides containing the active ingredients abamectin 180 ppm and profenofos 1500 ppm affect T. laeviceps bees.The odor bioassay of the aromatic insecticide abamectin 180 ppm was preferred compared to profenofos 1500 ppm.Based on mortality tests, the recommended concentrations of topical (contact) and oral insecticides abamectin and profenofos were very dangerous for bees because they can kill 100% of the bees but tend to be harmless when exposed to the residue.Based on the EPA 1346 (2024) 012027 IOP Publishing doi:10.1088/1755-1315/1346/1/0120278 classification, the LD50 value of abamectin and profenofos was included in insecticides with high toxicity to bees.

Figure 3 .
Figure 3.A simple conceptual model for pesticide exposure in the field based on mortality testing with topical (A), oral (B), and residual effect (C).

Table 1 .
Sources of aroma treatments for olfactory bioassay.

Table 2 .
Toxicity classification to bees based on LD50.

Table 4 .
Contact toxicity of abamectin and profenofos to Tetragonula laeviceps based on EPA classification.