Control of Varroa destructor in honey bees (Apis mellifera) with the aqueous extract of Petiveria alliacea and identification of its constituents by LC–MS

Controle de Varroa destructor em abelhas melíferas (Apis mellifera) com extrato aquoso de Petiveria alliacea e identificação de seus constituintes por LC-MS

Abstract

The aqueous extract of Petiveria alliacea L. (Caryophyllales, Phytolaccaceae) was found to control varroosis caused by Varroa destructor in honey bees (Apis mellifera). Adult female V. destructor mites were immersed in solutions of the aqueous extract at different concentrations (24, 60, 100, 140, 180 and 200 mg L^-1). After 24h of exposure, mortality was assessed and the median lethal concentration (LC₅₀) was estimated to be 105.2418 mg L^-1. In the direct contact acute toxicity experiments, 0.5 µL of each of the above extract solutions was applied to the thoraces of A. mellifera, and the estimated LC₅₀ was 165.4156 mg L^-1, demonstrating the minimal risk of the extract to this insect. Furthermore, the LC₅₀ in A. mellifera after residual contact was 104.904 mg L^-1, indicating a greater risk of toxicity. Furthermore, oral toxicity, determined by incorporating various concentrations of the extract (12, 30, 60, 75 and 92 mg L^-1) into the diet for 24 h, 48 h, 72 h and 96 h of exposure yielded LC₅₀ values of 153.1455, 12.0472, 50.5723 and 73.7513 mg L^-1, respectively, suggesting that ingestion poses the greatest risk of toxicity to bees. Finally, liquid chromatography–mass spectrometry (LC–MS) analysis revealed the presence of 16 compounds in the extract, of which 14 were identified, and the major compounds were two proline derivatives, trans-N-methyl-4-methoxyproline and N,N-dimethylproline, followed by N-α-tert-butyloxycarbonyl-valylproline-methyl ester. These results show the promising application potential of P. alliacea for the control of varroosis, but field bioassays are needed to establish the efficiency and possibility of using the acaricidal capacity of this plant to control V. destructor.

Keywords:
bioassays; botanical biopesticide; aqueous extract; LC₅₀; chromatography

Resumo

Foi demostrado que o extrato aquoso de Petiveria alliacea L. (Caryophyllales, Phytolaccaceae) controla a varroose causada por Varroa destructor em abelhas melíferas (Apis mellifera). Fêmeas adultas do ácaro V. destructor foram submersas em soluções do extrato aquoso em diferentes concentrações (24, 60, 100, 140, 180 e 200 mg L^-1). Após 24 h da exposição, a mortalidade foi avaliada e a concentração letal média (CL₅₀) foi estimada em 105,2418 mg L^-1. Nos experimentos de toxicidade aguda por contato direto, 0,5 µL de cada uma das soluções do extrato acima indicadas foram aplicadas no tórax de A. mellifera. Assim, a CL₅₀ estimada foi de 165,4156 mg L^-1, demonstrando risco mínimo do extrato para esse inseto. Em adição, a CL₅₀ em A. mellifera após contato residual foi de 104,904 mg L^-1, indicando maior risco de toxicidade. A toxicidade oral, determinada pela incorporação de várias concentrações do extrato (12, 30, 60, 75 e 92 mg L^-1) na dieta por 24, 48, 72 e 96 h de exposição, produziu valores de CL₅₀ de 153,1455; 12,0472; 50,5723 e 73,7513 mg L^-1, respectivamente, sugerindo que a ingestão representa o maior risco de toxicidade para as abelhas. Finalmente, a análise por Cromatografia Líquida-Espectrometria de Massas (LC-MS) revelou a presença de 16 compostos no extrato, dos quais 14 foram identificados, e os principais compostos foram dois derivados da prolina − trans-N-metil-4-metoxiprolina e N,N-dimetilprolina −, seguidos por N-α-terc-butiloxicarbonil-valilprolina-metil éster. Estes resultados mostram o potencial promissor de aplicação de P. alliacea para o controle da varroose, mas bioensaios de campo são necessários para estabelecer a eficiência e a possibilidade de usar a capacidade acaricida desta planta para controlar V. destructor.

Palavras-chave:
bioensaio; biopraguicida botânico; extrato aquoso; CL₅₀; cromatografia

1. Introduction

Varroasis is a health problem caused by mites (Varroa destructor) that occurs bee colonies and constitutes the greatest threat to the development of beekeeping worldwide. The impact V. destructor has been of such great magnitude that no other parasite has generated so many studies, applied research and monetary investments than that of any other pest since the total collapse of bee colonies occurs withing two to three years after contracting varroosis (Anderson and Trueman, 2000; Rosenkranz et al., 2010).

V. destructor is globally recognized as the principal driver of honey bee colony losses. Once attached to the body of the bees, the mite extracts essential nutrients, suppresses immune function, and transmits viruses, particularly Deformed Wing Virus (DWV). Ultimately, its continuous parasitic activity leads to severe weakening and colony collapse, an epidemic phenomenon documented across multiple regions of the world (Jeyapriya et al., 2025). Molecular evidence clearly demonstrates that the mite V. destructor disrupts honey bee immunity, metabolism, and neuronal function, thereby promoting the proliferation of associated viruses—most notably Deformed Wing Virus (DWV). This interaction triggers epidemic-level outbreaks within colonies, leading to behavioral alterations, reduced resilience, and ultimately colony collapse (Morfin et al., 2023; Warner et al., 2024).

However, until now, varroosis has not been able to be eliminated, considering that V. destructor, the parasite that most impacts Apis mellifera colonies, is an obligatory parasite that can attack during different stages of its development, which has become a main reason for the drastic decreases in the numbers of beekeepers and honey bee colonies and cross pollination in Europe, Peru and other regions of the world (Dávila and Ortiz, 1987; De la Rua et al., 2009; Hristov et al., 2020; Morfin et al., 2023).

The search for a sustainable solution to control V. destructor has led to the consideration of plant resources. One such plant resource of interest is mucura (P. alliacea), a perennial and herbaceous plant that is native to the Amazon rainforest and is widespread in Peru. It has been proven that P. alliacea extracts can function as botanical biopesticides, exhibiting both insecticidal and acaricidal properties (Johnson et al., 1997; Rosado-Aguilar et al., 2010, 2017).

The acaricidal activities of the crude extracts, fractions, essential oils and their components of P. alliacea have been demonstrated against the larvae and adults of the cattle tick Rhipicephalus (Boophilus) microplus and the two-spotted spider mite (Tetranychus urticae) (Rosado-Aguilar et al., 2010; Neves et al., 2011; Arceo-Medina et al., 2016; Flota-Burgos et al., 2021).

A recent publication that evaluated the toxicity of extracts obtained from P. alliacea against neonates of Daphnia magna used as a bioindicator revealed that the aqueous extract was the least toxic. Thus, given the importance of protecting the environment, the use of this aqueous extract as an acaricidal agent represents a sustainable alternative (Bracho-Pérez et al., 2024).

The objective of this work was to evaluate the use of the aqueous extract of P. alliacea to control the V. destructor mite, which damages honey bee (A. mellifera) colonies, as well as to study its chemical constituents via liquid chromatography–mass spectrometry (LC–MS).

2. Materials and methods

2.1. Plant material

P. alliacea leaves were collected in the district of Castillo, province of Leoncio Prado, in the Department of Huánuco (09° 16 'S, 76° 00' W), in August 2016. A sample was placed in the Herbarium of the National Agrarian University La Molina with voucher number 012-2019-HM-UNALM.

2.2. Biological material

2.2.1. V. destructor (Anderson and Trueman)

Adult female varroas were collected from hives with varroosis in an apiary containing 20 colonies via the roller method with the use of powdered sugar according to Dietemann et al. (2013).

2.2.2. A. mellifera Linnaeus

The bees that were used in this experiment were from 10 European honey bees (A. mellifera) colonies that are part of a healthy apiary and were free of varroosis and had not been subjected to any disease treatments. In addition, the apiary is located in a healthy ecosystem away from industrial and environmental pollution. The hives that are in this apiary are of the American standard type with a single body.

2.3. Methodology

2.3.1. Acute toxicity to V. destructor determined by immersion

The acute toxicity of the P. alliacea extract was determined via the use of adult female V. destructor mites collected from adult honey bees using the roller method with powdered sugar. Mites were collected before each toxicity bioassay. Contact toxicity bioassays were performed by exposing the mites to six different concentrations, ranging from 2.4% (24 mg L^-1) to 20% (200 mg L^-1), of the P. alliacea extract selected for 24 h (Dietemann et al., 2013).

In this bioassay, groups of five mites were immersed in the P. alliacea extract at various concentrations in glass vials followed by storage for a long duration to evaluate possible time-dependent acaricidal activity. Each group consisted of triplicate samples, including the control treatment and solvent control groups. Adult female mites were kept in a dark incubator at 34 °C and were observed with a stereoscope at the end of the exposure periods. Three types of mites were considered to evaluate final and effective lethality: 1. mobile varroas with coordinated or uncoordinated movement after stimulation; 2. paralyzed varroas with movement of one or more appendages; and 3. dead varroas without stimulation requirements (Dietemann et al., 2013).

2.3.2. Acute toxicity to A. mellifera

To estimate the acute toxicity of the P. alliacea extract to honey bees, individual bees were collected from the brood frames of a bee hive and kept in the dark overnight in a 50% sucrose solution at 34 °C. After harvesting, the bees were exposed to the P. alliacea extract by three methods: i. direct contact using a microapplicator; ii. residual contact; and iii. oral exposure by incorporation into the diet. The bioassays were carried out by exposing the bees to six different concentrations of the P. alliacea extract for the established durations.

1. Direct contact bioassay using a microapplicator

The direct contact bioassays were performed using serial dilutions (2.6–20% for a total of six concentrations) of the extract in the chosen solvent. The solutions (0.5 µL each) were topically applied to the dorsal thorax of the insects using a Burkard Arnold microapplicator. Control treatment consisted of applying 0.5 µL of solvent. Ten insects were treated with each concentration of extract or the solvent control, and the experiment was repeated six times. The treated and control insects were transferred to glass vials, which were incubated at 29-30 °C with 70–80% relative humidity, and mortality was evaluated after 24 h. The data were subjected to a probit analysis to determine the LD₅₀ values (Sakuma, 1998; Vu, 2016).

1. Residual contact bioassay

The methodology proposed by the United States Environment Agency (USEPA, 2012) was applied, with modifications. Bees were anesthetized with carbonic anhydride for 10 s, after which 10 individuals were placed in glass Petri dishes lined internally with filter paper that had been pretreated with the P. alliacea extract at concentrations ranging from 2.4% (24 mg L^-1) to 20% (200 mg L^-1) and dried by evaporation for 1 h. The bees were kept in environmental chambers at 34 °C and provided 50% sucrose syrup. Lethality was considered the endpoint for each bioassay, considering individuals that were unable to stand on their own, after 24 h of exposure.

1. Oral toxicity bioassay by incorporation into the diet

OECD test no. 213 was applied to determine the acute oral toxicity of the extract to both young and healthy adult A. mellifera collected in the morning. The bees were placed in a stainless steel mesh cage (40 cm × 20 cm × 20 cm), provided ad libitum access to an aqueous solution of sucrose (500 g L^-1, 50% w/v) in glass capillary tubes (50 mm long × 10 mm wide). The bees were deprived of food 2 h before the toxicity tests, and those that died were replaced before the experiment. The experiment was conducted in the dark at 25 ± 2 °C with a relative humidity of approximately 50–70% (OECD, 1998).

The aqueous extract was lyophilized until a dry powder, which was considered the ai, was obtained. Different amounts of the powder were mixed with a sucrose solution to obtain five ai solutions with concentrations of 12 mg L^-1, 30 mg L^-1, 60 mg L^-1, 75 mg L^-1 and 92 mg L^-1 supplied to the bees at a dose of 120 µL until they were entirely consumed, after which the sucrose solution was supplied alone ad libitum. Each bioassay was performed in triplicate. Mortality was recorded at 24 h, 48 h, 72 h, and 96 h, in which endpoint was recorded for those individuals who were completely immobile (OECD, 1998).

2.3.3. LC–electrospray ionization (ESI)–MS analysis

2.3.3.1. LC system conditions

Each sample was dissolved in 100 µL of methanol (MeOH) and analyzed on an Agilent 1200 Rapid Resolution high-performance liquid chromatography (HPLC) system connected to a Bruker maXis mass spectrometer. A Waters Atlantis T3 column (4.6 × 100 mm, 5 µm particle size) was used for component separation. The mobile phase was composed of two solvents, solvent A (H₂O:ACN (90:10)) and solvent B (H₂O:ACN (10:90)), both of which contained 13 mM ammonium formate and 0.01% trifluoroacetic acid (TFA). Gradient elution was performed as shown in Table 1. The injection volume was 2 µL.

Table 1
Gradient elution profile of the P. alliacea aqueous extract.

Time (min)	Solvent (%)		Flow rate (mL min-1)
	A1	B2
0.0	100	0	0.5
14.0	0	100	0.5
17.0	0	100	0.5
17.1	100	0	0.5
20.0	100	0	0.5

• ¹
  H₂O:ACN (90:10);
• ²
  H₂O:ACN (10:90).

2.3.3.2. MS conditions

Mass spectra were acquired in positive ESI mode with the following parameters: capillary voltage, 4 kV; drying gas flow, 11 L min^-1; nebulizer temperature, 200 °C; and nebulizer pressure, 2.8 bar.

2.3.3.3. LC–MS data analysis and processing

The chromatographic data were processed using Bruker's internal algorithm to extract the components, and the most intense peaks, both in the positive mode total ion chromatogram (TIC) and by absorbance at 210 nm, were selected for exact mass and molecular formula determination. The retention time and exact mass combinations were used as the criteria when searching the high-resolution mass spectrometry database and the Chapman & Hall dictionary of natural products, as well as other specialized primary sources such as the Base US National Institute of Standards and Technology (NIST) Standard Reference Data Number 69, NIST Chemistry WebBook and the open access PubChem Chemical Database of the US National Institute of Health. The official atomic weights of the chemical elements were updated by the researchers of the Committee of the Inorganic Chemistry Division of the IUPAC (National Institutes of Health (NIH)) (Petersen and Amstutz, 2008; Buckingham et al., 2015; Wallace, 2023; Kim et al., 2020; Meija et al., 2016).

2.3.4. Plant material processing and extract preparation

The sample was processed and the aqueous extract of P. alliacea leaves was obtained according to previously published experimental methods (Bracho-Pérez et al., 2024).

2.4. Statistical analysis

The acute toxicity bioassay results were processed to determine the median lethal concentration (LC₅₀), expressed in mg L^-1, with a confidence level of 95% (α = 0.05). Statistical significance and probit regression analyses were carried out by determining the angular coefficients of the slopes, chi-square (X²) values, and heterogeneity using the statistical program PoloSuite. These analyses determination of an appropriate effective concentration of the extract that would cause the least environmental impact (LeOra Software LLC, 2016; Robertson et al., 2017).

3. Results and discussion

3.1. Acute toxicity to V. destructor determined by immersion

Analyzing the plot of the bioassay results in Figure 1a, it can be observed that doses of P. alliacea extract above 140 mg L⁻¹, V. destructor mortality equaled or exceeded 80%, whereas at a dose of 100 mg L⁻¹, mortality reached values below 50% (46.66%). However, it is important to compare these data with those acquired via field assays.

Figure 1
Dose‒response curve of (a) V. destructor mortality caused by the P. alliacea aqueous extract and the exponential (b), linear (c) and logarithmic (d) segments.

Probit regression analysis revealed that the LC₅₀ was 105.2418 mg L^-1 with a 95% confidence interval of 97.092-115.6 mg L^-1 (Table 2), and this range contains the maximum acceptable concentration that guarantees the sustainable development of biopesticide. A previous study with D. magna determined 100 mg L^-1 P. alliacea aqueous extract as a suitable concentration (Bracho-Pérez et al., 2024).

Table 2
LC₅₀ values and 95% confidence intervals (mg L^-1) obtained from the Varroa destructor acute toxicity bioassay.

Extract P. alliacea	Varroa destructor	LC50 (mg L-1)	AC ± SE		DF	Heterogeneity
Aqueous	Susceptible	105.2418 (97.092-115.6)	5.1872 ± 0.0057	0.6007	4	0.1502

• AC, angular coefficient; SE, standard error of the mean; X², chi-square, DF, degrees of freedom.

The fit to the probit model was optimal, as indicated by the angular coefficient of the slope being greater than 1.96, showing that the response detected by probit analysis was significant, as was the reduction in V. destructor infestation upon application of the P. alliacea aqueous extract. In addition, the heterogeneity value was less than 1.0, showing the validity of the dose–mortality relationship and therefore the validity of the bioassay results (Robertson et al., 2017).

The dose–mortality regression curve constructed from the data from this bioassay (Figure 1) constitutes one of the possible responses of an organism upon the application of a chemical control agent. The probit analysis (Figure 1a) suggested three possible mathematical trends (Figure 1b, 1c, 1d), wherein at the beginning of treatment with the lowest two doses (24 and 60 mg L^-1), the effect was small, with mortality rates of 6.63% to 20%, followed by an exponential trend, indicating that the mite was resistant to the dose used.

At the third lowest dose (100 mg L^-1), the curve was linear with the highest quadratic correlation coefficient value (R² = 0.9597), and mortality increased rapidly to greater than 46.66% until the final stage of treatment, that is, the final portion of the curve (Figure 1a) exhibits logarithmic behavior. At the three highest doses (140, 180 and 200 mg L^-1), the mortality rates were the highest but the differences among them were the smallest. However, it is evident that, on the basis of the V. destructor mortality data, dosages of the P. alliacea aqueous extract in the range of 100 mg L^-1 to 200 mg L^-1 are needed to significantly reduce varroa infestation, adequate concentration of the aqueous extract of P. alliacea capable of causing the most intense mortality responses of V. destructor, requiring a reasonable time of 24 h, which allows adequate interaction of the mite with the botanical biopesticide.

3.2. Acute toxicity to A. mellifera

3.2.1. Direct contact bioassay using a microapplicator

In this bioassay, 0.5 µL of each of the six concentrations, ranging from 24 mg L^-1 (2.4%) to 200 mg L^-1 (20%), of the P. alliacea extract was applied to the thoraces of the bees with a Burkard Arnold microapplicator. The assay was performed as described below.

1. The P. alliacea aqueous extract was serially diluted with distilled water to obtain solutions with the following concentrations (doses): 24 mg L^-1 (C1), 60 mg L^-1 (C2), 100 mg L^-1 (C3), 140 mg L^-1 (C4), 180 mg L^-1 (C5), and 200 mg L^-1 (C6);

2. Each solution (0.5 µL) was applied to the thoraces of the bees with an Arnold hand-held microapplicator (Burkard, England);

3. Ten bees were placed in each semi-open test tube, with 6 replicate tubes at each concentration. The tubes were placed in an incubator at 29–30 °C with 70–80% relative humidity for 24 h;

4. After 24 h, the tubes were removed from the incubator and placed in different test tubes on the basis of the aqueous extract concentration (C1-C6) (Figure 2);

   

   Figure 2
   Bees were removed from the incubator for evaluation.
   

5. The LC₅₀ values were determined via probit analysis.

Qualitative analysis of the representative data in Figure 2 indicated the low susceptibility of the bees to C1, C2 and C3 on the basis of the low mortality rates, as most the bees climbed through the inner walls of the test tube. However, the bees treated with C4, C5 and C6 lost vitality and did not climb the inner wall of the test tube. Additionally, the highest mortality rates (30 - 70%) were observed at concentrations ranging from 140 – 200 mg L^-1 (Table 3 and 4).

Table 3
Results from the direct and residual contact toxicity bioassay with the aqueous extract of P. alliacea and A. mellifera after 24 h of exposure.

Treatment		Concentration (mg L-1)	Number of A. mellifera individuals	Direct contact (24 h)			Residual contact (24 h)
				Deaths	Mortality (%)	Deaths		Mortality (%)
Petiveria alliacea L. (mucura)	Aqueous extract	24	10	0	0.00	1		10.00
		60	10	1	10.00	2		20.00
		100	10	2	20.00	5		50.00
		140	10	3	30.00	7		70.00
		180	10	5	50.00	9		90.00
		200	10	7	70.00	10		100.00
Control	Water ed water	0.0	10	0	0.00	0			0.00

Table 4
LC₅₀ values and 95% confidence intervals (mg L^-1) obtained from direct contact, residual contact and oral acute toxicity bioassay after incorporation into the diet with the aqueous extract of P. alliacea.

Acute toxicity bioassay	Apis Mellifera	LC50 (mg L-1)	AC ± SE		DF	Heterogeneity
Direct contact (24 h)	Susceptible	165.4156 (152.651-180.765)	3.0087 ± 0.0075	0.2237	3	0.0746
Residual contact (24 h)	Susceptible	104.904 (96.647-113.293)	3.6499 ± 0.0077	0.1246	3	0.0415
Oral (48 h)	Susceptible	112.0472 (85.511-193.363)	3.2802 ± 0.0042	1.1477	3	0.3826
Oral (96 h)	Susceptible	73.7513 (60.429-96.462)	3.361 ± 0.0062	0.7483	3	0.2494

• AC, angular coefficient; SE, standard error of the mean; X², chi-square, DF, degrees of freedom.

Probit analysis of these data gave an LC₅₀ value of 165.4156 mg L^-1. The angular coefficient of the slope was acceptable (3.0087 ± 0.0075) and higher than 1.96, meeting the probit model conditions for a significant toxicological response upon application of the P. alliacea aqueous extract. The heterogeneity result was also acceptable and less than 1.0 (0.0746). These results demonstrate the good fit to the probit model and confirms the dose–mortality relationship and validity of the bioassay results, as shown in Table 4 (Robertson et al., 2017).

The plot of the bioassay results indicting the dose–mortality relationship shows that appreciable mortality occurred only at does much higher than 140 mg L^-1 after 24 h of exposure (Table 4 and Figure 3a).

Figure 3
Dose‒response curve of A. mellifera mortality in response to the P. alliacea aqueous extract in the direct contact (a), residual contact (b) and oral toxicity (c, d, e) bioassays after 24 h and 24 h, 48 h, 72 h, 96 h of exposure.

An analysis of the results of this bioassay revealed that the risk for the species A. mellifera is minimal and that very high doses are required to exceed the LC₅₀. Additionally, 100 mg L^-1 was considered the most appropriate dose on the basis of the previous bioassay data, which would lead to no more than 20% mortality in bees (Bracho-Pérez et al., 2024).

3.2.2. Residual contact bioassay

This bioassay consisted of placing filter papers that had been saturated with 6 concentrations of the P. alliacea extract and then dried in Petri dishes; the control treatment was distilled water. Bees (10 individuals) sedated with carbon dioxide before being placed in the Petri dishes. The bees were then maintained at 34 °C and assessed for mortality after 24 h of exposure. Individuals that were unable to stand on their own after treatment were also considered dead. High toxicity was observed at a dose of 140 mg L^-1, similar to those obtained from the direct contact bioassay, as shown in Table 4 and Figure 3b.

The dose–mortality relationship was much more intense in the residual bioassay than in the direct application bioassay, which may be related to the chemical composition of the P. alliacea extract. The residual bioassay method involved placing 10 mL of the aqueous extract on filter paper, placing the filter papers in Petri dishes, and evaporating the solvent at room temperature (USEPA, 2012).

The flowers, leaves and roots of P. alliacea contain sulfur-containing volatile organic compounds, such as dibenzyl trisulfide, allyl isobutyl sulfide, allyl propyl disulfide, diallyl disulfide, cis-dipropenyl disulfide, trans-dipropenyl disulfide, dibenzyl disulfide, allyl isobutyl disulfide, diisoamyl disulfide, allylmethyl trisulfide, propyl propenyl trisulfide, dibenzyl trisulfide, diallyl trisulfide, and allyl propyl tetrasulfide, as well as amino acid precursors and various benzyl and phenyl derivatives, such as S-benzyl-l-cysteine sulfoxide, S-benzylcysteine, S-methyl-cysteine, S-ethyl-cysteine, S-propyl-cysteine, S-(2-hydroxyethyl)-cysteine, S-(2-hydroxyethyl)-cysteine sulfoxide, and trans-N-methyl-methoxyproline, among others. These secondary metabolites are part of the volatile fraction of P. alliacea and are related to the insecticidal and acaricidal activities of this plant species (Sousa et al., 1990; Johnson et al., 1997; Kubec and Musah, 2005; Kubec et al., 2002; Das Gracas et al., 2002; Kim et al., 2006; USEPA, 2012).

The results of this bioassay (Table 4) were subjected to probit analysis, which yielded an LC₅₀ value of 104.904 mg L^-1. Compared with the results from the direct contact toxicity bioassay results, which gave an LC₅₀ value of 165.4156 mg L^-1, it can be inferred that the aqueous extract of P. alliacea is more toxic after residual contact than direct contact. This difference in toxicity may be related to the biological activities of the components of the volatile fraction responsible for its insecticidal and acaricidal activity. Like the previous bioassays, the residual contact bioassay demonstrated acceptable fit to the probit model after statistical analysis (Sousa et al., 1990; Johnson et al., 1997; Kubec and Musah, 2001; Kubec et al., 2002; Das Gracas et al., 2002; Kim et al., 2006).

The residual contact toxicity bioassay results were considered valid on the basis of the angular coefficient of the slope (3.6499 ± 0.0077, which exceeded 1.96, a necessary condition to fit the probit model) and the significant toxic effect of the P. alliacea aqueous extract. In addition, the value obtained for heterogeneity was 0.0415 (less than 1.0), demonstrating the good fit to the probit model, as well as the general accuracy of the dose–mortality relationship determined from this bioassay (Robertson et al., 2017).

On the other hand, the plot shown in Figure 3b for probit analysis shows that the LC₅₀ value of 104.904 mg L^-1 is very close to 100 mg L^-1, which was considered the optimal dose according to previous studies with D. magna. These data suggest that residual contact poses a greater risk of toxicity to bees (A. mellifera) than direct contact does. Therefore, the dose should be less than 100 mg L^-1. However, field bioassays should be performed to establish the true impact of the aqueous extract on bees in their natural environment, as well as the appropriate doses and the most effective and safe application methods to minimize bee toxicity (Bracho-Pérez et al., 2019; Bracho-Pérez et al., 2024).

3.2.3. Oral toxicity bioassay by incorporation into the diet

The guidelines and methodology of OECD test no. 213 were used to determine the acute oral toxicity of the P. alliacea aqueous extract to honey bees. Young, healthy adult worker bees (A. mellifera L.) were collected in the morning and placed in a stainless steel mesh cage (40 cm × 20 cm × 20 cm) and provided ad libitum access to an aqueous solution of 500 g L^-1 (50% w/v) sucrose, which was supplied in glass capillary tubes (50 mm long × 10 mm wide) with an open diameter of approximately 2 mm. The bees were deprived of food 2 h before the toxicity tests, and those that died were replaced before the test. The test was conducted in the dark at 25 ± 2 °C with a relative humidity of approximately 50–70% (OECD, 1998).

The previously prepared P. alliacea aqueous extract was lyophilized in the Environmental Chemistry Laboratory at the National Technological University of South Lima until a dry powder containing the ai was obtained. Different amounts of the powder were mixed with a sucrose solution to obtain five ai solutions with concentrations of 12 mg L^-1, 30 mg L^-1, 60 mg L^-1, 75 mg L^-1 and 92 mg L^-1. These mixtures were supplied to the bees at a dose of 120 µL until they were entirely consumed, after which the sucrose solution was supplied alone ad libitum. Each bioassay was performed in triplicate. Mortality was recorded at 24 h, 48 h, 72 h, and 96 h, in which fatality was recorded for those individuals who were completely immobile (OECD, 1998).

The results presented in Table 5 from 48 h and 96 h of exposure were fit to the probit model, while the data from 24 h to 72 h of exposure were discarded.

1. Oral toxicity after exposure for 24 h and 72 h

The oral toxicity data acquired after 24 h and 72 h of exposure fit linear (R² = 0.8743) and polynomial (R² = 0.9962) functions, respectively, on the basis of their quadratic correlation coefficients. The oral exposure of bees to the P. alliacea extract in the diet did not fit the probit model (Figure 3c).

Using the respective equations for the 24 h and 72 h data, the LC₅₀ values obtained did not comply with the probit model, and were as follows.

• Linear behavior (24 h): LC₅₀ = 143.1455 mg L^-1

• Polynomial behavior (72 h): LC₅₀ = 50.5723 mg L^-1

1. Oral toxicity after exposure for 48 h and 96 h

The results after exposure for 48 h and 96 h (Table 5) were subjected to probit analysis, and the LC₅₀ values were 112.0472 mg L^-1 and 73.7513 mg L ^-1, respectively. The LC₅₀ determined from the 48 h data was lower than that obtained from the 24 h data (LC₅₀ = 143.1455 mg L^-1) and much higher than that obtained from the 72 h data (LC₅₀ = 50.5723 mg L^-1). These data suggest that as the exposure duration increases, the mortality caused by and the toxic effects of the aqueous extract included in the diet increase. However, after the longest exposure duration (96 h), the LC₅₀ did not decrease. This is typical of a more severe toxic effect, which actually increased and then stabilized at a value slightly higher than that obtained at 72 h. However, in general, the highest mortality and toxicity were achieved after the longest exposure duration.

After 48 and 96 h of exposure, the behavior of the bioassays was optimal (Table 5), the angular coefficients of the slopes exceeded 1.96 (AC (48 h) = 3.2802 ± 0.0042; AC (96 h) = 3.361 ± 0.0062), which is necessary to fit to the probit model, and significant toxicity was observed. In addition, the heterogeneity (het.) values were also lower 1.0 after both exposure durations (het. (48 h) = 0.3826; het. (96 h) = 0.2494), demonstrating the good fit to the probit model and the general accuracy of the dose–mortality relationship in this bioassay (Robertson et al., 2017).

Table 5
Results from the oral toxicity bioassay in which the P. alliacea aqueous extract was incorporated into the diet and evaluation every 24 h for a total of 96 h of exposure.

Treatment		Concentration (mg L-1)	Number of A. mellifera individuals	24 h		48 h		72 h		96 h
				Deaths	Mortality (%)	Deaths	Mortality (%)	Deaths	Mortality (%)	Deaths	Mortality (%)
Petiveria alliacea L. (mucura)	Aqueous extract	12.0	30	5	16.66	3	10.00	6	20.00	7	23.33
		30.0	30	5	16.66	4	13.33	6	20.00	9	30.00
		60.0	30	7	23.33	5	16.66	8	26.66	11	36.66
		75.0	30	9	30.00	9	30.00	11	36.66	15	50.00
		92.0	30	10	33.33	13	43.33	16	53.33	19	63.33
Control	Distilled water water	0.00	30	0	0.00	0	0.00	0	0.00	0	0.00

The plot constructed from the probit analysis after exposure for 48 and 96 h shows that the mortality increases with increasing applied dose, reaching 50% mortality at a dose of 112.0472 mg L^-1 after 48 h and 73.3513 mg L^-1 after 96 h. However, although these data are similar, at 96 h, there was a greater trend toward linearity, wherein the LC₅₀ was much lower, indicating stronger toxicity (Figure 3d and 3e).

The LC₅₀ after the first 24 h of exposure was the lowest (143.1455 mg L^-1); that is, the dose needed to induce death in 50% of the individuals was the highest. Subsequently, the LC₅₀ values after 48 and 72 h of exposure reflect the toxicity of the extract to the internal organs of the bee after oral administration. After 72 h of exposure, the extract exhibited maximum toxicity (LC₅₀ = 50.5723 mg L^-1) and consequently, the highest mortality rate. Finally, the decreasing trend in LC₅₀ value stabilized at 96 h (73.7513 mg L^-1) while maintaining high toxicity.

These results demonstrate the importance of evaluating the effects of exposure every 24 h until 96 h because disturbances in living systems generally depend on the magnitude of the disturbance, which is related to the dose, time and duration of the disturbance and the susceptibility of the host to xenobiotics. Therefore, from these data, it can be inferred that supplying the aqueous extract to the colony feed is not a good option, as it may cause severe toxicity to this species.

Many biological systems can function normally within their homeostatic limits. When the dose of the aqueous extract is first low but then increases, clear biological responses, manifested as mortality, were produced. In this sense, the response obtained at 96 h (the longest exposure duration) is clearly the most important from the oral toxicity bioassay (Krewski et al., 2010).

3.2.4. Qualitative analysis of the components of the P. alliacea aqueous extract

The components of the P. alliacea aqueous extract were qualitatively examined via LC‒UV‒ESI–MS (λ = 280 nm). A representative HPLC chromatogram of the aqueous extract of P. alliacea is presented in Figure 4, where the identified peaks are numbered; their identities are presented in Table 6.

Figure 4
Representative LC–ESI–MS chromatogram of the aqueous extract of P. alliacea with detection at 280 nm. The identities of the peaks are given in Table 6.

Qualitative analysis of the P. alliacea aqueous extract showed the presence of 16 components, of which 14 were identified, as shown in Table 6. The extract dominated by polar components capable of establishing hydrogen bonds, which is typical after extraction into aqueous media and represents the physicochemical characteristics of these compounds that allows their entry into the aqueous fraction during extraction. The presence of four nitrogenous derivatives (P1, P2, P4, and P5), accounting for 25% of the total components of the aqueous extract, as well as four sulfur components equivalent to an additional 25% of extract components. These results are consistent with those of previous studies, such as that of Kim et al. (2006), which determined that sulfur derivatives, such as thiosulfonates, trisulfides and sulfinic acid derivatives, are responsible for the antibacterial and antifungal activity of the extract. Thus, it was established that thiosulfonates containing benzyl groups have the most potent biological activity. Other studies have also demonstrated the importance and presence of thiosulfonates in P. alliacea L. (Kim et al., 2006; Okada et al., 2008).

In addition to sulfur-containing amino acids, such as sulfur derivatives of cysteine and their sulfoxides, γglutamyl dipeptides were also identified, which have been isolated from the roots of P. alliacea and are present in the tissues of this plant species. The enzymatic breakdown of the cysteine derivatives in P. alliacea produces thiosulfonates in a similar way to that observed in Allium species. However, the biochemical role of dipeptides in plant tissue has not been fully established. These compounds are known only as intermediates in the formation of sulfoxides derived from cysteine present in P. alliacea, and various studies have associated them with nitrogen and sulfur storage in plants (Kubec and Musah, 2001, 2005; Kubec et al., 2002).

P8, P9, P10 and P11 in Table 6 were identified as secondary organosulfurized metabolites derived from thiophene, except for component P9, which is a sulfonate. Extracts of plants of other origins do not contain substituted sulfur derivatives of cysteine, which may be related to the direct dependence of the formation of these derivatives on various factors, such as the soil composition, climatic conditions and collection time. Thus, cysteine sulfoxides are not present because these compounds are precursors of the other organosulfides that are present in the leaves and are only found at very low concentrations, and the primary products of their decomposition, such as thiosulfonates, are formed by CS lyase (Fenwick et al., 1985; Block, 1992; Kubec and Musah, 2001; Kim et al., 2006).

On the other hand, Lyndon et al. (1997) demonstrated the powerful acaricidal activity of a fraction of the ethanolic extract of the roots of P. alliacea against ticks taken from infected cattle. Their bioassays revealed that the main component of this fraction was dibenzyl trisulfide, which has a high median lethal dose.

A subsequent study confirmed the acaricidal activities of extracts and fractions of the stems and leaves of P. alliacea, which led to the high mortality of cattle tick R. microplus larvae and adults after their immersion. The methanolic extracts of the stems and leaves of P. alliacea caused 100% mortality in these bioassays. Fractionation of the methanolic extract revealed that the best activity was achieved with the apolar n-hexane fraction, of which the main components were benzyldisulfide (BDS) and benzyltrisulfide (BTS), which may be responsible for the good acaricidal activity. These findings are consistent with the results obtained by Lyndon et al. with P. alliacea roots (Lyndon et al., 1997; Rosado-Aguilar et al., 2010).

Together, these studies demonstrate the powerful insecticidal and acaricidal activities of P. alliacea. However, the starting extracts in these studies were methanolic and ethanolic extracts, which were fractionated with low-polarity organic solvents to yield fractions enriched in DBD and DBT. However, this investigation with D. magna allowed the establishment of organic extracts as the most toxic extracts with the greatest effects on the environment; thus, these extracts have not been further applied, and the aqueous extracts have instead been used to control V. destructor due to their lower mortality and guarantee environmental safety. Furthermore, the most notable and novel finding of this study is that the aqueous extract has marked acaricidal activity, and LC–ESI–MS showed that the sulfurized components DBD and DBT are not in this extract (Table 6) (Lyndon et al., 1997; Rosado-Aguilar et al., 2010; Bracho-Pérez et al., 2019).

These results lead to two important findings. First, the aqueous extract of P. alliacea of Peruvian origin can act as a natural controller or acaricide of botanical origin of V. destructor, and second, the components of the aqueous extract of P. alliacea responsible for this biological activity are either new or related to the organic compounds (primary or secondary metabolites) previously identified in P. alliacea.

Previously published data have already shown the ability of the aqueous extract of this plant species of Peruvian origin to act as an insecticide, as it can cause appreciable mortality in third-stage larvae of Musca domestica; however, this is the first evidence of its ability to control V. destructor mites, which have caused the greatest infestation of honey bees worldwide. Unfortunately, there are very few molecules and products that are capable of at the very least reducing infestations in bee colonies with varroosis (Bracho-Pérez et al., 2019; Nöel et al., 2020).

The aqueous extract of P. alliacea contains nitrogen and sulfur compounds that constitute part of the nitrogen and sulfur reserves of the plant species. Among the four nitrogenous compounds present in the aqueous extract (P1, P2, P4, and P5), three are derivatives of proline (P1, P2, and P4). One of these identified derivatives, P1 (trans-N-methyl-4-methoxyproline), is a highly polar alkaloid previously isolated and identified by Sousa et al. (1990) from the inflorescences of P. alliacea collected in Minas Gerais, Brazil. The remaining two derivatives of proline are the previously unidentified N,N-dimethylproline (P2) and N-α-tert-butyloxycarbonyl-valylproline-methyl ester (P4), a novel dipeptide ester identified for the first time in P. alliacea.

The presence of γ-glutamyl dipeptides in the roots of P. alliacea shows that primary metabolites may exist in the tissue of this plant species. Thus, the identification of the dipeptide P2 and the tripeptide aspartyl-leucyl-leucine (P5) are novel constituents of this aqueous extract. The peptides in this plant species must have various biochemical roles, since peptides regulate a variety of processes related to plant development and defense. Specifically, peptides can act as molecular messengers during plant interactions with other organisms, alerting the plant to possible attacks and inducing defense mechanisms. Microbe-associated molecular patterns are molecular fragments recognized by plants that indicate a potential invasion, as peptides derived from microbial proteins are associated with plant-specific pattern recognition receptors, which elicit a cascade of defense responses (Zipfel et al., 2004, 2006; Matsubayashi and Sakagami, 2006).

In addition, peptides warn plants of attack by herbivorous insects; the inceptin peptide is one such molecular pattern that activates defenses in response to herbivore invasion. Thus, it is important to note the presence of endogenous plant peptides that regulate defenses and act as internal inducers (Schmelz et al., 2006; Ryan et al., 2007; Mithöfer and Boland, 2008).

The aqueous extract also contains aromatic compounds, such as n-propyl 3,4,5-trihydroxybenzoate (P7), 4-(4-hydroxy-3-methoxyphenyl)-2-butanone (P14) and 2'-hydroxy-2,3,4,4',5,5',6'-heptamethoxychalcone (P15), a simple phenolic ester, an aromatic ketone and a flavonoid precursor chalcone, respectively.

The constituents P7 and P14 are derivatives similar to phenylpropanoids that are found in the essential oils of the leaves and flowers of P. alliacea. Chalcone precursors of flavanones, such as leridal, have been widely found in the alcoholic extracts of P. alliacea aerial parts. However, the identified chalcone (P15) is often methoxylated and has not been previously identified in P. alliacea (Table 6) (Bastos Silva et al., 2018).

Table 6
Components identified in the mucura (P. alliacea L.) aqueous extract by LC–MS.

Peak	Compound	Concentration (mg kg-1)	tR (min)	m/z
1	trans-N-methyl-4-methoxyproline	6455	2.17	158.9544
2	N,N-dimethylproline	5271	2.32	144.1010
3	not identified	3200	2.55	-
4	N-α-tert-butyloxycarbonyl-valylproline-methyl ester	4597	2.80	228.1465
5	aspartyl-leucyl-leucine	134	3.25	360.2205
6	(2,4)-2-O-β-D-glucopyranosyl-2,4-pentanediol	409	5.39	266.1433
7	N-propyl 3,4,5-trihydroxybenzoate	182	5.56	212.2081
8	Ethyl 2-thiophenecarboxylate	117	5.89	156.0313
9	allyl benzenesulfonate 2-propen-1-ol	592	6.02	198.0420
10	butyl 2-thiophenecarboxylate	275	6.55	184.0629
11	4-methyldibenzo-thiophene	235	6.74	198.0596
12	not identified	220	7.44	-
13	β-D-mannopyranoside-19-isopimarane-7,15-dien-3β-ol	235	7.76	466.3014
14	4-(4-hydroxy-3-methoxyphenyl)-2-butanone	40	8.34	194.1011
15	2'-hydroxy-2,3,4,4',5,5',6'-heptamethoxychalcone	500	8.67	434.1643
16	6-Methoxy-1,2,3,4,5-pentahydroxy-cyclohexane	0.8	9.12	195.0863

As part of their defense mechanisms, plants induce the biosynthesis of chalcones and flavonoids, which are generally found in the leaves of plant species, including P. alliacea, which specifically protect the plant from predatory insects. After a leaf has been damaged by an insect, a cascade of enzymatic reactions occur that alter and stimulate the biosynthesis of chalcones and flavonoids. The metabolism of these compounds by insects generates byproducts that alter the palatability and suitability of the plant for an insect, cause weight loss, interfere with oviposition, and modify larval food selection, as larvae have neuronal sensory receptors that respond to a range of phagostimulants and deterrents such as carbohydrates, amino acids, alkaloids, diterpenoids and phenols (Simmonds, 2001).

Thus, the presence of complex phenols such as chalcones (e.g., P15), simple phenols such as the phenolic derivative 3,4,5-trihydroxybenzoate of n-propyl (P7), other metabolites such as nitrogenous compounds derived from amino acids (P1, P2, P4, and P5), and the diterpene glycoside virescenoside Q (P13) demonstrate the ability of the plant to control pests as a general defense mechanism against insects that could be related to the acaricidal effects of P. alliacea against V. destructor.

Finally, (2,4)-2-O-β-D-glucopyranosyl-2,4-pentanediol) (P6) and β-D-mannopyranoside-19-isopimarane-7,15-dien-3β-ol (P13) are highly polar and high solubility in water due to the presence of OH groups that can form hydrogen bonds with water and indicates their propensity to be glycosylated. The component of the P. alliacea aqueous extract with the longest LC retention time (t_R = 9.12 min; Figure 4) was identified as 6-methoxy-1,2,3,4,5,6-pentahydroxy-cyclohexane (Table 6). This monomethylated cyclohexitol, also known as D-pinitol, exhibited the strongest affinity for the mobile phase because its five active hydroxyl groups (Sousa et al., 1990; Urueña et al., 2008).

4. Conclusions

The aqueous extract of P. alliacea exhibited an LC₅₀ of 105.2418 mg L^-1 against V. destructor and reduced V. destructor infestation by 46.66, 80.00, 86.66 and 93.33% concentrations of 100, 140, 180 and 200 mg L^-1, respectively, after 24 h of exposure.

The application of 100 mg L^-1 aqueous extract, which has LC₅₀ values after direct contact and residual contact of 165.4156 mg L^-1 and 104.904 mg L^-1, respectively guarantees the minimum impact on honey bees (A. mellifera L.). The direct contact bioassays revealed minimal impact on bees, and when applied at a dose of 100 mg L^-1, a mortality rate of less than 20% is guaranteed. However, the results from the residual contact and oral toxicity by incorporation into the diet bioassays revealed a risk of toxicity to bees since the optimal dose (100 mg L^-1) caused 50% mortality in the residual contact bioassay and 63.33% mortality in the oral toxicity study after 96 h of exposure.

Therefore, we suggest that the aqueous extract of P. alliacea should not be supplied to bees in their food. However, it is vital to carry out field studies to establish how direct and residual contact with the extract components and application methodologies affect the colonies to reduce the impact on bees while adequately controlling V. destructor.

The chemical composition of the aqueous extract contains new components not previously identified in P. alliacea aqueous extracts, as well as other components that have been previously or are of a chemical nature similar to others previously identified, that demonstrate the acaricidal potential of the aqueous extracts of this plant species. However, it is critical to continue studies that establish a direct relationship between the chemical composition and the acaricidal activity to determine the biologically active components.

Therefore, field bioassays should be performed to determine the optimal dose of the extract and conditions to control V. destructor infestation with a minimal impact on A. mellifera colonies.

• Data Availability Statement
  All data analyzed in the research are available at Repositorio Institucional UNHEVAL and can be accessed via hyperlinks: https://hdl.handle.net/20.500.13080/7125

References

• ANDERSON, D.L. and TRUEMAN, J.W., 2000. Varroa jacobsoni (Acari: Varroidae) is more than one species. Experimental & Applied Acarology, vol. 24, no. 3, pp. 165-189. https://doi.org/10.1023/A:1006456720416 PMid:11108385.
  » https://doi.org/10.1023/A:1006456720416
• ARCEO-MEDINA, G.N., ROSADO-AGUILAR, J.A., RODRÍGUEZ-VIVAS, R.I. and BORGES-ARGAEZ, R., 2016. Synergistic action of fatty acids, sulphides and stilbene against acaricide-resistant Rhipicephalus microplus ticks. Veterinary Parasitology, vol. 228, pp. 121-125. https://doi.org/10.1016/j.vetpar.2016.08.023 PMid:27692312.
  » https://doi.org/10.1016/j.vetpar.2016.08.023
• BASTOS SILVA, J.P., MARTINS DO NASCIMENTO, S.C., HIDEMI OKABE, D., GODINHO PINTO, A.C., RODRIGUES DE OLIVEIRA, F., PORTAL DA PAIXÃO, T., SOUZA SIQUIERA, M.L., BAETAS, A.C. and ATAÍDE DE ANDRADE, M., 2018. Antimicrobial and anticancer potential of Petiveria alliacea L. (Herb to “Tame the Master”): a review. Pharmacognosy Reviews, vol. 12, no. 23, pp. 85-93. https://doi.org/10.4103/phrev.phrev_50_17
  » https://doi.org/10.4103/phrev.phrev_50_17
• BLOCK, E., 1992. The organosulfur chemistry of the genus Allium implications for the organic chemistry of sulfur. Angewandte Chemie International Edition in English, vol. 31, no. 9, pp. 1135-1178. https://doi.org/10.1002/anie.199211351
  » https://doi.org/10.1002/anie.199211351
• BRACHO-PÉREZ, J., TACZA-VALVERDE, I. and VÁSQUEZ-CASTRO, J., 2019. Peruvian botanical biopesticides for sustainable development and protection of the environment. Peruvian Journal of Agronomy, vol. 3, no. 3, pp. 126-133. https://doi.org/10.21704/pja.v3i3.1206
  » https://doi.org/10.21704/pja.v3i3.1206
• BRACHO-PÉREZ, J.C., TACZA-VALVERDE, I., CHÁVEZ-ROJAS, D., AQUIJE, C., HARO, J. and VÁSQUEZ-CASTRO, J.A. 2024. Toxicity of Mucura (Petiveria alliacea) extracts from the peruvian amazon against Daphnia magna for environmental protection and sustainable development.Brazilian Journal of Biology = Revista Brasleira de Biologia, vol. 84, pp. e278758. https://doi.org/10.1590/1519-6984.278758
  » https://doi.org/10.1590/1519-6984.278758
• BUCKINGHAM, J., COOPER, C.M. and URCHASE, R., 2015. Natural products desk reference Boca Raton: CRC Press. https://doi.org/10.1201/b19137
  » https://doi.org/10.1201/b19137
• DAS GRACAS, M., ZOGHBI, B., ANDRADE, H.A.E. and MAIA, G.S.J., 2002. Volatile constituents from Adenocalymma alliaceum Miers and Petiveria alliacea L., two medicinal herbs of the Amazon. Flavour and Fragrance Journal, vol. 17, no. 2, pp. 133-135. https://doi.org/10.1002/ffj.1051
  » https://doi.org/10.1002/ffj.1051
• DÁVILA, M. and ORTIZ, M., 1987 [viewed 30 May 2025]. Presencia del ácaro Varroa jacobsoni, ectoparásito de la abeja de la miel en el Perú. Revista Peruana de Entomología [online]. vol. 28, pp. 79-80. Available from: https://sisbib.unmsm.edu.pe/BVRevistas/entomologia/v28/pdf/a17v28.pdf
  » https://sisbib.unmsm.edu.pe/BVRevistas/entomologia/v28/pdf/a17v28.pdf
• DE LA RUA, P., JAFFE, R., DALL’OLIO, R., MUÑOZ, I. and SERRANO, J., 2009. Biodiversity, conservation and current threats to European honeybees. Apidologie, vol. 40, no. 3, pp. 263-284. https://doi.org/10.1051/apido/2009027
  » https://doi.org/10.1051/apido/2009027
• DIETEMANN, V., NAZZI, F., MARTIN, S.J., ANDERSON, D.L., LOCKE, B., DELAPLANE, K.S. and ELLIS, J.D., 2013. Standard methods for varroa research. Journal of Apicultural Research, vol. 52, no. 1, pp. 1-54. https://doi.org/10.3896/IBRA.1.52.1.09
  » https://doi.org/10.3896/IBRA.1.52.1.09
• FENWICK, G.R., HANLEY, A.B. and WHITAKER, J.R., 1985. The genus Allium. Part 2. Critical Reviews in Food Science and Nutrition, vol. 22, no. 4, pp. 273-377. https://doi.org/10.1080/10408398509527417 PMid:3902371.
  » https://doi.org/10.1080/10408398509527417
• FLOTA-BURGOS, G., ROSADO AGUILAR, J.A., RODRÍGUEZ-VIVAS, R.I. and BOLIO-GONZÁLEZ, M., 2021. Contribuciones científicas de la FMVZ-UADY sobre Petiveria alliacea: alternativa de control de garrapatas y nematodos en animales domésticos. Tropical and Subtropical Agroecosystems, vol. 24, no. 3, pp. 1-14. https://doi.org/10.56369/tsaes.3902
  » https://doi.org/10.56369/tsaes.3902
• HRISTOV, P., SHUMKOVA, R., PALOVA, N. and NEOV, B., 2020. Factors associated with honey bee colony losses: A mini-review. Veterinary Sciences, vol. 7, no. 4, pp. 166. https://doi.org/10.3390/vetsci7040166 PMid:33143134.
  » https://doi.org/10.3390/vetsci7040166
• JEYAPRIYA, G., SUMATHI, E., SAMINATHAN, V.R., RENUKADEVI, P., SASIKALA, R., PRIYA, S.S., KOWSIKA, S. and PRADEEP, S., 2025. Parasitic Mites of Honey Bees (Apis spp.): a detailed review of varroa destructor in parasitism, pathogen transmission and its management. Acta Parasitologica, vol. 70, no. 5, pp. 184. https://doi.org/10.1007/s11686-025-01124-w PMid:40828485.
  » https://doi.org/10.1007/s11686-025-01124-w
• JOHNSON, L., WILLIAMS, L.A.D. and ROBERTS, E.V., 1997. An insecticidal and acaricidal polysulfide metabolite from the roots of Petiveria alliacea. Pesticide Science, vol. 50, no. 3, pp. 228-232. https://doi.org/10.1002/(SICI)1096-9063(199707)50:3<228::AID-PS575>3.0.CO;2-J
  » https://doi.org/10.1002/(SICI)1096-9063(199707)50:3<228::AID-PS575>3.0.CO;2-J
• KIM, S., CHEN, J., CHENG, T., GINDULYTE, A., HE, J., HE, S., LI, Q., SHOEMAKER, B.A., THIESSEN, P.A., YU, B., ZASLAVSKY, L., ZHANG, J. and BOLTON, E.E., 2020 [viewed 30 May 2025]. PubChem 2020 update: improved access to chemical data [online]. Available from: https://pubchem.ncbi.nlm.nih.gov/search/search.cgi
  » https://pubchem.ncbi.nlm.nih.gov/search/search.cgi
• KIM, S., KUBEC, R. and MUSAH, R.A., 2006. Antibacterial and antifungal activity of sulfur-containing compounds from Petiveria alliacea L. Journal of Ethnopharmacology, vol. 104, no. 1-2, pp. 188-192. https://doi.org/10.1016/j.jep.2005.08.072 PMid:16229980.
  » https://doi.org/10.1016/j.jep.2005.08.072
• KREWSKI, D., ACOSTA JUNIOR, D., ANDERSEN, M., ANDERSON, H., BAILAR 3rd, J.C., BOEKELHEIDE, K., BRENT, R., CHARNLEY, G., CHEUNG, V.G., GREEN JUNIOR, S., KELSEY, K.T., KERKVLIET, N.I., LI, A.A., MCCRAY, L., MEYER, O., PATTERSON, R.D., PENNIE, W., SCALA, R.A., SOLOMON, G.M., STEPHENS, M., YAGER, J., ZEISE, L. and STAFF OF COMMITTEE ON TOXICITY TEST, 2010. Toxicity testing in the 21st century: a vision and a strategy. Journal of Toxicology and Environmental Health. Part B, Critical Reviews, vol. 13, no. 2-4, pp. 51-138. https://doi.org/10.1080/10937404.2010.483176 PMid:20574894.
  » https://doi.org/10.1080/10937404.2010.483176
• KUBEC, R. and MUSAH, R.A., 2001. Cysteine sulfoxide derivatives in Petiveria alliacea. Phytochemistry, vol. 58, no. 6, pp. 981-985. https://doi.org/10.1016/S0031-9422(01)00304-1 PMid:11684199.
  » https://doi.org/10.1016/S0031-9422(01)00304-1
• KUBEC, R. and MUSAH, R.A., 2005. γ-Glutamyl dipeptides in Petiveria alliacea. Phytochemistry, vol. 66, no. 20, pp. 2494-2497. https://doi.org/10.1016/j.phytochem.2005.06.029 PMid:16146637.
  » https://doi.org/10.1016/j.phytochem.2005.06.029
• KUBEC, R., KIM, S. and MUSAH, R.A., 2002. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II. Phytochemistry, vol. 61, no. 6, pp. 675-680. https://doi.org/10.1016/S0031-9422(02)00328-X PMid:12423888.
  » https://doi.org/10.1016/S0031-9422(02)00328-X
• LEORA SOFTWARE LLC, 2016 [viewed 30 May 2025]. LeOra software polosuite for windows and OS [online]. Parma, MO. Available from: http://www.LeOra-Software.com
  » http://www.LeOra-Software.com
• LYNDON, J., LAWRENCE, A.D.W. and EARLE, V.R., 1997. An insecticidal and acaricidal polysulfide metabolite from the roots of Petiveria alliacea. Pesticide Science, vol. 50, no. 3, pp. 228-232. https://doi.org/10.1002/(SICI)1096-9063(199707)50:3<228::AID-PS575>3.0.CO;2-J
  » https://doi.org/10.1002/(SICI)1096-9063(199707)50:3<228::AID-PS575>3.0.CO;2-J
• MATSUBAYASHI, Y. and SAKAGAMI, Y., 2006. Peptide hormones in plants. Annual Review of Plant Biology, vol. 57, no. 1, pp. 649-674. https://doi.org/10.1146/annurev.arplant.56.032604.144204 PMid:16669777.
  » https://doi.org/10.1146/annurev.arplant.56.032604.144204
• MEIJA, J., COPLEN, T.B., BERGLUND, M., BRAND, W.A., DE BIÈVRE, P., GRÖNING, M., HOLDEN, N.E., IRRGEHER, J., LOSS, R.D., WALCZYK, T. and PROHASKA, T., 2016. Atomic weights of the elements 2013 (IUPAC Technical Report). Pure and Applied Chemistry, vol. 88, no. 3, pp. 265-291. https://doi.org/10.1515/pac-2015-0305
  » https://doi.org/10.1515/pac-2015-0305
• MITHÖFER, A. and BOLAND, W., 2008. Recognition of herbivory-associated molecular patterns. Plant Physiology, vol. 146, no. 3, pp. 825-831. https://doi.org/10.1104/pp.107.113118 PMid:18316636.
  » https://doi.org/10.1104/pp.107.113118
• MORFIN, N., GOODWIN, P.H. and GUZMAN-NOVOA, E., 2023. Varroa destructor and its impacts on honey bee biology. Frontiers in Bee Science, vol. 1, pp. 1272937. https://doi.org/10.3389/frbee.2023.1272937
  » https://doi.org/10.3389/frbee.2023.1272937
• NEVES, I.A., DA CÂMARA, C.A., DE OLIVIERA, J. and DE ALMEIDA, A.V., 2011. Acaricidal Activity and Essential Oil Composition of Petiveria alliacea L. from Pernambuco (Northeast Brazil). The Journal of Essential Oil Research, vol. 23, no. 1, pp. 23-26. https://doi.org/10.1080/10412905.2011.9700426
  » https://doi.org/10.1080/10412905.2011.9700426
• NOËL, A., LE CONTE, Y. and MONDET, F., 2020. Varroa destructor: how does it harm Apis mellifera honey bees and what can be done about it? Emerging Topics in Life Sciences, vol. 4, no. 1, pp. 45-57. https://doi.org/10.1042/ETLS20190125 PMid:32537655.
  » https://doi.org/10.1042/ETLS20190125
• OKADA, Y., TANAKA, K., SATO, E. and OKAJIMA, H., 2008. Antioxidant activity of the new thiosulfinate derivative, S-benzyl phenylmethanethiosulfinate, from Petiveria alliacea L.Organic & Biomolecular Chemistry, vol. 6, no. 6, pp. 1097-1102. https://doi.org/10.1039/b715727d
  » https://doi.org/10.1039/b715727d
• ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 1998. Test No. 213: Honeybees, Acute Oral Toxicity Test. OECD Guidelines for the Testing of Chemicals, Section 2 Paris: OECD Publishing. https://doi.org/10.1787/9789264070165-en
  » https://doi.org/10.1787/9789264070165-en
• PETERSEN, F. and AMSTUTZ, N., 2008. Natural compounds as drugs, Volume 1 Berlin: Birkhäuser Verlag AG Basel, vol. 65, Progress in Drug Research. https://doi.org/10.1007/978-3-7643-8117-2
  » https://doi.org/10.1007/978-3-7643-8117-2
• ROBERTSON, J.L., JONES, M.M., OLGUIN, E. and ALBERTS, B., 2017. Bioassays with arthropods 3rd ed. Boca Raton: CRC Press. https://doi.org/10.1201/9781315373775
  » https://doi.org/10.1201/9781315373775
• ROSADO-AGUILAR, J.A., AGUILAR-CABALLERO, A., RODRIGUEZ-VIVAS, R.I., BORGES-ARGÁEZ, R., GARCÍA-VÁZQUEZ, Z. and MÉNDEZ-GONZÁLEZ, M., 2010. Acaricidal activity of extracts from Petiveria alliacea (Phytolaccaceae) against the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Veterinary Parasitology, vol. 168, no. 3-4, pp. 299-303. https://doi.org/10.1016/j.vetpar.2009.11.022 PMid:20042296.
  » https://doi.org/10.1016/j.vetpar.2009.11.022
• ROSADO-AGUILAR, J.A., ARJONA-CAMBRANES, K., TORRES-ACOSTA, J.F.J., RODRÍGUEZ-VIVAS, R.I., BOLIO-GONZÁLEZ, M.E., ORTEGA-PACHECO, A., ALZINA-LÓPEZ, A., GUTIÉRREZ-RUIZ, E.J., GUTIÉRREZ-BLANCO, E. and AGUILAR-CABALLERO, A.J., 2017. Plant products and secondary metabolites with acaricide activity against ticks. Veterinary Parasitology, vol. 238, pp. 66-76. https://doi.org/10.1016/j.vetpar.2017.03.023 PMid:28385541.
  » https://doi.org/10.1016/j.vetpar.2017.03.023
• ROSENKRANZ, P., AUMEIER, P. and ZIEGELMANN, B., 2010. Biology and control of Varroa destructor. Journal of Invertebrate Pathology, vol. 103, suppl. 1, pp. S96-S119. https://doi.org/10.1016/j.jip.2009.07.016 PMid:19909970.
  » https://doi.org/10.1016/j.jip.2009.07.016
• RYAN, C.A., HUFFAKER, A. and YAMAGUCHI, Y., 2007. New insights into innate immunity in Arabidopsis. Cellular Microbiology, vol. 9, no. 8, pp. 1902-1908. https://doi.org/10.1111/j.1462-5822.2007.00991.x PMid:17593247.
  » https://doi.org/10.1111/j.1462-5822.2007.00991.x
• SAKUMA, M., 1998. Probit analysis of preference data. Applied Entomology and Zoology, vol. 33, no. 3, pp. 339-347. https://doi.org/10.1303/aez.33.339
  » https://doi.org/10.1303/aez.33.339
• SCHMELZ, E.A., CARROLL, M.J., LECLERE, S., PHIPPS, S.M., MEREDITH, J., CHOUREY, P.S., ALBORN, H.T. and TEAL, P.E., 2006. Fragments of ATP synthase mediate plant perception of insect attack. Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 23, pp. 8894-8899. https://doi.org/10.1073/pnas.0602328103 PMid:16720701.
  » https://doi.org/10.1073/pnas.0602328103
• SIMMONDS, M.S., 2001. Importance of flavonoids in insect--plant interactions: feeding and oviposition. Phytochemistry, vol. 56, no. 3, pp. 245-252. https://doi.org/10.1016/S0031-9422(00)00453-2 PMid:11243451.
  » https://doi.org/10.1016/S0031-9422(00)00453-2
• SOUSA, J.R., DEMUNER, A.J., PINHEIRO, J.A., BREITMAIER, E. and CASSELS, B.K., 1990. Dibenzyl trisulphide and trans-n-methyl-4-methoxyproline from Petiveria alliacea. Phytochemistry, vol. 29, no. 11, pp. 3653-3655. https://doi.org/10.1016/0031-9422(90)85294-P
  » https://doi.org/10.1016/0031-9422(90)85294-P
• UNITED STATES ENVIRONMENTAL PROTECTION AGENCY – US EPA, 2012 [viewed 1 March 2025]. Ecological effects test guidelines: OCSPP 850.3030: honey bee toxicity of residues on foliage [online]. Washington: United States Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention. EPA-HQ-OPPT-2009-0154-0017. Available from: https://nepis.epa.gov/Exe/ZyPDF.cgi/P100IRF8.PDF?Dockey=P100IRF8.PDF
  » https://nepis.epa.gov/Exe/ZyPDF.cgi/P100IRF8.PDF?Dockey=P100IRF8.PDF
• URUEÑA, C., CIFUENTES, C., CASTAÑEDA, D., ARANGO, A., KAUR, P., ASEA, A. and FIORENTINO, S., 2008. Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells. BMC Complementary and Alternative Medicine, vol. 8, no. 1, pp. 60-77. https://doi.org/10.1186/1472-6882-8-60 PMid:19017389.
  » https://doi.org/10.1186/1472-6882-8-60
• VU, P., 2016 [viewed 1 March 2025]. Toxicological analysis of acaricides for varroa mite management [online]. Available from: https://vtechworks.lib.vt.edu/server/api/core/bitstreams/d2b61cbb-51e9-40c3-a64b-8436981875ad/content
  » https://vtechworks.lib.vt.edu/server/api/core/bitstreams/d2b61cbb-51e9-40c3-a64b-8436981875ad/content
• WALLACE, W.E., 2023. "Mass Spectra" en NIST Chemistry WebBook, NIST Standard Reference Database Number 69 Gaithersburg: National Institute of Standards and Technology.https://doi.org/10.18434/T4D303
  » https://doi.org/10.18434/T4D303
• WARNER, S., POKHREL, L.R., AKULA, S.M., UBAH, C.S., RICHARDS, S.L., JENSEN, H. and KEARNEY, G.D., 2024. A scoping review on the effects of Varroa mite (Varroa destructor) on global honey bee decline. The Science of the Total Environment, vol. 906, pp. 167492. https://doi.org/10.1016/j.scitotenv.2023.167492 PMid:37778563.
  » https://doi.org/10.1016/j.scitotenv.2023.167492
• ZIPFEL, C., KUNZE, G., CHINCHILLA, D., CANIARD, A., JONES, J.D., BOLLER, T. and FELIX, G., 2006. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell, vol. 125, no. 4, pp. 749-760. https://doi.org/10.1016/j.cell.2006.03.037 PMid:16713565.
  » https://doi.org/10.1016/j.cell.2006.03.037
• ZIPFEL, C., ROBATZEK, S., NAVARRO, L., OAKELEY, E.J., JONES, J.D., FÉLIX, G. and BOLLER, T., 2004. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature, vol. 428, no. 6984, pp. 764-767. https://doi.org/10.1038/nature02485 PMid:15085136.
  » https://doi.org/10.1038/nature02485

Edited by

• Editor: Takako Matsumura Tundisi

Data availability

All data analyzed in the research are available at Repositorio Institucional UNHEVAL and can be accessed via hyperlinks: https://hdl.handle.net/20.500.13080/7125

Publication Dates

• Publication in this collection
  13 Mar 2026
• Date of issue
  2026

History

• Received
  30 May 2025
• Accepted
  12 Jan 2026