Introduction
Sucking lice are wingless insects that live as permanent obligate ectoparasites of various mammals, including humans (Fu et al., Reference Fu, Yao, Deng, Elsheikha, Shao, Zhu and Liu2022). Among these, the blood-sucking louse Haematopinus tuberculatus (Burmeister, 1839) is the key ectoparasite infesting the water buffalo (Bubalus bubalis) (L.) breeding (Shakya et al., Reference Shakya, Kaveri, Jamra, Singh, Fular, Agrawal, Jatav, Jayraw and Kumar2022), causing direct damages characterized by various symptoms such as skin irritation, anaemia, anorexia, restlessness and loss of body condition breeding (Da Silva et al., Reference Da Silva, Lopes, Diaz, Tonin, Stefani and Araújo2013), or also indirect damages as a vector of several pathogens, including Brucella abortus or anaplasmosis, as observed in preliminary studies conducted by Da Silva et al. (Reference Da Silva, Lopes, Diaz, Tonin, Stefani and Araújo2013) and Neglia et al. (Reference Neglia, Veneziano, De Carlo, Galiero, Borriello, Francillo, Campanile, Zicarelli and Manna2013), respectively. As an additive subsequent impairment attributable to lice infestations (pediculosis), there is the reduced productivity of buffalo herds, which play an important role in livestock farming in several countries (Bosco et al., Reference Bosco, Morgoglione, Amadesi, Masiello, Antenucci, Cringoli and Rinaldi2018; Morgoglione et al., Reference Morgoglione, Bosco, Maurelli, Alves, Saralli, Bruni, Cringoli and Rinaldi2020). Ectoparasitic infestations occur more frequently in the winter, as animals tend to move closer together to keep warm (Shakya et al., Reference Shakya, Kaveri, Jamra, Singh, Fular, Agrawal, Jatav, Jayraw and Kumar2022).
The Italian Mediterranean water buffalo breed is mainly reared in Italy to produce high-quality milk, mandatory for the production of the ‘buffalo mozzarella’, a fresh cheese with a protected designation of origin, according to European Union legislation (Zicarelli, Reference Zicarelli2004). Although modern intensive water buffalo farming has almost completely replaced the traditional free range/semi-wild buffalo herd and has greatly reduced helminth infections on farms, at the same time it has led to a higher incidence of protozoal and ectoparasitic diseases, due to the high density of animals (Cringoli et al., Reference Cringoli, Musella, Maurelli, Morgoglione, Santaniello, Condoleo, Guariglia and Rinaldi2009; Bosco et al., Reference Bosco, Rinaldi, Maurelli and Cringoli2017; Morgoglione et al., Reference Morgoglione, Bosco, Maurelli, Alves, Saralli, Bruni, Cringoli and Rinaldi2020).
The control of pediculosis caused by H. tuberculatus in buffaloes is a key issue in order to prevent economic losses (Veneziano et al., Reference Veneziano, Neglia, Buono, Pacifico, Manna, Bassini, Miotto, Santoro and Gokbulut2017). Common solutions include the use of drugs (i.e. ivermectin and eprinomectin) at the same dose and recommended intervals as those used in cattle breeding (Veneziano et al., Reference Veneziano, Neglia, Cimmino, Balestrieri, Rufrano, Bastianetto, Santoro and Gokbulut2013). Pyrethroid-based pesticide insecticides (deltamethrin, cypermethrin and flumethrin) are also sometimes used, but with low control effects due to the emergence of resistances in the target parasites (Shakya et al., Reference Shakya, Kaveri, Jamra, Singh, Fular, Agrawal, Jatav, Jayraw and Kumar2022). Insects can although rapidly develop resistance to drugs due to their genetic plasticity, with rapid mutations manifested in target genes and leading to the emergence of resistant alleles, that are passed on to subsequent generations (Durand et al., Reference Durand, Bouvresse, Andriantsoanirina, Berdjane, Chosidow and Izri2011; McNair, Reference McNair2015). The search for alternative control strategies for pediculosis in the veterinary sector is of paramount importance to limit insecticide residues in milk, environmental pollution and the emergence of resistances in target organisms, but still relatively low explored.
Recently, the wood-combustion derived products such as biochar and wood vinegar (WV) have shown promising results against many different pests in agricultural sector (Dewi et al., Reference Dewi, Hastuti and Nuraeni2020; Bonanomi et al., Reference Bonanomi, Zotti, Cesarano, Sarker, Saulino, Saracino, Idbella, Agrelli, D’Ascoli and Rita2021; Jesu et al., Reference Jesu, Laudonia, Bonanomi, Flematti, Germinara, Pistillo, Giron, Bèzier and Vinale2022). The WV is a by-product of wood pyrolysis and has attracted considerable interest due to its potential applications in agriculture, including plant growth promotion, and both pest and disease control (Mhamdi, Reference Mhamdi2023), which antifungal, biopreservative, antioxidant and insecticidal effects are derived from the acidic compounds, phenols and carbonyls (Faisal et al., Reference Faisal, Utari, Hayvia and Maulana2019). The studies conducted have shown WV has a repellent action against numerous species of insects including flies and termites (Mhamdi, Reference Mhamdi2023; Ouattara et al., Reference Ouattara, Niamké and Yao2023), and in this study the possibility of using this product for the control of ectoparasites was explored and evaluated.
The aim of this study was to evaluate in vitro the possibility of using WV as a repellent against the louse H. tuberculatus in buffaloes, in attempt to find an alternative to chemicals. In order to evaluate the possible practical applications, WV was chemically characterized for both the organic and inorganic fractions while the cytotoxicity was assessed by cultured kidney cells. The novel in vitro bioassay was developed ad hoc during the study as a replacement for in vivo tests to reduce the use of animals for scientific experiments, in accordance with European regulations based on animal welfare and ethical principles (Verderio et al., Reference Verderio, Lecchi, Ciniselli, Shishmani, Apolone and Manenti2023).
Material and methods
Collection of lice on the farm
Lice were collected from the skin of 22 animals using a proper comb in a commercial buffalo farm located in the Campania region, southern Italy, early in the morning and 1 h before the start of each in vitro bioassay replication. In this farm, no resistance of lice to pyrethroids has been recorded in the past. The lice were placed in test tubes under controlled and optimal conditions (27 °C) and transported to the entomological laboratories of the Regional Center for Monitoring Parasitic Infections (CREMOPAR, Campania Region, Italy), where the adults were selected and sexed under a stereomicroscope before the experiments.
In vitro bioassay
The in vitro bioassay was performed in glass Petri dishes (ø 19 cm) divided into 3 separate sections, each covered with a layer of bibulous paper soaked with 3 mL of different solutions. The first (starting) and the third (finishing) area layers were soaked with distilled water. The layer of finishing area was previously vigorously scrubbed on the host skin and then covered with buffalo fur after soaking, to increase the attraction to the lice (Figure 1). The central (obstacle) area was impregnated with different concentrations of the WV to be tested (100%, 75%, 50% and 25%). In the positive and negative controls, the central layer was soaked with deltamethrin solution (BUTOX® 7,5 Pour-On, MSD Animal Health Srl) and distilled water, respectively (Figures 1 and 2).
Figure 1. Division in 3 areas of the glass Petri dish for the in vitro bioassays.
Figure 2. Three petri dishes to evaluate repellent effect of wood vinegar against Haematopinus tuberculatus confronted to negative (distilled water) and positive (deltamethrin) controls.
For each biological replication, which included a test trial and 2 controls, 3 Petri dishes were set up at the same time, each with 10 adult lice (n = 5 males + 5 females) placed in the starting area. Experiments were carried out in a dark, controlled room at optimal condition (27 °C, 75% relative humidity), with observations made after 5, 15, 30, 60, 90, 120 and 180 min to evaluate the movement, the living/dead ratio and the overall ability to overcome the obstacle area of the lice in terms of comparative avoidance (%). In addition, in vitro cytotoxicity bioassay tests for all the tested concentrations were also performed, as well as a preliminary chemical characterization of WV.
Preparation of WV and chemical characterization
The pyrolysis process took place at a facility in Ferrara (Northern Italy), using pruning residues from pome and stone fruit trees. The woody residues were cleaned from soil residues, chipped with pieces of maximum size 8 cm and subsequently subjected to pyrolysis at a temperature of 800 °C. During the process, the gases were directed into a steel pipe for condensation, and the resulting pyrolytic liquid, referred to here as WV, was collected in a plastic container for further use.
The WV was chemically characterized for the following parameters: total organic carbon (method: UNI EN 15936 2012), acetic acid (method: IS 08.03/161 2017), propionic acid (method: IS 08.03/161 2017), total phenols (method: EPA 3510C 1996 + EPA 8270E 2018), pH (method: APAT CNR IRSA 2060 Man 29 2003), electrical conductivity (method: APAT CNR IRSA 2030 Man 29 2003), total suspended solids (method: APAT CNR IRSA 2090 B Man 29 2003), ammonia nitrogen (method: UNI 11669:2017), sulphates (method: APAT CNR IRSA 4020 Man 29 2003), sulphites (method: APAT CNR IRSA 4150 Man 29 2003), sulphides (method: APHA Standard Methods 4500), chlorides (method: APAT CNR IRSA 4020 Man 29 2003), total phosphorus (method: M.U. 2252:08), nitrates (method: APAT CNR IRSA 4020 Man 29 2003), aluminium (method: EPA 3015A 2007 + EPA 6020B 2014), arsenic (method: EPA 3015A 2007 + EPA 6020B 2014), iron (method: EPA 3015A 2007 + EPA 6020B 2014), manganese (method: EPA 3015A 2007 + EPA 6020B 2014), hydrocarbons (method: UNI EN 14039:2005) and acetone (method: EPA 5021A 2014 + EPA 8260D 2018).
To extract non-polar metabolites from WV, the procedure described by Flematti et al. (Reference Flematti, Ghisalberti, Dixon and Trengove2009) was followed. Briefly, 1 L of WV was extracted 3 times using 200 mL of dichloromethane (DCM) per extraction. The combined extracts were dried with anhydrous sodium sulphate (Na2SO4) and evaporated under vacuum at 40 °C using a rotary evaporator. The resulting extract was characterized via mass spectrometric analysis. For this, the DCM extract was reconstituted in methanol (MeOH) to achieve a final concentration of 1 mg mL−1. A 7 µL aliquot was injected into an Agilent HP 1260 Infinity Series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a quadrupole time-of-flight mass spectrometer (Agilent Technologies) and a diode array detector (Agilent Technologies). Sample analysis was conducted following the protocol by Staropoli et al. (Reference Staropoli, Vassetti, Salvatore, Andolfi, Prigigallo, Bubici, Scagliola, Salerno and Vinale2021).
The DCM extract was also analysed using gas chromatography–mass spectrometry (GC-MS). Before analysis, an aliquot of the extract was resuspended in ethyl acetate to reach a final concentration of 100 ppm and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (Fluka, Buchs, Switzerland). The derivatization reaction was carried out in an ultrasonic bath (Sonorex, Bandelin Electronic GmbH & Co. K, Berlin, Germany) for 30 min. The sample was then injected into an Agilent 8890 GC system (Agilent Technologies) coupled with an Agilent 5977B MSD system (Agilent Technologies). The temperature gradient and other chromatographic and spectrometric conditions were adjusted based on the method described by Staropoli et al. (Reference Staropoli, Vassetti, Salvatore, Andolfi, Prigigallo, Bubici, Scagliola, Salerno and Vinale2021).
Cell culture and cytotoxicity assay
The Madin Darby Bovine Kidney (MDBK) cells were selected as a model for the in vitro cytotoxicity assay. This choice is justified by the fact that this cell line originates from bovine and can therefore be considered a good model for predicting the effect of WV on buffalo host cells. In addition, MDBK cells are a well-established and widely used cell line in cytotoxicity and toxicology studies. This means that a large amount of existing data and standardized protocols are available for their use. Finally, MDBK cells are epithelial cells, which make them a suitable model for the assessment of potential toxic effects on host tissue.
MDBK cells (CCL22, American Type Culture Collection CLS Cat# 600396/p848_MDBK_(NBL-1), RRID:CVCL0421) were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 2% fetal calf serum, 1% l-glutamine, 1% penicillin/streptomycin and 0.2% sodium pyruvate, and maintained in an incubator at 37 °C (in 5% CO2/95% air). Cells (2 × 104 cells/well) in 96-well plates were treated at confluence with WV (0.03%, 0.1%, 0.3% or 1% in DMEM) and incubated for 24, 48 or 72 h.
Cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (SERVA Electrophoresis GmbH Cat# 20395.02). The principle of this method is that MTT, a soluble tetrazolium salt (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg mL−1), is converted to insoluble formazan by active mitochondrial dehydrogenases of living cells. This conversion of yellowish soluble tetrazolium into purple formazan can be determined spectrofluorimetrically (Pagnini et al., Reference Pagnini, Montagnaro, Pacelli, De Martino, Florio, Rocco, Iovane, Pacilio, Gabellini, Marsili and Giordano2004). In brief, 100 μL of MTT reagent (0.5 mg mL−1; SERVA) was added to each well and maintained at 37 °C. After 1 h, 100 μL of solubilization buffer (dimethyl sulphoxide; SERVA Electrophoresis GmbH Cat# 39757.0) was added to dissolve the formazan crystals produced by the viable cells. After 3 h at 37 °C, the optical absorbance was measured at 570 nm using a spectrophotometer. Data are calculated as mean ± standard deviation (SEM) of 3 independent experiments performed in triplicate.
Statistical analyses
The Cox proportional hazard model was used to evaluate the times to reach the finishing area of the treated individuals compared to the control ones (Daher et al., Reference Daher, Varghese, Gruschkus, Jimenez, Waguespack, Bedrose, Altameemi, Bazerbashi, Naing, Subaiah, Campbell, Shah, Zhang, Sheth, Karam, Wood, Perrier, Graham, Lee and Habra2022). In the present study, this model was designed for the time to reach finishing area of the Petri dish arena, overcoming the presence of the repulsive treatment. In this context, the regression coefficient (β), standard error (SE), significance (P-value), relative risk (Exp(β)), as well as lower and upper limits of the confidence interval for the hazard ratio of each experimental treatment (i.e. the WV concentrations), compared to the control, are calculated. Three replicate Petri dishes, each consisting of 10 lice (experimental units), for a total of 30 experimental units, were used for each treatment.
Statistical analyses were performed with GraphPad Prism 7.0 for Windows (GraphPad Software). Statistically significant differences between the means of multiple matched groups were determined by one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test. P < 0.05 was considered statistically significant.
Results
In vitro bioassay
The 100% concentration of WV had a similar repellent effect on 77% of adult H. tuberculatus of both sexes as deltamethrin, 30 min after exposure (P < 0.05). For the other WV concentrations, a dose-dependent effect was observed (Figure 3). However, deltamethrin led to the death of all exposed individuals during this period, while WV had not shown any topic effect on the tested adults, even when they stopped moving. In the negative control, all lice managed to reach the finishing area within 15 min. Regression coefficient (β), standard error (SE), significance (P-value), relative risk (Exp(β)) and lower and upper limits of the confidence interval for the hazard ratio of each experimental treatment, compared to the control, are presented in Table 1. The negative β values, derived from the statistical analysis, confirm the direct effect of the treatments on the increasing difficulty of overcoming the repulsive effect of WV and reaching the finishing area. In addition, the in vitro bioassay cytotoxicity tests showed no toxic effects.
Figure 3. Trend of the percentage of the total lice exposed reaching the finishing area of the arena during the in vitro bioassay, in time. Purple (CNT): negative control, obstacle area imbue with distilled water; blue (deltamethrine): obstacle area imbue with deltamethrin solution; red (WV 25%): obstacle area imbue with wood vinegar diluted at 25% in distilled water; orange (WV 50%): obstacle area imbue with wood vinegar diluted at 50% in distilled water; green (WV 75%): obstacle area imbue with wood vinegar diluted at 75% in distilled water; black (WV 100%): obstacle area imbue with non-diluted wood vinegar. The purple line (negative control) ends before the timestone of 50.
Table 1. Summary of cox proportional hazard model for time to reaching the finishing area of the Petri dish arena, overcoming the presence of the repulsive treatment
WV, wood vinegar; β, regression coefficient; SE, standard error; CI, confidence interval.
To determine the effect of WV on cell viability of the MDBK cell line, the MTT assay with increasing concentrations (0.03%, 0.1%, 0.3% and 1%) of WV for 24, 48 and 72 h was performed. In MDBK, cell viability decreases only at a concentration of 1% WV and after 48 and 72 h (P < 0.001) (Figure 4).
Figure 4. The MTT test was performed at different hours after treatment and absorbance was measured as described in the Materials and methods section. The results are presented as mean ± SD of 3 independent experiments performed in triplicate. Statistical differences between untreated cells and treated groups were analysed by One-way analysis of variance (ANOVA) followed by Tukey post-hoc test and expressed as probability P. *P < 0.05, **P < 0.01 and ***P < 0.001.
WV chemical characterization
The WV was acidic (pH 3.2) and had an electrical conductivity of 1389.10 μS cm−1. The phenol concentration was high (54.00 mg L−1), as the concentration of acetic acid (27 840.16 mg L−1), and propionic acid (72 447.06 mg mL−1). The total suspended solids were 26.00 mg L−1, ammonium nitrogen was 8.30 mg L−1, while sulphates, sulphites and sulphides were 53.70, 0.32 and 0.12 mg L−1, respectively. Chlorides were 29.20 mg L−1, total phosphorus 0.97 mg L−1, nitrates 5.20 mg L−1, aluminium 0.92 mg L−1, arsenic 0.02 mg L−1, iron 183.00 mg L−1, manganese 1.11 mg Lv, total organic carbon 15 600.00 mg L−1, hydrocarbons 77.10 mg L−1 and acetone 320.00 mg L−1 (Table 2).
Table 2. Chemical and biochemical parameters of the wood vinegar
Additional analysis of the organic extract using LC-MS identified 54 compounds, with 2 of them putatively matched to an internal database of plant secondary metabolites. These were identified as nicotinic acid (also referred to as niacin or vitamin B3) and malvalic acid. The DCM extract of the WV was further analysed using GC-MS, which revealed 133 chromatographic peaks. Of these, 23 compounds were identified through comparison of their mass spectra with the NIST library and verification of their retention indices (Table 3). The detected compounds spanned several categories, including phenolic derivatives (e.g. guaiacol, syringol, eugenol, catechol), fatty acids (such as oleic, stearic and palmitic acids), polyalcohols (glycerol) and pyrimidine derivatives (2,5-dimethyl-4-pyrimidinamine).
Table 3. Wood vinegar compounds identified by GC-MS analysis
RT, retention time; RI, retension index.
Discussion
The present study is the first to demonstrate the potential use of WV against ectoparasites in buffaloes. Specifically, the results of the in vitro bioassay highlight WV’s repellent effect on H. tuberculatus without the necessity to kill them, a species with reported resistance to the commonly used pesticide deltamethrin (Shakya et al., Reference Shakya, Kaveri, Jamra, Singh, Fular, Agrawal, Jatav, Jayraw and Kumar2022). Considering the well-documented role of lice as vectors for certain diseases (Da Silva et al., Reference Da Silva, Lopes, Diaz, Tonin, Stefani and Araújo2013; Neglia et al., Reference Neglia, Veneziano, De Carlo, Galiero, Borriello, Francillo, Campanile, Zicarelli and Manna2013) and their widespread distribution (Štrbac et al., Reference Štrbac, Krnjajić, Stojanović, Ratajac, Simin, Orčić, Rinaldi, Ciccone, Maurelli, Cringoli and Bosco2023), controlling these ectoparasites in farms has proven to be more critical than previously thought. Moreover, exclusive reliance on ectoparasiticides poses significant risks to public health due to residue accumulation in animal-derived products and potential environmental contamination. In this context, natural products like WV offer a more sustainable and safer alternative (Sarfraz et al., Reference Sarfraz, Jamil, Ali, Ullah, Jabeen, Ramzan, Khan, Khan, Khan, Shakirullah, Khan and Khan2023; Baz et al., Reference Baz, Alfagham, Al-Shuraym and Moharam2024; Ratajac et al., Reference Ratajac, Pavlićević, Petrović, Stojanov, Orčić and Štrbac2024), as their biodegradability and low toxicity demonstrated in cytotoxicity tests make them suitable for integrated parasite management.
The WV has garnered significant attention not only for its efficiency in control pests and diseases but also for its positive effects in agriculture, such as promotion of plant growth (Mhamdi, Reference Mhamdi2023). Its ability to stimulate soil microbial activity while inhibiting pathogen growth is largely attributed to its antimicrobial compounds, particularly acetic acid. The high concentrations of acetic and propionic acid in WV have recently been exploited for the control of weeds (Liu et al., Reference Liu, Zhan, Li, Li, Feng, Bagavathiannan, Zhang, Qu and Yu2021). Furthermore, the presence of nicotinic acid in WV connects its applications to pharmaceutical and commercial uses aimed at improving mammalian health (Rasheed et al., Reference Rasheed, Shiha, Mohamed and Ibrahim2023; Yang et al., Reference Yang, Bao, Song, Zhao, Liang, Yu and Qu2024). The sulphate content in WV, known to affect the biology of species like Daphnia magna (Palma et al., Reference Palma, Palma, Matos, Fernandes, Bohn, Soares and Barbosa2009) and Myzus persicae (Douglas, Reference Douglas1988), may also influence H. tuberculatus, though this requires further investigation. Additionally, the high volatility and significant repellent properties of methyl isoeugenol (Scolari et al., Reference Scolari, Valerio, Benelli, Papadopoulos and Vaníčková2021) known to deter pests across various orders (He et al., Reference He, Zhang, Shen, Wang, Qian, Lyu, Cong, Cai, Chen, Wen, Wen and Cai2023; Niassy et al., Reference Niassy, Mohamed, Cheseto, Omuse, Ochola, Khamis, Badji, Ndlela, Ombura, Okun, Kupesa, Dubois, Belayneh, Subramanian and Ekesi2023; Santos et al., Reference Santos, Silva, Santos, Dantas, Tavares and Andrade2023) enhance WV’s potential. Propionic acid, the most abundant compound in WV, is a well-known lethal and repellent agent effective against ectoparasites and useful in packaging to prevent infestations (Germinara et al., Reference Germinara, Conte, Lecce, Di Palma and Del Nobile2010). Its dose-dependent effects, as demonstrated in this study, align with findings from similar natural products (Germinara et al., Reference Germinara, Rotundo and De Cristofaro2007).
The WV has also shown promising results against mosquitoes, with lethal effects reported by Paulraj et al. (Reference Paulraj, Reegan and Ignacimuthu2011). Its dose-dependent properties are consistent with those observed in other natural extracts (Malheiro et al., Reference Malheiro, Casal, Cunha, Baptista and Pereira2016; Bonanomi et al., Reference Bonanomi, Zotti, Cesarano, Sarker, Saulino, Saracino, Idbella, Agrelli, D’Ascoli and Rita2021) supporting its versatility. Additionally, WV has been studied for wood preservation (Akkuş et al., Reference Akkuş, Akçay and Yalçın2022), its efficacy against stored-product pests (Hashemi et al., Reference Hashemi, Safavi and Estaji2014; Othman et al., Reference Othman, Elias and Zainalabidin2023) and general in pest control (Kim et al., Reference Kim, Seo, Lee and Lee2008; Kiarie-Makara et al., Reference Kiarie-Makara, Yoon and Lee2010). In a recent study, Jesu et al. (2023) demonstrated that a similar by-product could alter the fitness of Bactrocera oleae on field and also the fly microbiome upon ingestion, despite its congenital plasticity (Jesu et al., Reference Jesu, Vinale, Lorito and Laudonia2024), suggesting that similar effects might occur in H. tuberculatus. However, further studies are needed to confirm this, given the differences in feeding behaviour between the 2 species. Derivatives from biochar production have shown broad pesticidal (Bonanomi et al., Reference Bonanomi, Zotti, Cesarano, Sarker, Saulino, Saracino, Idbella, Agrelli, D’Ascoli and Rita2021; Jesu et al., 2023; Oramahi et al., Reference Oramahi, Permana, Diba and Indrayani2023) and defensive (Chenari et al., 2023; El-Fawy et al., Reference El-Fawy, Abo-Elyousr, Sallam, El-Sharkawy and Ibrahim2023; Gama et al., Reference Gama, Pimenta, Feijó, Santos, Castro, Azevedo and De Medeiros2023) potential, often with low environmental impact while supporting agricultural systems (Darwesh and Elshahawy, Reference Darwesh and Elshahawy2023; Essa et al., Reference Essa, Afifi, Thalooth and El-Ashry2023). These characteristics make WV an excellent candidate for integrated parasite management strategies under both controlled and field conditions. A recent study by Iacomino et al. (Reference Iacomino, Idbella, Staropoli, Nanni, Bertoli, Vinale and Bonanomi2024) further explored WV’s multifaceted potential, investigating its bioactivity in various settings, including field trials, greenhouse experiments and in vitro assays: in olive groves, WV failed to exhibit repellent effects against B. oleae when applied as an aerosol bu, conversely, field trials showed that a 1% WV concentration significantly reduced Meloidogyne incognita nematode infections in strawberry plants by 15%. These findings underscore the critical need to optimize WV’s concentration and application methods to maximize its benefits while minimizing adverse effects. WV’s varying efficacy across different scenarios highlights its adaptability, serving both as a pest control agent and a soil conditioner and growth promoter. With its abundant bioactive compounds and environmentally friendly production, WV aligns with the principles of a circular economy, presenting a valuable resource for organic and integrated farming systems. The results of the present study confirm the hypothesized repellent effect of WV against H. tuberculatus and indicate the possibility of using this natural product to control pediculosis in buffaloes. In addition, the in vitro bioassay developed proved to be reliable in evaluating the repellent effect of molecules against lice. However, despite the in vitro bioassay cytotoxicity tests showed no toxic effects, further and thorough analyses must be carried out to better understand the mechanisms of action before an actual and safe use of WV on field.
Author contributions
GJ, AB, GP, AN, GM and LC performed bioassay on H. tuberculatus at the laboratory of the CREMOPAR. AB and GJ designed the ad hoc set-up of the in vitro bioassay and analyzed the results. SM performed cytotoxicity assay. SM, FV and GI have carried out the chemical characterization of WV. EB and GP provided to collect lice by farm. GC, AB, GJ and LR conceived, designed and coordinated the study. FŠ reviewed the draft of a paper and formulated its final version.
Financial support
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Competing interests
The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The final article has been approved by all the authors.
Ethical standards
We obtained verbal informed consent from the owners of farm to collect the lice (Haematopinus tuberculatus) from buffaloes.
Open access
