Evaluation of the in vitro acaricidal activity of ethanol extracts of seven Chinese medicinal herbs on Ornithonyssus sylviarum (Acari: Macronyssidae)

Ornithonyssus sylviarum (Acari: Macronyssidae) is a common ectoparasite that feeds on the blood of poultry. Following infestation, this mite will cause symptoms such as weight loss, anemia, and decreased egg production. To explore green and safe drugs for the prevention and treatment of O. sylviarum, this study evaluated the effects of ethanol extracts of seven Chinese medicinal herbs—Leonurus artemisia (motherwort), Illicium verum (star anise), Cinnamomum cassia (cinnamon), Hibiscus syriacus, Artemisia argyi (Chinese mugwort), Taraxacum sp. (dandelion), and Syzygium aromaticum (clove)—on O. sylviarum at different life stages. The results showed that different methods of administration affected the acaricidal efficacy of these plant extracts on O. sylviarum. After 6 h of administration with the fumigation method, the acaricidal efficacy of S. aromaticum on adults, nymphs and larvae of O. sylviarum reached 100%. 30 min after administration with the infiltration method, S. aromaticum, H. syriacus and L. artemisia showed acaricidal effects on adults and nymphs of O. sylviarum reaching 100%. In another experiment evaluating the inhibition of egg hatching of O. sylviarum with alcohol extracts of these seven herbs, at 48 h after treatment, A. argyi and C. cassia showed inhibition rates of 19.4%. The results of this study indicate that S. aromaticum induced mortality at all stages of O. sylviarum, whereas A. argyi was found to be the most effective at inhibiting the mite’s egg hatching among the seven herbs. These herbs can therefore be used as potential substitutes for chemical pesticides to prevent and control O. sylviarum. These results provide practical knowledge for the control of O. sylviarum.


Introduction
The northern fowl mite (NFM), Ornithonyssus sylviarum (Canestrini & Fanzago) (Acari: Macronyssidae), is a permanent ectoparasite that feeds on the blood of birds (incl. poultry) and humans. The NFM often parasitizes the vent region of the host and completes its entire life cycle on the host (Murillo and Mullens 2017). Although there have been no reports of NFM bites on humans, Dermanyssus gallinae and Ornithonyssus bursa bites have been reported, and people who are bitten can experience skin diseases such as skin itching, rash, dermatitis and urticaria (Castelli et al. 2015;Sioutas et al. 2021). Infested birds have clinical manifestations of restlessness, feather pecking, skin inflammation, anemia, slowed growth, and decreased egg production and quality (Murillo et al. 2020;Vezzoli et al. 2016). In severe infestations, the number of NFMs on a chicken can reach 50,000, a level that will cause the infested chickens to lose up to 6% of their blood every night (DeVaney 1981). The NFM is also one of the most prevalent ectoparasites on poultry farms in most parts of the world (Jansson et al. 2014;Knee and Proctor 2007;Waap et al. 2017). According to previous reports, > 70 species of wild birds in North America can serve as hosts of the NFM, including starlings and house sparrows (Knee and Proctor 2007). China is considered the world's largest egg producer, and poultry farming is an important industry in this country. According to an epidemiological survey of ectoparasites in China (Wang et al. 2010), 91.3% of poultry farms are infested with ectoparasites, among which O. sylviarum accounted for 46.9%.
At present, the most common method used to control ectoparasites is the spraying of chemical insecticides. In China, pyrethroids, organophosphates, cypermethrin or other acaricides are used to control ectoparasites in 95% of laying hens and 74.9% of breeders (Wang et al. 2010). There are two problems with spraying acaricides to prevent and control NFMs. First, NFMs can survive for several weeks in an environment without a host, and chemical acaricides must be applied to large areas in chicken coops, which is likely to create problems with chemical residues (Gokbulut et al. 2019). In addition, mites are small, and they attach to the roots of chicken feathers to lay eggs and reproduce, causing the feathers to become densely covered with mite bodies and a large quantity of mite feces. When infestations are high, the feathers soiled by the mites will stick together, which greatly increases the difficulty of applying medication. In this situation, it is necessary to apply the acaricides multiple times, increasing the probability of pesticide resistance, which generally leads to problems such as decreased efficacy (Marangi et al. 2009). To reduce the use of chemical pesticides and address the problems associated with insecticide residues and insecticide resistance, physical and biological methods have been used in some European countries for prevention and control, including the use of inert dust, diatomaceous earth, synthetic silica (Kilpinen and Steenberg 2009), and fungi such as Metarhizium anisopliae, Beauveria bassiana, Aspergillus oryzae and others (Wang et al. 2019;Steenberg and Kilpinen 2014). Furthermore, natural plant essential oils have shown promise as substitutes for synthetic pesticides. According to reports, many plant essential oils, such as carvacrol, thymol, and the essential oils of fennel and lemongrass, have shown in vitro efficacy against D. gallinae and Sarcoptes scabiei (Baran et al. 2020;Li et al. 2020;Nechita et al. 2015).
To explore safe and effective potential insecticides, extracts of seven plants used as Chinese herbal medicines, including those obtained from clove and motherwort, were used in mortality tests against NFMs.

Ethanol extraction of herbal medicines
Fifty grams of the above herbs was crushed and passed through 20-mesh screens. Next, 200 mL of a 90% ethanol solution was added; the samples were soaked for 1 week and then filtered through six layers of gauze. The residue was then added to 100 mL of a 90% ethanol solution and soaked again for 24 h. This solution was filtered through six layers of gauze, and then the two filtrates were combined. The filtrate was centrifuged at 3000 rpm for 10 min, and the ethanol was evaporated from the supernatant in a boiling water bath to concentrate it into a paste. This paste was diluted to 50 mL with 0.9% sodium chloride; i.e., 50 g of each herb was used to make 50 mL of the experimental insecticide. This insecticide was then stored at 4 °C for later use.

Acquisition of mites
A suspected case of chicken mite infestation occurred at a chicken farm in Yuanyang County, Xinxiang city, Henan Province, China, with dense, fast-moving mites appearing in the vent region of the sick chickens ( Fig. 1). A small brush was used to transfer the mites into a No. 4 seal bag, which was taken to the Parasitology Laboratory of Henan Agricultural University. Under a stereomicroscope, using the identification method of Di Palma et al. (2012), the morphology of the mouth organs, horns, and vent region of the mites was examined, and the molecular method of Bhowmick et al. (2019)  sylviarum was then transferred to a 9-cm Petri dish, and a layer of Vaseline was applied around the dish to prevent the mites from crawling out until they were used in subsequent trials.

Infiltration method
All herbal medicine extracts were diluted to 0.5 g/mL. Ten viable mites were selected under a stereomicroscope (mites were grouped into adults + nymphs and larvae, 10 mites in both groups) and placed into the wells of a 24-well cell culture plate (Nest Biotechnology, Shanghai, China), to which 0.3 mL of an herbal extract (0.5 g/mL) was added. There were three replicates per set of experiments. The negative control was treated with 0.9% NaCl, and the positive controls were treated with 100 mg/mL ivermectin and 5% cypermethrin. The wells were observed for 3 h, and the numbers of dead and surviving mites were recorded at 0.5 h, 1 h and 3 h. If a mite remained immobile after being continuously stimulated with a needle for 1 min, it was considered to be dead.

Fumigation
All herbal medicine extracts were diluted to 0.5 g/mL, and healthy viable mites at different stages (adults + nymphs are in a group, and larvae in another, 10 mites in both groups) were selected under a stereomicroscope and placed in a 2-mL Eppendorf tube. Each tube was sealed with a cotton ball to which 0.8 mL of herbal liquid (0.5 g/mL) had been added. At the beginning of the evaluation of each herb extract, the negative control was treated with 0.9% NaCl, and the positive controls were treated with 100 mg/mL ivermectin and 5% cypermethrin. Three replicates were used for each set of experiments. Mite mortality was observed at 6 h, 12 h, and 24 h under a stereomicroscopy. If a mite remained immobile after being continuously stimulated with a needle for 1 min, it was considered to be dead.

In vitro evaluation of the ovicidal activity of alcohol extracts of herbs
Twenty healthy, viable female adults were selected and transferred to 36-well cell culture plates (Nest Biotechnology). The plates were placed in a constant temperature and humidity incubator, and observed every 24 h to collect fresh, healthy eggs. The incubator was at 26 °C, 65-70% RH, and completely dark. Groups of 10 eggs were collected. There were three replicates of each group. A 0.2 mL aliquot of the diluted herbal liquid (0.5 g/mL) was placed in full contact with the eggs for 3 min; the eggs were then transferred to fresh nonherbal solution in a 36-well cell culture plate, and the plate was placed in a constant-temperature and -humidity incubator. Observations were made at 24 h and 48 h. Positive and negative controls were also established. The positive controls were 100 mg/mL ivermectin and 5% cypermethrin, whereas the negative control was 0.9% NaCl. A lower hatching rate indicates a stronger inhibition of egg hatching.

Statistical analysis
The experimental data were sorted with Excel, and mortality of the test group was calculated: mortality (%) = [no. dead mites/total no. tested mites] × 100%. The mortality in the treatment groups was corrected to take into account control mortality using Abbott's formula (Abbott 1925): corrected mortality (%) = [1 -% dead mites in the treated plate/% dead mites in the untreated control plate] × 100%. SPSS v.20.0 was used to perform oneway ANOVA on the experimental data, followed by least significant difference (LSD) tests.

Adult mites/nymphs
After 30 min of treatment, the mortality of adult and nymphal mites associated with clove, hibiscus and motherwort reached 100%, similar to the mortality in the positive control groups (ivermectin, 100%; cypermethrin, 93%); these values were significantly higher than those associated with Chinese mugwort (77%), dandelion (73%), star anise (57%), and cinnamon (13%) ( Table 2). No mortality occurred in the negative control group. After 1 h of treatment, the mortality rate associated with cypermethrin in the positive control reached 100%, which was significantly higher than that observed for the extracts of cinnamon (83%), Chinese mugwort (80%), dandelion and star anise (both 73%) ( Table 2). After 3 h, the corrected mortality rate associated with Chinese mugwort reached 100%, and the mortality rates associated with dandelion and cinnamon (both 85%) were significantly higher than that observed for star anise (69%) and the negative control group (13%) ( Table 2).

Larvae
After 30 min of treatment, the larval mortality caused by clove, motherwort and ivermectin (positive control) reached 100%, whereas that caused by hibiscus and dandelion reached 90%-all were significantly higher than the mortality observed for cypermethrin (70%), cinnamon (50%), and Chinese mugwort (47%) ( Table 2); star anise and the negative control caused no larval mortality at all. After 1 h of treatment, the mortality associated with hibiscus (93%) and dandelion (90%) was significantly higher than that associated with cypermethrin (70%), Chinese mugwort (63%), and cinnamon (60%); there was no statistical significance between the star anise group (3%) and the negative control group (0%) ( Table 2). After 3 h, the mortality caused by hibiscus reached 100% and that caused by dandelion 90%, both significantly higher than the mortality caused by cypermethrin,

In vitro evaluation of the ovicidal activity of herb extracts
After 24 h, the lowest hatching rates were observed of motherwort, Chinese mugwort and dandelion (9%, 10% and 13%, respectively) followed by cinnamon and clove (16% and 20%, respectively); the results for these herbs were significantly different from those for the negative control (43%) ( Table 4). There were no significant differences in the hatchability rates of eggs exposed to star anise and hibiscus (both 37%), or the negative control (43%). The hatchability of eggs exposed to cypermethrin and ivermectin (both 13%) were significantly lower than that of star anise and hibiscus (both 37%) and negative control (43%) ( Table 4).

Discussion
In this study, the different administration methods and life stages significantly affected the mortality rate, as summarized in Fig. 2. The results showed that hibiscus, clove and motherwort had better acaricidal efficacy on NFMs using the infiltration method, whereas cinnamon had higher and more stable repellent activity on NFMs using the fumigation method. Among the herbs, star anise (infiltration) and Chinese mugwort (fumigation) only had acaricidal effects on the adult and nymphal stages of NFMs, and dandelion (fumigation) only on the larval stage. Clove and hibiscus had 100% mortality to adults, nymphs and larvae of NFMs using both infiltration and fumigation methods. This study is the first to find that motherwort and hibiscus have acaricidal activity against NFMs. In the in vitro evaluation 1 3 of the effects of alcohol herb extracts on the egg hatching rate of the NFMs, after 48 h, Chinese mugwort was found to have the lowest hatching rate of 19%. According to previous studies, terpenoids, flavonoids, sugars, phenols and alkaloids can inhibit the activity of acetylcholinesterase, causing toxicity in the mite nervous system. In addition, these chemicals can act on other targets in the mite nervous system such as nicotinic acetylcholine receptors (Nacr), octopamine receptors, tyramine receptors, sodium channels and gamma-aminobutyric acid (Gba)-gated chloride channels (Pritchard et al. 2015). These may be the mechanism of action of herbal remedies against mites, as the active ingredients in herbs are complex and abundant. These results have been confirmed in a number of studies on herbal mite control. Lee et al. (2019) reported that clove contains a volatile oil (15-20%) and that this oil is mainly composed of eugenol (78%-95%), acetyl eugenol (7.3%), and ß-caryophyllene (9%). At a concentration of 1.3 µg/m 2 , it has a 100% acaricidal effect on D. gallinae (Lee et al. 2019;Tabari et al. 2020). In a contact test of the effects of plant essential oils on mites, clove was found to have the highest contact toxicity, with an LC 50 value of 8.9 µg/ mL; in addition, clove has been reported to have repellent and acaricidal activity on various arthropods, such as the whitefly Bemisia tabaci and the ticks Rhipicephalus microplus and Dermacentor nitens (Kim et al. 2011;Zeringota et al. 2013). Studies have reported that Chinese mugwort contains a variety of biologically active compounds such as phenols, flavonoids, eucalyptol, β-caryophyllene, camphor and other components (Xiao-Yan et al. 2020). This herb has antibacterial and antiviral effects, can stop bleeding and pain, and can be used externally to reduce dampness and relieve itching (Dib and El Alaoui-Faris 2019). Chinese mugwort is widely used in China not only for moxibustion and foot bathing but also for consumption, as in the traditional Chinese food 'Qing Tuan'. Chinese mugwort has often been reported to have insecticidal activity; for example, it has obvious acaricidal effect on Demodex folliculorum, D. brevis and the cabbage aphid Brevicoryne brassicae (Ahmed et al. 2020;Du et al. 2021). According to previous research (Zhang et al. 2014), the four compounds it contains (β-caryophyllene, eucalyptol, β-pinene and camphor) have strong acaricidal activity on adults of Lasioderma serricorne. These compounds might also act on NFM eggs, which would explain the inhibitory effect of Chinese mugwort found in the current study.
Hibiscus (Malvaceae) contains many components such as alkenes, esters, aldehydes, alcohol compounds, and alkanes (Wei et al. 2015). At present, there are no reports on the acaricidal effect of hibiscus. Only one study addresses its insecticidal effects. For the larvae and adults of the pollen-feeding phytoseiid mite Amblyseius swirskii, eating hibiscus pollen can cause 100% mortality (Goleva and Zebitz 2013).
Motherwort is mainly distributed in Russia and China. It promotes blood circulation, regulates menstruation, has detoxification effects, and reduces fever, diuresis, and swelling (Wojtyniak et al. 2013). A variety of compounds such as monoterpenes, flavonoids, phenolic acids, volatile oils, and sterols have been identified in motherwort (Shang et al. 2014). The acaricidal activity of motherwort has not been previously reported.
Many plant essential oils and herbal extracts contain various volatile compounds with fumigation effects such as alkanes, alcohols, aldehydes, and especially terpenes and monoterpenes (Bordin et al. 2021). The main component of cinnamon bark oil is cinnamaldehyde (Lee et al. 2019), which may explain the difference in NFMs mortality between the infiltration method (13% after 30 min exposure) and the fumigation method (87% after 6 h).
Therefore, when extracting herbs, a higher extracted content may result in a higher toxicity in mites. However, plant components may vary due to many factors, such as the plant location, extraction method, plant age, cultivation conditions and harvest time (Fernandes et al. 2019;JerkovicÂ and Milos 2001). For example, it has been reported that star anise exhibits a substantial mortality effect on D. gallinae (Tabari et al. 2020), but the effect observed in our experiment was not substantial. This may be related to the extraction method and place of origin.

Conclusions
In this study, S. aromaticum (clove), A. argyi (Chinese mugwort), H. syriacus (hibiscus) and L. artemisia (motherwort) extracts caused significant mortality of O. sylviarum at different life stages. The mechanisms and compositions associated with the effects on O. sylviarum are currently unknown, so further in-depth study is needed. In addition, if these herbs are mixed at appropriate treatment dosages and used together with appropriate control methods (spraying or fumigation), greater mortality and stability could be achieved.