Study on Liquid Chromatography and Mass Spectrometry of Memnoniella echinata Metabolites against Vector of Dengue and Chikungunya

Research Article

Austin Chromatogr. 2014;1(3): 6.

Study on Liquid Chromatography and Mass Spectrometry of Memnoniella echinata Metabolites against Vector of Dengue and Chikungunya

Gavendra Singh1* and Soam Prakash1

1Department of Zoology, Dayalbagh Educational Institute, India

*Corresponding author: Gavendra Singh, Environmental and Advanced Parasitology and Vector Control Biotechnology, Biomedical Laboratories, Department of Zoology, Dayalbagh Educational Institute, Agra-282005, India

Received: October 08, 2014; Accepted: October 27, 2014; Published: October 29, 2014

Abstract

Aedes aegypti and Ae.albopictus are vectors for transmitting Dengue, Chikungunya, and Yellow fever. These fevers are growing public health problem globally. Memnoniella echinata is a cosmopolitan fungus. This fungi is exceptionally good model for bio control and more significant as bio agent. Recently, especially fungi, has emerged as a current and future candidate for mosquito control. In the study M. echinata was cultured in the Potato Dextrose Broth (PDB) medium. The metabolites were filtered with the Whatman-1 filter paper. These purified metabolites were tested in the statistically significant concentrations of 50, 100, 150, 200, 250 ppm against first, second, third, and fourth instar larvae of Ae. aegypti and Ae. albopictus. The bioactive compounds have been isolated with the Thin Layer Chromatography (TLC) plates with chloroform and methanol used as solvent system for analysis. These compounds were detected with the Liquid Chromatography Mass Spectrometry (LC-MS/MS) analysis. The LC50, LC90 and LC99 values were 84, 188, 205 ppm against first and second instar. However, in case of third instar 115, 191, 209 ppm and 125, 196, 214 ppm was observed against fourth instarlarvae of Ae. aegypti. Thus, the LC50, LC90 and LC99 values were 84, 188, 205 ppm against first and second instar of A. albopictus. The third instar LC50, LC90 and LC99 values were 84, 192, 210 ppm, and fourth instar 89.2, 196, and 215 ppm respectively after exposure for 24 hours. These new results significantly support broadening the current dengue and Chikungunya vectors control paradigm beyond chemical larvicides.

Keywords: Memnoniella echinata; Metabolites; LC-MS/MS analysis; Aedes aegypti, Ae. Albopictus

Introduction

Aedes aegypti and albopictus are vectors for dengue Chikungunya, and Yellow fever. In recent years, dengue and Chikungunya transmission has increased predominantly in urban and semi-urban areas as major public health concern. Globally, the incidence of dengue has grown in recent decades. Approximately 2.5 billion people over40% of the world’s population is now at risk from dengue. Currently there may be 50–100 million dengue infections worldwide every year [1]. The entomopathogenic fungus to control mosquito adults and larvae is not a new issue [2]. In contrast to bacteria, fungi are adulticidal agents that could be developed for domestic use to reduce vector densities and impair their vectorial capacity. Globally, chemical spraying methods have used for control Ae. aegypti and Ae. albopictus during dengue and Chikungunya epidemics. Therefore new alternative is urgently needed. The entomopathogenic fungi have used for control of Ae. aegypti and Ae. albopictus. The virulence of entomopathogenic fungi has confirmed against adult Ae. aegypti [3,4]. Metarhizium anisopliae is well known entomopathogenic fungi. M. anisopliae strain IP 46 was isolated from soil which shows pathogenicity against all stages of Ae. aegypti including the egg [5- 8]. Field studies have encouraged because significant and rapid larval mortality caused by Leptolegnia chamanii against Ae. Aegypti [9]. Lecanicillium muscarium has isolated from a dead culicid mosquito, this found pathogenic to adults of Ae. aegypti, An. arabiensis and Cx. quinquefasciatus under laboratory conditions demonstrating how naturally occurring fungal pathogens of culicids might have potential for mosquito control [10]. Aspergillus clavatus isolated from an African locust causes >95% mortality after 24 hours against An. gambiae, Ae. aegypti, and Cx. quinquefasciatus larvae [11]. The oil based formulations of conidia of M. anisopliae have enhanced ovicidal of activity at high humidity in the control of Ae. aegypti [12].

The effectiveness and deploy ability of such fungi under field conditions have yet to be explored. The fungal toxins are the true chemical in nature. The entities of fungal metabolism are not known until recent times. The fungi have isolated, a Glomeromycete (possibly Entrophospora sp.) and a Dothideomycete (possibly Phaeosphaeria sp.), for bioactive secondary metabolites. The six new compounds consisting of clearanols and disulochrin have purified [13]. A new fungi Chrysosporium lobatum strain BK-3 has isolated from Assam India. This fungal strain has produced two bioactive compounds [14]. Several metabolites from entomopathogenic Deuteromycetes have been investigated. Moreover, Beauveria spp. was known to produce beauvericin, a depsipeptide metabolite which has shown toxicity to a number of invertebrates [15]. Similarly, proteases have produced by entomopathogenic fungi to degrade cuticle and assist entry into the host were similar to proteases used by insects to degrade their own cuticle during molting [16]. A number of enzymes have known from entomopathogenic fungi, such as the proteases, lipases, and chitinases that assist in cuticular breakdown. These enzymes can be thought of as bioactive and there has been increasing interest in use of these enzymes in mosquito control. Entomopathogenic fungi also produce insecticidal toxins. The early literature on toxins from entomopathogenic fungi was reviewed by Roberts and Stressed [15,17].

Memnoniella echinata is a cosmopolitan fungus. This commonly found in tropical and sun tropical areas especially on cellulose based materials. The maximal spore germination temperature is 37 °C. M. echinata releases different mycotoxins. Accordingly in present study we investigated the efficacy of M. echinata metabolites against Ae. aegypti and Ae. albopictus larvae in laboratory and Liquid Chromatography Mass Spectrometry (LC-MS/MS) analysis for compounds.

Material and Methods

Collection and culture of Memnoniella echinata (MTCC 604)

The discovery of bioactive compounds usually begins with isolation and identification of the fungi and growing them at various temperature regimes in a variety of selective and non-selective culture media. The M. echinata (MTCC 604) was obtained from Microbial Type Culture Collection and Gene Bank, Institute of Microbiology, Chandigarh, India. The Potato Dextrose Broth (purchased from Himedia) M403, 24.0 gm was suspended in sterile water. The medium was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes. The broth was supplemented with 50 μg/ml chloramphenicol as a bacteriostatic agent. M. echinata colonies grown on the Sabouraud Dextrose Agar plates were transferred to each flask using the inoculation needle. The conical flasks inoculated with M. echinata were incubated at 24±2°C for 15 days (Figure 1).