Novel Pesticidal Efficacy of Araucaria heterophylla and Commiphora molmol Extracts against Camel and Cattle Blood-Sucking Ectoparasites

Botanical insecticides are promising pest control agents. This research investigated the novel pesticidal efficacy of Araucaria heterophylla and Commiphora molmol extracts against four ectoparasites through treated envelopes. Seven days post-treatment (PT) with 25 mg/mL of C. molmol and A. heterophylla, complete mortality of the camel tick, Hyalomma dromedarii and cattle tick, Rhipicephalus (Boophilus) annulatus were reached. Against H. dromedarii, the median lethal concentrations (LC50s) of the methanol extracts were 1.13 and 1.04 mg/mL and those of the hexane extracts were 1.47 and 1.38 mg/mL, respectively. The LC50 values of methanol and hexane extracts against R. annulatus were 1.09 and 1.41 plus 1.55 and 1.08 mg/mL, respectively. Seven days PT with 12.5 mg/mL, extracts completely controlled Haematopinus eurysternus and Hippobosca maculata; LC50 of Ha. eurysternus were 0.56 and 0.62 mg/mL for methanol extracts and 0.55 and 1.00 mg/mL for hexane extracts, respectively, whereas those of Hi. maculata were 0.67 and 0.78 mg/mL for methanol extract and 0.68 and 0.32 mg/mL, respectively, for hexane extracts. C. molmol extracts contained sesquiterpene, fatty acid esters and phenols, whereas those of A. heterophylla possessed monoterpene, sesquiterpene, terpene alcohols, fatty acid, and phenols. Consequently, methanol extracts of C. molmol and A. heterophylla were recommended as ecofriendly pesticides.


Effect of the Plant Resin Extracts on Arthropods
Bloodsucking arthropods have an elegant method of delivery for a wide range of infectious agents [4], and their safe control is very crucial. This work evaluated two plant extracts of A. heterophylla and C. molmol against four arthropods, H. dromedarii (camel tick), R. annulatus (cattle tick), Hi. maculata (cattle louse fly), and Ha. eurysternus (cattle louse). The data expressed dose and time-dependent efficacy, a similar response was observed [52,65].
All plant extracts in this study showed moderate to high toxic effects against cattle and camel ectoparasites after 24 h of exposure, and methanol extracts were more effective than hexane extracts. The mortality percent (MO%) seven days PT of H. dromedarii with 12.5 mg/mL methanol extracts of C. molmol and A. heterophylla were 100% with LC 50 (50%, median lethal concentration) = 1.13 and 1.04 mg/mL, respectively); whereas those of hexane extracts were 100% PT with 25 mg/mL (LC 50 = 1.47 and 1.38 mg/mL, respectively (Tables 1 and 2). 93.33 ± 3.33 aC 100.0 ± 0.00 aB 100.0 ± 0.00 aA 76.67 ± 3.33 aC 90.00 ± 10.00 aB 100.0 ± 0.00 aA * letters refer to significant difference; a-f: There is no significant difference (p > 0.05) between any two means, those within the same column have the same superscript letter; A, B & C: There is no significant difference (p > 0.05) between any two means for the same solvent, those within the same row have the same superscript letter. Three replicates were used for each concentration and 10 adult pests per replicate were used. Similar to the response of camel ticks, the results of this work showed that plant extracts effectively controlled the cattle tick, R. annulatus because 100% mortality% was reached seven days PT with 12.5 mg/mL methanol extracts of C. molmol and A. heterophylla (LC 50 = 1. 09 and 1.41 mg/mL, respectively) whereas those of hexane extracts were reached PT with 25 mg/mL (LC 50 = 1.55 and 1.08%, respectively) (Tables 3 and 4).   60 , and 95 values = lethal concentration that kills 50, 90, and 95% of the exposed ectoparasite; (95%CL) = lower and upper confidence limit; ** Regression line equation; X 2 = chi-square; Significant at p < 0.05 level.
Commiphora swynnertonii (Burtt) exudate had a parallel strong acaricidal effect against ticks such as Rhipicephalus appendiculatus and Amblyioma variegatum (LC 50 = 1.72 and 1.91 mg/mL, respectively, and LC 99 were 3.5 and 3.7 mg/mL, respectively) and adversely affected their reproduction capability [66]. C. swynnertonii (Burtt) stem bark exudate also induced an acaricidal effect against Rhipicephalus appendiculatus and exhibited a significant (p < 0.05) mortality and inhibition of laid eggs of ticks PT with concentrations over 25 and 90 mg/mL, respectively, and no hatching of eggs was observed in all treated groups [67]. A similar study revealed the adulticidal effect of the C. swynnertonii stem bark ethyl acetate, petroleum ether, and methanolic extracts against R. appendiculatus and A. variegatum. The petroleum ether extract exhibited higher acaricidal activity (LC 50 = 72.31 and 71.67 mg/mL, respectively) and its MO%, 156 h PT, were 100 and 87% against Amblyomma variegatum and Rhipicephalus appendiculatus, respectively [67].
A related study showed that the methanol extract of neem and Citrullus colocynthis produced an acaricidal effect against adult females, eggs, and larvae, and neem was more effective against H. dromedarii [72]. Some other materials are also effective in vitro acaricides such as peracetic acid against Boophilus annulatus and the fowl tick, Argas persicus [17] and A. persicus, infesting laying hens [18]. Moreover, some photosensitizers such as safranin and rose bengal had a strong acaricidal effect against H. dromedarii and suppressed the reproductive potential of its engorged females [16].
Lice infestation in cattle is mainly controlled by conventional insecticides [73], and to the best of our knowledge, there are no natural treatments for controlling such pests as. Data from this work showed that the methanol extracts of C. molmol and A. heterophylla effectively controlled the cattle lice, Ha. eurysternus, reaching 100% mortality PT with 6.35% of methanol extracts (LC 50 = 0.56 and 0.62 mg/mL, respectively, and 96.67 and 83.33%, respectively, PT with 6.3% hexane extracts (LC 50 = 0.55 and 1.00 mg/mL, respectively) ( Tables 5 and 6). * letters refer to significant difference; a-f: There is no significant difference (p > 0.05) between any two means, within the same column they have the same superscript letter; A, B & C: There is no significant difference (p > 0.05) between any two means for the same solvent, within the same row they have the same superscript letter. Three replicates were used for each concentration and ten numbers of adult pests per replicate were used. Studies about using botanicals against lice infesting large animals are very rare. A comparable study indicated that essential oils had in vitro and in vivo lousicidal potential against the buffalo louse, Haematopinus tuberculatus (Burmeister, 1839), in Egypt. Through filter paper contact bioassays, the LC 50 values, four minutes PT, were 2.74, 12.35, 7.28, 22.79, and 18.67% for camphor (Cinnamomum camphora, Laurales: Lauraceae), peppermint (Mentha piperita L., Lamiales: Lamiaceae), onion (Allium cepa, Asparagales: Amaryllidaceae), rosemary oils (Rosmarinus officinalis Linn, Lamiales: Lamiaceae), and chamomile (Matricaria chamomilla L., Asterales: Asteracea), respectively, and oils induced ovicidal effects except rosemary, which was not applied [33]. Moreover, essential oils of garlic, clove, pumpkin, onion, and marjoram effectively controlled the dog louse, Trichodectes canis in vitro [51] and camphor oil controlled the slender pigeon louse, Columbicola columbae, in vitro and in vivo [49].
This investigation indicated that complete mortalities were reached seven days PT for the cattle louse fly, Hi. maculata, with 12.5 mg/mL extracts of C. molmol and A. heterophylla (LC 50 values PT with methanol extract were 0.67 and 0.78 mg/mL, respectively, whereas those of hexane extracts were 0.68 and 0.32 mg/mL, respectively. After treatment with a lower concentration, 6.3%, MO% reached 100 and 93.33% PT with methanol extracts and 90 and 100% PT with hexane extracts (Tables 7 and 8). 100.0 ± 0.00 aB 100.0 ± 0.00 aA * letters refer to significant difference; a-f: There is no significant difference (p > 0.05) between any two means, within the same column they have the same superscript letter; A, B & C: There is no significant difference (p > 0.05) between any two means for the same solvent, within the same row they have the same superscript letter. Three replicates were used for each concentration and ten numbers of adult pests per replicate were used. Parallel studies of using botanicals against Hi. maculata were also recorded. The leaf of Ricinus communis, Malabarica malabarica, and Gloriosa superba (methanol, chloroform, and chloroform extracts, respectively) effectively controlled Hi. maculata and the tick Haemaphysalis bispinosa [74]. The aqueous crude leaf extracts of Catharanthus roseus had insecticidal efficacy against the adults of Hi. maculata and the sheep-biting louse, Bovicola ovis (LD 50 = 36.17 and 30.35 mg/L, respectively) [75].
Likewise, in our findings, some Oil-resins had larvicidal activity against Culex pipiens such as C. molmol, A. heterophylla, Boswellia sacra, Pistacia lentiscus, and Eucalyptus camaldulensis. After treatment for 24 and 48 h PT with 1500 ppm, the best effect was observed PT with acetone extracts of C. molmol, 83.3% and 100% with LC 50 values were 623.52 and 300.63 ppm, as well as A. heterophylla, 75% and 95% with LC 50 values, were 826.03 and 384.71 ppm, respectively. On the other hand, the aqueous extract of A. heterophylla was highly effective against Cx. pipiens (LC 50 = 2819.85 and 1652.50 ppm) followed by C. molmol (LC 50 = 3178.22 and 2322.53 ppm) 24 and 48 h PT, respectively [59]. As mosquito larvicides, A. heterophylla and Azadirachta indica (gum polysaccharides) were used for encapsulation of cyfluthrin-loaded superparamagnetic iron oxide nanoparticles [61].

Biochemical Analysis
It was noticed that most of the compounds belong to sesquiterpene, fatty acid esters and phenols were the most common compounds found in the methanol and hexane extracts of the myrrh, C. molmol plant while monoterpene, sesquiterpene, terpene alcohols, fatty acid, and phenols were found in in methanol and hexane extracts of A. heterophylla plant in larger amount.
Phytochemical analysis of this work revealed that the constituents of C. molmol and A. heterophylla extracts were identified by GC-MS analysis (Tables 9-12)      Parallel studies demonstrated that the Araucariaceae family including A. heterophylla, produces several monoterpenes, such as pinene, camphene, and limonene as common compounds [78]. Araucaria spp. contains various sesquiterpenes like humulanes, cadinanes, caryophyllanes, and other compounds [79]. The resin of Araucaria columnaris is rich in aromadendrene and bicyclogermacrene and contains sesquiterpene hydrocarbons and oxygenated sesquiterpenes [80,81]. Similar studies indicated that A. heterophylla contained flavonoids, sesqui and di-terpenes, and phenylpropanoids [81]; two monoterpene resins, b-pinene and a-pinene, were commonly found in wood found in Araucaria angustifolia and such compounds were detected in Norway spruce with many monoterpenoids in wood and bark [82].
Finally, our data and others confirm that the presence of many secondary metabolites such as sesquiterpenes, phenols, aromatic terpenoids, fatty alcohol, eugenol, and many other bio-effective compounds may explain the effectiveness of A. heterophylla and C. molmol resin extracts against insect pests [82,84,85].
Phenolics are linked to toxicity against because they are important in plant-herbivore and pathogen interactions. Antioxidant characteristics were found in phenolic chemicals, which are thought to be the primary cause of the pesticide effect in nature [86]. The mode of action of C. molmol extract was revealed through histopathological and transmission election microscope of treated A. persicus via penetrating the cuticle towards the body cavity of treated ticks, destroying the epithelial gut cells, and ultimately resulted in the death of ticks. Moreover, lysing of epithelial gut cells with an irregularly distributed nucleus was commonly PT with low concentrations and rarely PT with high concentrations of C. molmol, whereas lysed epithelial gut cells (without nucleus or with aggregated one beside the basal lamina) were commonly observed PT with high concentrations and rare recorded PT with low concentrations [65,69]. Using plant-based pesticides had minimum or low toxicity for non-target organisms [5]. Specifically, the safety of Commiphora spp. was confirmed after oral toxicity in mice and rats [63].

Pest Collections
The collection of the adult stage of four pests of mixed sex was done from May to July 2021. The camel tick, Hyalomma dromedarii (Koch, 1844) and cattle tick, Rhipicephalus (Boophilus) annulatus, formerly Boophilus annulatus (Say, 1821), (Acari: Ixodidae), were collected from areas around infested camel and cattle, respectively, at the slaughterhouse in Jazan Province, Saudi Arabia. The adult cattle louse fly, Hippobosca maculata Leach (Diptera: Hippoboscidae) was collected from infested cattle mainly in the ears and tails. The cattle louse, Haematopinus eurysternus, was collected from the dewlap, cheeks, neck, flank, withers, and back of infested cattle. Pests were collected from and around animals that had no previous exposure to pesticides.

Collection of Plant Materials
A. heterophylla and C. molmol were collected from different areas in Saint Catherine (28 •

Preparation of Plant Extracts
Stock solutions of the plant oil-resins A. heterophylla and C. molmol were extracted by mechanically grinding 50 g of both plant oil-resins using a stainless-steel electric mixer and placing the powder in a Soxhlet apparatus for 6-8 h according to the type of solvent. Methanol and hexane were used as solvent, individually. The solution was filtered using Whatman No. 1 filter paper through a Buchner funnel, and the extracts were dried in an oven at 30 • C for 6 h. The extracts were stored in a dark bottle in a refrigerator at −5 • C for 24 h prior to the experiment [52].

Bioassays
The pesticide effectiveness of methanol and hexane extracts of A. heterophylla and C. molmol was evaluated against four ectoparasites, H. dromedarii, R. annulatus, Hi. maculate, and Ha. eurysternus. Preliminary experiments each containing 30 adult pests, grouped in three replicates, were made to evaluate the range of concentrations used for each pest. Treated envelopes were used [74]. The adult cattle and camel ticks were treated with the following concentrations: 1.6, 3.1, 6.3, 12.5, 25 mg/mL, while adult cattle louse fly and cattle louse were treated with the following concentrations: 0.8, 1.6, 3.1, 6.3, 12.5 mg/mL. Three replicates (each contained ten adult pests) were used for each concentration.
Each group of pests were added to a filter paper envelope, Whatman filter paper No.1, 125 mm diameter, and treated with a single concentration of the plant extracts as 3 mL test solution uniformly distributed with a pipette on internal surfaces of the envelopes. The control envelopes were impregnated with distilled water. The opening of the envelopes was folded and secured with a metallic clip with its identification marks like tested solution and concentration. Each treated replicate of pests was transported to a Petri dish lined with a filter paper. Treated pests were kept at 28 ± 2 • C and a relative humidity of 80 ± 5%. Mortalities were recorded one, three and seven days post-treatment (PT).

Biochemical Analysis
Biochemical analyses were made using GC/MS, a Thermo Scientific Trace GC Ultra/ISQ Single Quadrupole MS, TG-5MS fused silica capillary column, 0.1 mm, 0.251 mm, and 30 m thick. An electronic ionizer with 70 eV ionization energy was used. Helium gas was utilized as a carrier gas (flow rate = 1 mL/min). The injector and MS transmission line were set at 280 • C. The oven temperature was set at 50 • C, then increased to 150 • C at a rate of 7 • C per minute, then to 270 • C at a rate of 5 • C per minute (wait for 2 min), and finally to 310 • C at a rate of 3.5 • C/min (continued for 10 min). To investigate the quantification of all components found, a relative peak area was used. By comparing the retention periods and mass spectra of the chemicals with those of NIST, Willy Library data from the GC-MS instrument, and the chemicals were tentatively identified. The collective spectra of user-generated reference libraries were used for identification. Single-ion chromatographic reconstructions were used to assess peak homogeneity. Co-chromatographic analysis of reference compounds was performed whenever possible to confirm GC retention times [87,88].

Data Analyses
The data were analyzed by the software, SPSS V23 (IBM, New York, NY, USA), for doing the Probit analyses to calculate the lethal concentration (LC) values and the one-way analysis of variance (ANOVA) (Post Hoc/Turkey's HSD test). The significant levels were set at p < 0.05.

Conclusions
It is crucial to safeguard livestock and domesticate animals from blood-feeding ectoparasites and vector-borne diseases. Worldwide, pest control is dependent on conventional pesticides, but resistance has developed to almost all classes of pesticides. Botanicals as eco-friendly pesticides represent conspicuous alternatives because of the wide diversity and high effectiveness of several plant-borne compounds. This study revealed, for the first time according to our knowledge, the efficacy of methanol and hexane extracts of C. molmol and A. heterophylla against four camel and cattle blood-sucking arthropods.
Our results confirmed that cattle lice and the louse fly were more susceptible (12.5 mg/mL) than cattle and camel ticks (25.0 mg/mL) to A. heterophylla and C. molmol extracts. Both methanol extracts were recommended as an ideal eco-friendly and inexpensive pest control approach that could be incorporated into integrated pest management used for the protection of large animals from vectors and vector-borne diseases. Further studies could be directed towards the field application and safety profile of C. molmol and A. heterophylla against non-target organisms as well as studying the synergistic effects of surfactants.