Effect of Metarhizium anisopliae on Different Group Size of Coptotermes heimi

Coptotermes heimi is a destructive subterranean termite species commonly found on the Indian subcontinent. Environmental concerns of chemical insecticides usage forced scientist to concentrate on alternative methods. Metarhizium anisopliae is observed to have pathogenic effects against various arthropods. This study focused on the interaction of M. anisopliae with C. heimi to check its virulence against the termites. Specific spore concentration of M. anisopliae (1.1×10 7 conidia/ml) was applied on different group sizes of termites to check if termite population has any effect on its mortality. Results showed that with the increase of group size of termites, mortality rate decreases.


INTRODUCTION
Termites are an important group of insects that are extensively distributed in temperate, tropical and subtropical countries of the world [1]. Termites have seven families which then further divided in to more than 2,600 define species [2,3]. Despite having common traits, termites

Original Research Article
have adapted wide range of ecological habitats showing great diversity in cast physiology and morphology, nesting behavior and association with different kinds or microbial community [4]. Their main food is cellulose based material. Most of the termites do not have efficient cellulose digestion system [5] they tend to make symbiotic association with microbial communities that can break cellulose into simpler sugar units [ 21]. Despites the great advantageous nature of these chemical applications, one cannot deny their effect on environment which begs the question of having alternate, eco-friendly approaches to control termite growth [22][23][24].
Termites controls using biological methods have gained much attention as an alternate technique to chemical control usages. Biological based techniques were started in 1960's with special focus on fungal pathogens [25][26][27][28][29]. The use of fungi as biological control of termites was built on the view of classical biological technique [30] with the use of fungi virulence that can replicate itself in the nest of termites and can spread from one termite to other by high social interaction, thus cause an epizootic and destroy the whole colony. Most of the researchers focused on the entomopathogenic fungi nature of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin [23,24,31]. The fungi are broadly spread in soil and broad range of host. Studies showed that these fungal species have potential against termites, however have less successful trial in fields [22,32].
M. anisopliae is a soil fungi generally lived in temperate, arctic, tropical and subtropical parts of world [33]. Even though, the fungi have ability to survive in soil [34,35], it is assumed to be the fungi which has pathogenic nature against wide range of insects such as beetles, moths, flies, cockroaches, locusts and other arthropods [36]. Pathogenic activity of Metarhizium strains varies from host to host [37][38][39]. This study explores the interactions of C. heimi with the entomopathogenic fungus M. anisopliae to assess the effective candidacy of this pathogenic fungus as biological control.

Collection and Identification of Termites
C. heimi (Wasmann) were collected from verminous trees of Populus euramericana at the of Forman Christian |College, Lahore. Termites was identified using morphological traits for soldiers and workers [40].

Conservation of Termites at Controlled Laboratory Conditions
Laboratory conditions at 25°C and 70% Rh was provided for termite's acclimatization prior to experimentations. Termites were kept on Whatmann filter paper for three days in Petri plates during experiments.

Culturing of M. anisopliae on Selected Media
The culture of Metarhizium anisopliae was provided by entomology laboratory at Forman Christian College, Lahore. The fungus was cultured on potato dextrose agar (PDA) media at 27 or 30 C in the dark for 15-20 days.

M. anisopliae Effects on Different Group Size of Termites
An estimate number of spores were made using a hemacytometer. Spores of fungus M. anisopliae were distributed on 10 petri dishes having Whatman No. 1 filter paper moistened with water. All plates contained different group size of termites 1.e 5, 10, 15, 20, 25,30,35,40,45,50. The experiment was conducted for 2 weeks and termites' mortality was checked every day. While examining, dead termites were separated in another petri plate from healthy ones. Fungus growth was checked in petri plate having dead termites. Control group in this experiment was plate without the fungus.
The impact of standard fungal concentration (1.1×10 7 conidia/ml) on group size of termite was significant (P=0.05; F=91.87, DF, 1-18 ANOVAs one way) on mean termite % mortality. 100% mortality was achieved after day 8,10,12,14,11 and 10 against termite group size ranged from 5-30 respectively. However, a pronounced effect was recorded where termite mortality decreased with the increase in population size from 83% to 67% and group size ranged 35-50. The root square adjusted was 82.71% (Table 1). With the increase of group size of termites, mortality rate decreases. This can be possible because of the social behavior of termites [49,50] such as avoiding infected termites. Moreover, they also develop chemical mediated defenses against pathogen which include their ability to sense the toxins released by pathogenic fungi such as Beauveria and Metarhizium species. Because of this ability, they avoid the contact with fungus spores or any toxin contaminated substrates [51]. Rosengaus