International Journal of Zoology and Applied Biosciences	ISSN: 2455-9571
Volume 3, Issue 3, pp: 269 -274, 2018	http://www.ijzab.com
Research Article
ASSEMBLAGE OF NEST SOIL BACTERIA AND EVALUATE The ANTIBACTERIAL ACTIVITY OF CARPENTER ANT (CAMPONOTUS COMPRESSUS) MANDIBULAR EXTRACTS AGAINST SELECTIVE GRAM STAINING BACTERIA *P. Mohana¹, C. Gunasekaran² and P. Selvarasu²** ¹Department of Zoology, Vivekanandha College of Arts and Sciences for Women (Autonomous), Elayampalayam, Tiruchengode – 637 205, Tamil Nadu, India. ²Conservation Biology laboratory, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore – 641 046, Tamil Nadu, India.
Article History: Received 2^nd April 2018; Accepted 25^th April 2018; Published 25^th June 2018
Abstract The present study was under taken to explore the assemblage of ant nest soil microbes and potential of mandibular gland extraction of carpenter ant Camponotus compressus. The microbial assemblages in nest soil from different habitats like Grasslands, Shola forest, Wattle plantation, Pine and Tea plantations were investigated. Among the isolated microbes from the nest soil bacteria, Staphylococcus aeureus was robust in three sites of the study area. Disc diffusion method was used to evaluate the antimicrobial activity of mandibular gland extraction against two robust microbial strains like S. aeureus and Escherichia coli. The result revealed that the maximum zone of inhibition was observed against the S. aeureus in both 10 µl and 20 µl of the mandibular sample and the minimum in gram negative bacteria Escherichia coli. The presences of mandibular gland proved have good capability more than commercially available antimicrobial products to kill or inhibit the growth of microbes inside the nests. Keywords: Antibacterial, Ant nest, Camponotus compressus, Mandibular gland, Mukurthi National Park.

INTRODUCTION

The ant nests are frequently accumulated by the stored food and waste materials, other organic debris by the foragers. This kind of nest alterations leads to determine the changes, size and activity of soil microbial assemblages inside the ant nests (Boulton & Amberman, 2006; Savin, et al., 2004). Microbes play an important role in ant communities like mutualists, commensals or pathogens. Ants are able to stimulate a broad range of physiologically different groups of microorganisms (Dauber & Wolters, 2000; Jakubczyk et al., 1972). This microbial-rich environment may lead to the risk of infections and disease transmissions in ants. In such pathogenic risk conditions, ants use their unique defense mechanism to cope with the diseases (Hughes, 2005; Poulsen et al., 2002). According to (Wilson Rich et al., 2008) the social insects have evolved a variety of adaptations to cope with the intense pathogenic pressures in their environments. The secretions of metapleural and mandibular glands of ants contain antimicrobial substances that defend infections. Bot et al. (2002) and Poulsen et al. (2002) found many ants are secret the metapleural gland, spreading them over the cuticle, while termites secret antibiotics from the salivary glands and sternal gland.

The Camponotus ant genus the metapleural gland is clearly absent (Ayre & Blum, 1971; Holldobler & Engel Siegel, 1984) possibly making more susceptible to infection and disease transmission. However, orally associated mandibular glands and salivary glands are also known to produce the antimicrobial factors. The ant mandibular glands are a pair of thin-walled sacs filled with mixture of alcohol, aldehydes and ketones (Blum & Hermann, 1978). These volatile compounds also have sturdy effects on ant behavior. The madibular gland secretions are the main source of nest mate recognition odor and modulators of alarm signals (Bradshaw et al., 1975;Cammaerts et al., 1983; Powell & Clark, 2004). High microbial parasites and pathogen pressure have led to the evolution of immune proteins in social insects including ants (Viljakainen et al., 2009). Antibiotic secretions are also associated with the salivary glands of arthropods including ticks, mosquitoes and termites have also been reported by Lu et al. (2005).

The available literature claims that the mandibular and metapleural gland are protecting the colony against the invaded micro-organisms of leaf-cutting ants (Hughes et al., 2002; Yanagawa et al., 2008). These compounds were investigated for potential activities against human pathogens particularly resident to conventional antibiotics (de Lima Mendonca et al., 2009). The study reported that the E.coli is resistant to common antibiotics Ampicillin and Cefoxitin (Souza et al., 2015) and the S.aureus was resistant to Chloramphenicol, Cefalotin, Erythromycin, Sulphonamide and Vancomycin (de Lima Mendonca et al., 2009). Junior et al. (2001) found that the mandibular gland secretion of leaf-cutter ant Atta sexdens rubropilosa inhibits the germination of necrotrophic fungus Botrytis cinerea which causes the diseases in economically important crops like Wine grapes and strawberries. Brough,(1983) discovered antimicrobial secretions from the mandibular gland in a Formicine ant (Calomyrmex sp.). Very few reports were documented about the bacterial diseases in ants. The Pseudomonas genus bacteria killed and fed all Solenopsis invicta larvae vegetative cells with-in five days (Lofgren et al., 1975). It is estimated that 66% of insect species and 30% ants are infected by Wolbachia (Hilgenboecker et al., 2008; Russell et al., 2012). Wolbachia infections cause detrimental effects on colony fitness of Formica truncorum (Wenseleers, Sundström, & Billen, 2002) and also affects Dorylinae, Formicinae, Myrmicinae, Ponerinae and Pseudomyrmecinae subfamilies (Van Borm et al., 2001). There are some beneficial microbes associated with ants, for example Blochmannia provides nutritional benefits in carpenter ants and leaf cutter ants use actinomycete bacteria to maintain stable ant-fungus relationship (Feldhaar et al., 2007; Mueller et al., 2008). Developing a better understanding of the associations between carpenter ants and microorganisms can reveal the role of Camponotus in forest ecosystems (Mankowski & Morrell, 2004). Hence the present study focuses on microbial assemblage in nest soil and the respective defense mechanisms mounted by C. compress in various habitats.

MATERIALS AND METHODS

Study site

The study area Mukurthi National Park (MNP: 11 º 26' to 76º 10' to 11º22' N and 76º38' E) is about 78.46 Sq.km and has the elevation of 2400m and it is protected under UNESCO since 1^st July 2012. The study area comprises the patches of evergreen forest surrounded by grasslands. The park has a wide variety of flora and fauna. But most of the grasslands have been replaced by exotic species namely wattle (Acacia spp.), pine (Pinus roxburghii), tea (Camellia sinensis) and bluegum (Eucalyptus globulus). The climate is highly seasonal, with a dry season extending from December to February and a wet season between June and November. The soil sample was collected from five habitats, namely Grasslands, Shola forest, Wattle plantation, Pine and Tea plantations. The carpenter ant C. compressus is one of the most abundant species of ants in South India. They live in enormous communities, travelling long distances from their nests, and are active during both day and night. C. compressus is one of the best known robust species in Mukurthi National Park. Three replicates of ant nest soil were sampled to a depth of 5, 10 and 15 cm from ten randomly selected ant nests from five habitats. The collected soil samples with ants were immediately transferred to the laboratory for the further study.

Mandibular gland extraction

The re-sealable plastic bags contains workers of C. compressus were placed in a freezer for 1 hr to immobilize the ants. Decapitation and preservation of ant heads in solvents probably 2 ml dichloromethane is a standard method for extraction of mandibular gland secretion of ants when they cannot be analyzed immediately. Decapitation ant heads about 1 gm were homogenized well using 2 ml of 70% alcohol and subsequently the mixture was centrifuged at 5000 rpm at 4˚C for 15 minutes. The pure supernatant was tested against the pathogens in a culture plates. The residuary samples were stored in a defreezer at -20⁰C for long-term storage.

Screening of ant nests soil microbes

The pre-treated soil samples were serially diluted using 9 ml of sterilized phosphate buffer saline and 1 gm of the soil sample was added to it in a test tube and thoroughly mixed. The suspension was then serially diluted by transferring 1 ml of the suspension to a series of test tubes containing 9 ml of sterilized phosphate buffer till 10^-6 dilutions. The swab sticks used for the collection of the samples and the samples were streaked directly on the labeled agar plates and incubated at 37°C for 24 h. After incubation, cultures were examined for significant bacterial colony growth. Subcultures were then made into plates of nutrient agar and incubated for another 24 hours. The primary identification of the bacterial isolates was made based on colonial appearance and pigmentation. Biochemical tests such as standard Catalase test, Citrate utilization, Oxidase, Methyl red, Voges Proskauer, Indole production, Motility, Glucose, Sucrose, Maltose, Lactose were performed to identify microbes, Characterization and identification of the isolates was done using the methods of Bergey’s manual of determinative bacteriology (Buchanan & Gibbons, 1974).

Disc diffusion method

Kirby-Bauer disc diffusion method was used to determine the antibacterial activities (Fazeli et al., 2007; Molan, 1992; Samy et al., 2006) which has been widely used by several researchers. The Escherichia coli and Staphylococcus aeureus inoculums were inoculated on the surface of the

separate nutrient agar plates with a help of sterile cotton swabs. The disc of antibacterial mandibular extraction of the C.compressus was placed on the surface of the agar plates. Further, the plates were incubated at 37˚C for 24 hours to determine on the basis of zone of inhibition. Using nutrient agar disk diffusion method, the isolated microbes were tested by the mandibular extraction. The selected isolated microbes were treated with various combinations of 10, 20 µl samples using petri disc. Double distilled water was used as a control. The same test was done with the standard commercial product Ampicillin. After incubation, the antibacterial activity was evaluated by measuring the inhibition zone (mm) ± SD of three replicates.

RESULTS AND DISCUSSION

The carpenter ant, C. compressus is a large colonial and social insects consisting of reproductive and sterile castes. The moist soil of the C. compressus ant nest is a habitat of high microbial abundance in the field, and the microbial biomass differs among the habitats. A total of 13 bacterial isolates were obtained and the maximum number of microbial isolates with 1X 10³(cfu/g) from ant nests in various habitats of Mukurthi National Park. The study identified 13 species of microbes namely Bacillus alvei, Bacilllus clausii, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, Klebsiella pneumonia, Lactobacillus acidophilus, Micrococcus luteus, Proteus mirabilis.

Table 1. List of isolated dominant microbes in ant nests of the study area.

Sample plots	Soil code	Isolates (CFU)		Dominative isolate name
Grassland	GL	10^-3	644	Proteus mirabilis
		10^-4	713	Staphylococcus aeureus
		10^-5	121	Bacillus clausii
Shola Forest	SF	10^-3	1022	Bacillus subtilis
		10^-4	747	Lactobacillus acidophilus
		10^-5	500	Pseudomonas fluorescens
Wattle plantation	WP	10^-3	1017	Staphylococcus aeureus
		10^-4	212	Pseudomonas sp.
		10^-5	321	Klebsiella pneumonia
Pine plantation	PP	10^-3	742	Enterococcus faecalis
		10^-4	128	Bacillus sp.
		10^-5	79	Pseudomonas aeruginosa
Tea plantation	TP	10^-3	1789	Escherichia coli
		10^-4	798	Proteus vulgaris
		10^-5	230	Staphylococcus aeureus

Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aeureus and Streptococcus salivarius in nest soil of C.compressus. Among these microbes Staphylococcus aeureus was the most frequently isolated bacteria in three various habitats (Table 1). Ant mounds consist primarily of sand-sized particles, resulting in lower water holding capacity, increased drainage and aeration relative to bulk soil and conditions that can errand particular for microbial populations (Amador & Gorres, 2007).

The present result revealed that the occurrence of microbial biomass is varied greatly according to the condition of soil among the habitats. The composition of the organic materials is not the same throughout the whole nest volume (Coenen stass et al., 1980; Horstmann & Schmid, 1986). Nest conditions, such as high humidity and stable temperatures, favor the growth of microbes. The soil moisture affects the microbial activity because it controls both the substrate and oxygen diffusion. If soil is too dry, substrates may not be sufficiently mobile to support the microbial activities. The coarse texture and lower moisture content of ant mound soil could be responsible for the low microbial biomass and activities. In Pine plantation the ant nest soil contains low quantity of microbes and this may be due to resin which inhibits the growth of potentially pathogenic bacteria and fungi in the nest (Christe et al., 2003). Froz et al. (2005) reported that the microbial communities in European red wood ant Formica polyctena ant nest differ from that in the surrounding soil in part because of differences in pH, food availability and soil quality.

In the carpenter ant have the large mandibles and powerful tools for prey catching, fighting, digging, seed crushing, wood-scraping, grooming, brood care and trophallaxis (Muscedere et al., 2011), also protecting the colony against pathogenic pressures. In the present work, it has been demonstrated that the main constitutions of the mandibular gland secretions of ants possess strong inhibitory activities against two robust grams positive and gram negative bacteria isolated from ant nests (Figure 1). Antibacterial potency of mandibular extraction of C.compressus tested against robust bacterial species Escherichia coli (gram negative) and Staphylococcus aeureus (gram positive). The maximum zone of inhibition was observed against the Staphylococcus aeureus (10 mm, 12 mm) in both 10 µl and 20 µl of the mandibular extracts. The zonal inhibition against the gram negative bacteria Escherichia coli was 8 mm and 10 mm.

S.No	Bacterial strains	Commercial product	Concentration of Camponotus compressus mandibular extraction
S.No	Bacterial strains	AMP	MN 10 µl	MP 20 µl
1	Escherichia coli	6 mm	8 mm	10 mm
2	Staphylococcus aureus	6 mm	10 mm	12 mm