Chapter 2 | Group 6 Del Mundo

BROWN DOG TICKS

Brown dog ticks (Rhipicephalus sanguineus) are the most prevalent ixodid tick worldwide (Gray et al., 2013). Given their name, they appear in colors ranging from yellowish-brown to uniform reddish-brown and are external parasites in dogs. They are minute arachnids that only grow from four to five millimeters and belong to the subclass Acari—where ticks and mites also reside (University of Saskatchewan, 2016). They are mostly found on the ears and toes of dogs, seldom on the back.

Larval ticks, which only come in body dimensions of 0.54 millimeters in length and 0.39 millimeters in width, only carry six legs but grow two additional legs during their nymphal stage (University of Florida, 2018). Furthermore, nymphs only grow from 1.14 to 1.30 mm in length and 0.57 to 0.66 mm in width, while adult brown dog ticks grow from 2.28 to 3.18 mm in length and 1.11 to 1.68 mm in width. Moreover, R. sanguineus has festoons and a hexagonal basis capitulum, characteristics that set them apart from other ticks (Abdel-Shafy et al., 2015). Following the aforementioned study by the University of Florida, male and female brown dog ticks only differ in their scutum. Scutums are darkened shield-like appendages that cover the dorsal surface in males while protecting only the anterior dorsal in females. Ticks with a splash of white color on their dorsal surface can not be considered brown dog ticks (University of Florida, 2018). These collections of characteristics set brown dog ticks apart from other organisms. Their morphological structure capacitates them to infest domestic animals like dogs, cats, pigs, and cattle (D’Souza, 2016; Weaver et al., 2022; Maggi et al., 2013).

MORPHOLOGY

Photo credits: ticksafety.com

T
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Despite only being considered a nuisance because of their size, brown dog ticks pose a huge threat to their hosts, which are common domestic animals. Brown dog ticks may cause intense allergic reactions and anemia in their hosts (Ybañez et al., 2018). They are also vectors of protozoan parasites, including Babesia and Theileria that wreak babesiosis and theileriosis, respectively (Llera & Ward, 2018; Rosa et al., 2014). Brown dog ticks are also vectors of microorganisms, such as the bacteria Anaplasma and Ehrlichia canis that cause anaplasmosis and ehrlichiosis, respectively (Longley, 2020; Livestock Biosecurity, 2022). The impact of brown dog ticks as ectoparasites does not stop with domestic animals; they occasionally cause harm to humans as well. As IGeneX Inc. (2019) stated, although there are rare cases of brown dog tick bites in humans, they may still spread if there are no more canines for them to feed on. Also, according to this literature, they can spread the fatal Rocky Mountain Spotted Fever by carrying the bacteria Rickettsia rickettsii. The threat that brown dog ticks constitute to both humans and animals necessitates the need to address the issue behind it. However, the solution to this problem is not that easy to come up with as they have characteristics that make them still abundant and prevalent.

ABUNDANCE

Infestations of brown dog ticks are difficult to manage because the parasites are not the only ones that cause harm to other organisms. Being a vector of pathogens, their feces, eggs, and dirt add up to the harm they cause to animals and humans (Coates, 2019). Based on Beugnet & Chalvet-Monfray (2013), temperature and humidity are also factors that contribute to the adversity of solving this problem. Although the ideal temperature for brown dog ticks only ranges from 75° to 85°F with at least 85% humidity, they can still adapt to cold surroundings. This makes their infestations last throughout the year. The Philippines being a tropical country makes it the perfect host for these parasites.

CONTROL:
HOME MANAGEMENT

CONTROL:
ACARICIDES

Although brown dog tick infestations can be controlled by doing the following steps: bathing the animals, cleaning the house, mowing the lawn, and vacuuming the house to sanitize it (University of Rhode Island, 2022), the number of steps required and time allotment for each step hinders pet owners from managing brown dog tick infestations (Coles & Dryden, 2014). All of these added obstacles that make the domination of these parasites arduous only prompted scientists and manufacturers to provide more innovative and effective solutions to the problem.

labour-intesive & time-consuming

Scientists and manufacturers formulated anti-ticks (acaricides) using synthetic chemicals and compounds to provide a solution to different domestic animal ectoparasites. Anti-ticks commonly have pyrethroids, one of the most practical components of acaricides (Onyango, 2016). However, despite the formulations of synthetic acaricides against brown dog ticks, they are only partially successful, since they require chemicals that are heavy for the environment, which necessitates the need to address another issue (Basu & Charles, 2017).

Synthetic acaricides are expensive and detrimental to the environment. Basu & Charles (2017) indicated the downside of utilizing synthetic acaricides which is the harmful after-effects they pose on dairy products and meat intended for human and animal consumption. Nevertheless, Quadros et al., (2020) determined that numerous plant species contain natural acaricidal properties such as cultivated tobacco and the caster oil plant. However, there have been instances of natural acaricides' inconsistent effectiveness (Gashout et al., 2020). In addition, some brown dog ticks have eventually developed resistance to commercial anti-tick products (Staffa, 2018).

non-eco-

friendly

harmful if

consumed

inconsistent

effectiveness

developed

resistance

In summary, this paper tackles the following problems: brown dog tick infestations and their treatment, the drawbacks of synthetic and commercial acaricides, the erratic effectiveness of natural acaricides, and the acaricidal resistance of the aforementioned parasites. Even so, providing a solution to these brown dog tick infestations is challenging because a flaw is always observed in the process. With this, the production of acaricidal products must implement more effective and modernized methods and processes such as the growing concept of nanotechnology.

COPPER NANO-PARTICLES

From physical and mechanical to therapeutic applications, nanotechnology has been regarded as a fast-developing field of technology that plays an essential role in various fields of science (Youssef et al., 2019). Nano, from Latin origins nanus, meaning very little in size is emphasized in its materials’ properties with sizes ranging from only 1 to 100 nm (Choudhury et al., 2021). Nanoparticles, because of their extremity in size, possess unique features such as their increased surface area, absorption rate, and ability to carry compounds like drugs, probes, and proteins (De Jong & Borm, 2008). Because of this, they are now being utilized as an enhancer of the solubility and bioavailability of insoluble or poorly soluble drugs (Bandgar et al., 2018). Nanoparticles also safeguard drugs from gastrointestinal enzymes that may wear them down and lessen their effectiveness (Khatra et al., 2013). Aside from treatments targeting the well-being of humans, nanoparticles have also been used in the field of veterinary medicine—mostly, in vaccine formulations (Osama et al., 2020). Some uses of it also include cryopreservation of gametes, gonadal tissues, and embryos that are essential for reproduction (Choi & Jang, 2022).

Nanoparticles can be classified according to their structure, synthesis, shape, and purpose. The two main classifications of nanoparticles are organic and inorganic nanoparticles. Organic nanoparticles are nanoparticles from microdroplets, polymer and organic microcrystals, swollen micelles, and unimolecular and polymer fine particles. The main reasoning behind the sudden bloom of interest regarding organic nanoparticles mostly revolves around them having different characteristics from their large organic molecule origins (Masuhara et al., 2003; Tsuzuki et al., 2010; Zhang et al., 2015). In contrast, inorganic nanoparticles are nanoparticles synthesized commonly from inorganic materials such as metals and metal oxides. Inorganic nanoparticles can be further classified into metal oxide-based, carbon-based, and metal-based nanoparticles. Metal oxide-based nanoparticles are similar to metal-based nanoparticles however with the addition of an oxidizing agent to change the features of the resulting nanoparticles while carbon-based nanoparticles are nanoparticles made wholly from carbon molecules and further organized into more subcategories such as fullerenes, graphene, carbon nanotubes, etc. (Khan et al., 2021; Haq & Jafry, 2022). On the other hand, metal nanoparticles are nanomaterials produced from the construction or destruction of bulk metals such as silver, cadmium, aluminum, cobalt, copper, iron, lead, gold, and zinc (Reverberi et al., 2016). Among the aforementioned metals, copper is the most abundant in the Philippines with approximately 4 billion MT reserves making the country the fourth largest contributor of copper in the world (Philippine Statistics Authority, 2020).

SYNTHESIZING COPPER NANOPARTICLES

Due to its cost-efficiency and higher surface-to-volume ratio, copper has been regularly investigated as a substitute for other noble metals in terms of nanoparticle production (Umer et al., 2012). Even with low cost, copper still provides promising results in terms of conductivity as compared to noble metals (Jeong et al., 2008 as cited by Muthu & Kamalanathan, 2022). Following the aforementioned papers, copper nanoparticles are often fabricated using physical (top-down) and chemical (bottom-up) methods, however, one of the major drawbacks of copper nanoparticles is the rapid oxidation to Cu2+ ions in the air or liquid matter. Some of the most common processes involving the chemical synthesis of copper nanoparticles revolve around reduction, emulsion, the use of electromagnetic energies, and the use of pressure and temperature. Among these methods and according to several studies involving the use of chemical synthesis of copper nanoparticles, chemical reduction presented the widest range of nanoparticle sizes from 2 nm to 100 nm. Physical synthesis of copper nanoparticles, on the contrary, involves laser ablation which produces colloidal solutions that prevent oxidation, milling, pulsed wire discharge, and sonochemical method (Ranjbar et al., 2017). Among these four, laser ablation produced the smallest nanoparticles with sizes ranging only from 3-9 nm (Xiong et al., 2011, Wang et al., 2006, and Muniz-Miranda et al., 2011 as cited by Khodashenas & Ghorbani, 2014). The decrease in the particle size of nanoparticles increases their surface area, and ability to interact with other particles, and amplifies their efficiency as an antimicrobial agent (Karthik & Geetha, 2013). This is also in line with Sanchez-Sanhueza (2016) who concluded an inversely proportional relationship between the size and antimicrobial properties of nanoparticles.

In 2014, Chatterjee et al. conducted a study involving gelatin-mediated copper nanoparticles against the growth of gram-negative bacteria and found that the concentration of copper nanoparticle solution was directly proportional to the cell filament size, which prohibits cell reproduction and shows high potentiality in cell killing. Nanoparticles also possess the ability to inhibit the antibiotic resistance of bacteria since they can target multiple biomolecules simultaneously. Zaheer et al. (2022) tackle how metal nanoparticles kill ticks. Silver nanoparticles function by binding with the P and S regions of nucleic acids and proteins which causes the cell membrane permeability to decrease. This degrades the enzyme the nanoparticles were attached to and kills the cell. Silver nanoparticles can also diminish the acetylcholinesterase activity of the cell, which denatures the enzymes they are attached to and causes the cells to die. The same literature stated that gold nanoparticles bind with trypsin enzymes and replace its substrate, causing inactivity of the cell. This results in the poor development and malabsorption of ticks.

Additionally, copper nanoparticles synthesized using chemical reduction of copper ions exhibit high antifungal activity against Neofusicoccum sp., F. Oxysporum, and F. solani through damages found on the cell membranes and the intracellular production of reactive oxygen species (Crisan et al., 2022). In the field of parasitology, various nanoparticles have also shown various effects on different kinds of parasites (Kandeel et al., 2022). Some of the most distinctive nanoparticles used are silver against Fasciola (Brodaczewska et al., 2013), chitosan against Trichinella spiralis (Abulaihaiti et al., 2015), gold against Leishmania major (Lopes et al., 2018), and copper against Plasmodium (Obisesan et al., 2020). Nanoparticles cannot completely replace all antiparasitic drugs at once, however, they can still be used to aid the production of pre-existing antiparasitic drugs that may need support in absorption or protection from degradation (Sarangi & Padhi, 2018; da Silva & Dreiss, 2016). Copper nanoparticles are foreseen to have resistant effects, especially against hematophagous ectoparasites that ingest blood from hosts (Ramyadevi et al., 2011 as cited by Sandhu et al., 2022).

NANOPARTICLES IN ACARICIDES

Chemical acaricides are one of the most essential treatments against tick infestation. Despite this, the downsides of the remedy almost outweigh the advantages, especially in how some of its residues end up on consumable meat and milk (Banumathi et al, 2017). The addition of nanoparticles in the formulation of chemical acaricides opened a wider range of more promising insecticides and acaricides (Benelli, 2018). However, while the exploration of the concept is being continued up to the present, a gap lies in the variety of pests and vectors that are being experimented on using nanoparticle acaricides (Rai et al., 2014).

While different kinds of nanoparticles have already demonstrated their properties and capabilities, the process of fabricating these materials can sometimes still be seen as cost-inefficient and environmentally heavy. Physical and chemical syntheses of nanoparticles require substances and machinery that may be harmful to the environment. Copper nanoparticles often require high concentrations of stabilizers or reducing agents to decompose into nanoparticles, but this process results in harmful byproducts for humans and the environment (Din & Rehan et al., 2016). Because of this, more methods were developed aiming to create nanoparticles but with less environmental burden such as biosynthesis.

BIOSYNTHESIS OF GUYABANO

The biological synthesis of nanoparticles, also known as biosynthesis or green synthesis, has been garnering attention in current research studies within the field of nanotechnology. Its prominence is raised by the method’s proven efficiency in nanoparticle production, further improved with the advantage of fewer failure chances, lower costs, and easier characterization (Gour & Jain, 2019). This is highly compatible with applications in the pharmaceutical, medical, agronomical, and environmental sectors. Furthermore, the biological approach proposes a solution to the disadvantages of other methods of synthesis in which both chemical and physical means are known to be destructive to the environment and dangerous to public health (Messaoudi & Bendahou, 2020). Rather than toxic chemicals, green synthesis promotes eco-friendliness and safety through its utilization of numerous biological sources available in nature, like fungi, bacteria, actinomycetes, yeast, algae, and plants (Singh et al., 2018).

PLANT-MEDIATED BIOSYNTHESIS

Among the natural sources for biosynthesis, the use of plants is presently being popularized. Diverse plant parts, including the stem, root, fruit, seed, callus, peel, leaf, and flower, are utilized to generate nanoparticles of various shapes and sizes (Kuppusamy et al., 2016). From these parts, it is favorable to operate with the extracts, rather than the tissue, because it is simpler, cheaper, and readily scalable (Iravani, 2014). These extracts contain a wide range of metabolites that are involved in the redox reaction process, resulting in them being responsible for the reduction of metal ions into metallic nanoparticles (Aromal & Philip, 2012). Namely, the types of metabolites primarily associated with this function are terpenoids (eugenol), flavonoids (luteolin, quertcetin), a reducing hexose with the open-chain form, and amino acids (tryptophan and tyrosine) (Makarov, et al., 2014). Following the preceding literature, aside from being a reducing agent, plant extracts have also been suggested to be a good support in the stability of the nanoparticles, preventing their agglomeration. This can be considered an advantage because there will no longer be a need to add other external capping and stabilizing agents (Rajeshkumar & Bharath, 2017).

ADVANTAGES OF USING PLANTS

There are more benefits to the plant-mediated biosynthesis of nanoparticles that make it more notable than the others. Compared to the other biomaterials, it is considered one of the most practical approaches because of its availability and ease of material gathering, making it an important prospect in expanding the scale of nanoparticle generation (Rastogi et al., 2017). Plant-mediated synthesis is also particularly advantageous because of its easier and less time-consuming process wherein it avoids the elaborate maintenance of cell cultures occurring in the other types of biosynthesis, specifically the microbe-based ones (Mustapha et al., 2022). In the same context, it is generally a simple and fast process made possible by the various metabolites contained in plants that catalyze ion reduction into nanoparticles (Iravani, 2014).

EXPERIMENT PROTOCOL OF
PLANT-MEDIATED BIOSYNTHESIS

The rapid rate of synthesis using plant biomass adheres to an experiment protocol illustrated in Figure 1 which is based on Vijayaraghavan and Ashokkumar (2017). The starting plant particles are typically in the powdered or extracted form (Mohamad et al., 2013). To obtain this extract, the plant is first added to a solvent that will dissolve or dilute the material. Ethanol is regularly chosen here as it has good extraction effectiveness while exhibiting low toxicity, allowing it to be safe in food and medicinal applications (Chaves et al., 2020). The acquired extract will then be filtered and mixed with a metal solution, taking into consideration the target pH level and temperature. Without the need for more complicated steps, it will be left until the suspension reaches a state of equilibrium. After a short period, the synthesis will already be accomplished within the aforementioned basic procedures. The obtained nanoparticles can then be characterized using various methods, such as Ultraviolet-Visible spectrophotometry (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and X-ray powder diffraction (XRD). These analyses ensure that the incorporation of plants indeed reduced the metal ions into metallic nanoparticles (Modena et al., 2019).

Photo credits: Vijayaraghavan & Ashokkumar (2017)

GUYABANO

An example of a plant currently being explored as a mediator in the reduction of metals into nanoparticles is the Annona muricata, better known as guyabano. It is native to South America, and is commonly harvested in a wide range of tropical locations, such as in Southeast Asia which includes the Philippines (Solís-Fuentes et al., 2021). From the family Annonaceae, guyabano or soursop is a species of tree that bears heart-shaped fruits with green skin and white flesh (de la Cruz, 2013). With regards to nanoparticle synthesis, it contains the four compounds mentioned above, specifically tannins, flavonoids, terpenoids and reducing sugars (Agu & Okolie, 2017).

With these phytochemicals considered, researchers have explored the usage of guyabano in producing copper oxide nanoparticles. The green synthesis method of (Mahmood et al., 2022) incorporates a mixture of Copper II Nitrate and deionised distilled water with Annona muricata peel extract. It was then subjected to a hot air oven, which resulted in burnt brown nanoparticles. These were characterized through UV-Vis spectroscopy where it was found to have peaked in absorbance at 260 nm. Further, SEM analysis proved it to be nanoparticles with size outcomes ranging between 33.24 ± 6.49 nm. On the other hand, Kayalvizhi et al. (2020) utilized guyabano leaf extract to also synthesize copper oxide nanoparticles. The extract was mixed with CuSO4 solution and constantly stirred with a magnetic stirrer until the color changed to brick red. It was then centrifuged and air dried. In accordance with the characterization analyses done, the resultant particles were 30-40 nm in size, crystal in nature, and spherical in shape.

BENEFICIAL CHARACTERISTICS OF
GUYABANO

Beyond the ability to synthesize nanoparticles, the extracts of guyabano also comprise proven benefits for numerous medicinal fields, such as being antioxidant, anti-microbial, anti-inflammatory, insecticidal, larvicidal, anticancer, and anti-tumoral (Gavamukulya et al., 2017). In addition, a notable characteristic of guyabano is its acaricidal potential (Merritt & Villacorta, 2015). This property is supported by the natural acaricidal chemicals it encompasses which are Goniothalamicin (Alkofahi et al., 1987) Gigantetrocin A, annomontacin, and bullatalicin (Alali et al., 1998) Squamocin (Guadano et al., 2000; Borges, 2011). Furthermore, a paper by Alagan et. al (2016) proves the acaricidal potential existing in A. muricata Linn. Specifically, the study experimented on the seed extract of guyabano against lice infestations in backyard poultry. Results indicated that the plant extract showed strong pediculicidal activity which denotes that it is also a good source of acaricidal properties. Research from Maciel et. al (2017) agrees with this finding wherein it has been found that ethanolic extract of A. muricata seeds exhibited its capability to reduce reproduction of mites, another type of arachnids. Considering all the aforementioned properties of guyabano, the diverse features of A. muricata make its utilization in an extensive range of applications possible.

With regard to the inherent characteristics of guyabano as a basis, its extracts have been used in a variety of nanotechnological studies, aiming to incorporate its useful properties into the production of nanoparticles. As previously stated, guyabano extracts are discovered to have antioxidant properties. From there, Sobretodo et al. (2019) tested the antioxidant properties of silver nanoparticles (AgNPs) synthesized through the mediation of A. muricata leaf ethanolic extracts in Ag+ ions contained in silver nitrate (AgNO3). The end product was confirmed to be nanoparticles through a UV-Vis spectrophotometer. In the results, the guyabano-mediated AgNPs were reported to have higher antioxidant levels than commercialized AgNPs, proving their claim that the AgNPS on its own has no significant antioxidant properties but the addition of guyabano extracts increased the nanoparticles’ potential to exhibit them. On the other hand, antitumor properties were explored in a study conducted by Gonzales-Pedroza et al. (2021). Similarly, guyabano was a component in the methodology, and specifically, its stem and leaves were experimented on in separate set-ups. The synthesis facilitated by the guyabano parts successfully produced nanoparticles with sizes smaller than 50 nm and these exhibited potent in vitro anti-tumor effects. Furthermore, Badmus et al. (2020) have also used guyabano in mediating the synthesis of AgNPs and they have presented that the results exhibited an effective anti-diabetic and antimicrobial material.

CONCEPTUAL FRAMEWORK

It has been observed in several studies that guyabano has exhibited mediating properties that prove to be effective in the reduction of metal ions into plant-mediated nanoparticles. Thus, its extract was utilized in the biosynthesis of copper nanoparticles. Specifically, the guyabano leaf ethanolic extract was analyzed through phytochemical screenings to ensure the presence of chemical constituents required to successfully mediate the process. Rhipicephalus sanguineus was subjected to different concentrations of the resulting guyabano-mediated CuNPs. This study aimed to evaluate the guyabano-mediated CuNPs to see how their effects on mortality rate differ across five different concentrations (5%, 10%, 20%, 30%, 40%) and how it performs in comparison with water (negative control), and Amitraz acaricide (positive control). The independent variables are the guyabano-mediated CuNPs while the dependent variable is the mortality rate of Rhipicephalus sanguineus.

HYPOTHESES

H01

RESEARCH HYPOTHESIS

This study aimed to determine the acaricidal potential of guyabano-mediated CuNPs against brown dog ticks to address the following problems: brown dog tick infestations, downsides of synthetic and commercial acaricides, uncertain efficacy of natural acaricides, and acaricide resistance of the parasites. The researchers hypothesized that the five different concentrations (5%, 10%, 20%, 30%, 40%) of guyabano-mediated CuNPs will result in high mortality rates of brown dog ticks because of the natural acaricidal components found in guyabano leaves and its incorporation to the synthesis of copper nanoparticles.

HA1

H02

HA2

STATISTICAL HYPOTHESIS

There is no significant difference in the mortality rate of Rhipicephalus sanguineus within the experimental groups (5%, 10%, 20%, 30%, and 40% of guyabano-mediated CuNPs).

There is a significant difference in the mortality rate of Rhipicephalus sanguineus within the experimental groups (5%, 10%, 20%, 30%, and 40% of guyabano-mediated CuNPs).

There is no significant difference in the mortality rate of Rhipicephalus sanguineus between the experimental groups (5%, 10%, 20%, 30%, and 40% of guyabano-mediated CuNPs) and control groups (Amitraz and distilled water).

There is a significant difference in the mortality rate of Rhipicephalus sanguineus between the experimental groups (5%, 10%, 20%, 30%, and 40% of guyabano-mediated CuNPs) and control groups (Amitraz and distilled water).