New insight in to transcriptome data

In search of a mighty formulation for accelerated degradation of pesticide residue in fruit and
vegetables: A sustainable alternative for agriculture and food safety

Characterization of enzymes in gut microbiome of pesticide

resistant insects from crop field and testing its efficacy degradation
of organic molecule employed in commonly used commercial
pesticides: A valuable alternative for sustainable agriculture and
food safety

Characterization of gut microbiome of pesticide resistant insects

from vegetables, field crops and purification of enzymes involved in
degradation of organic molecules employed in commonly used
commercial pesticides: A valuable alternative for sustainable
agriculture and food safety

Fresh fruits and vegetables are known to be packed with vitamins, minerals, fibers and range of disease
preventing phyto constituents which makes them an integral component of healthy diet. The choice for
minimally processed, prepackaged, ready to eat fruits and vegetables has greatly reduced among
consumers in recent past. At the same time, consumption of leafy, raw vegetables such as celery,
lettuce, cucumber, tomato, carrot, beet root, cabbage and fresh fruits has increased to a greater extent
due to increased health concern and nutritional benefit. On the other hand the risk factor for several life
threatening diseases is commonly accepted as crop fields are regularly sprayed with insecticide,
pesticide and herbicides which raises serious threat on human health.

In India pesticide intoxication from agriculture has steadily increased over the recent years due to its
increased usage ranging from seed germination, cultivation, storage of fruit and vegetables. According
to World health organization (WHO) unintentional pesticide poisoning is a significant global public
health problem in developing countries. Based on the hazard assessment pesticides are reported as
carcinogenic, neurotoxic and teratogenic. Improper knowledge on pesticide handling, lack of sufficient
research on degradation of pesticides in to a less or non toxic leads to endanger non targeted aquatic
life forms e.g. fishes, crabs, snails etc.

The pesticides are mainly categorized in to organochlorines (e.g Lindane, Endosulfan),

organophospahtes (e.g Clorpyriphos, Diazinon, Azinphos, Parathion), organocarbamates (e.g Carbaryl,
Aldicarb), pyrethroids (e.g Allethrin, Permethrin, Tetramethrin) based on their chemical composition.
Among the different categories of pesticides (organochlorines, organophospahtes, organocarbamates)
Organophosphorous compound based synthetic pesticides constitute a major portion of modern
insecticide, herbicide and pesticide and are the most widely used in agriculture, home, garden. Though
they are proved to be more biodegradable compared to organochlorine based pesticide little amount of
organophosphate can cause potential damage to human nervous system. The organophosphate group
of pesticide phosphorylate acetylcholinestarase, an enzyme necessary for controlling nerve impulse
transmission leads to accumulation of acetylcholine causing neurobehavioural and neurological damage.
Similarly carbamates, pyrethrines are potent nerve poison used for controlling different insects and
pests also work in the same principle to organophsphates also most frequently used around the world
today. The mode of action of chlorpyrifos and diazinon pesticides is by inhibiting axonogenesis,
synaptogenesis and also inhibit RNA synthesis in neurons and glial cells.

On the other hand pesticides have made such a great impact on its application in several ways that it is
inevitable to completely stop its use. Therefore finding an alternative method for its safer use is the one
and only approach. Finding an agent to degrade the recalcitrant and toxic molecules in the pesticides in
to a non toxic product and to reduce its bioaccumulation potential in environment has been constantly
shouted by researchers. Biobeds with different microbes intended to promote microbial degradation
activities on pesticide and reducing low risk of leaching has been implemented by farmers of Sweden
and other countries (Castillo et al., 2012).

The aim of current research is develop a novel formulation with the concept 1. pesticide resistant
insects could be the most potential source of enzymes that can readily catabolize the toxic molecule in
pesticide to non toxic form. Therefore we want to focus on exploitation of enzymes in gut and gut
associated microbiome of pesticide resistant insects collected from different crop fields for enhanced
degradation process of organic molecule in commonly used pesticide.

2. Probiotics of human intestinal microflora which able to servive in various adverse environment and
provide beneficial effect could be another potent source of the enzymes involved the degradation of
pesticides. Though several report suggests their efficiency in pesticide degradation but their mode of
action is not understood till date. Here along with their efficiency we want to characterize the detailed
mechanism of action involved in pesticide degradation and the end product metabolite formed and their
toxicity lebel will be analysed using expression of markers in model cell lines.

Relevence to Field

Increase in resistance towards pesticides among insects becomes the common phenomenon. At the
same time role of symbiotic microflora of pesticide resistant insects has draws attention of scientist
community for biotechnological exploitation such as i) strategies for control of pests and disease
originating from insect vector ii) conversion of synthetic bioactive molecules of pesticides to a lesser or
non toxic compounds and reducing their contamination by leaching or spills iii) metabolisation of
xenobiotics from environment. The increased tolerance towards pesticide in insects indicates the gut
microbiota are capable of catabolising the insecticide to survive. In general the bacteria associated with
insect and the enzymes produced by them plays a great role in detoxification of toxic molecules in
pesticides and also greatly contribute towards the fitness trait of host insects. Insects use various
strategies for detoxifying the complex toxic compounds in gut lumen by employing a reducing
environment and producing potent complex enzymes involved in detoxification. There is also indication
that gut microbiota in due course of evolution significantly contributes in such enzyme production.
These enzymes are mainly involved in degradation process of pesticide ingested by the host insect and
obtaining its nutrient for growth by hydrolysis of these compounds. Characterization of these enzymes
and detail investigation of gut microbial flora in degrading the complex organic molecule that are
employed in most commonly used commercial pesticide is the prime focus here. The current research
will find a valuable resource that will contribute for reducing pesticide intoxication, maintaining safer
nutritional ecology, sustainable agriculture, and food safety. Similarly we also want to investigate the
potential of probiotics food grade bacteria like Lactobacilli, Bifidobacteria and the enzyme produced by
them in reducing pesticide contamination in fruits and vegetables. Their mechanism of action in
different pesticide degradation will also be studied.

Methodology

Collection of insects

The insects from various cultivation field (paddy, vegetables E.g Cauliflower, brinjal, Cucumber, leafy
green vegetables E.g Spinach, mustard green, lettuce etc.) sprayed with insecticides organophosphate,
pyrethroid, organocarbamates were collected with farmers consent. The selected resistant lines will be
maintained with artificial diet and controlled condition in our laboratory.

Preliminary screening and Selective isolation of Gut microbiome and enzymes for pesticide
metabolism

The in house maintained insects were surface sterilized in 4% sodium hypochloride followed by 80%
ethyl alcohol in a conical flask and washed repeatedly in distilled water until the complete removal of
sterilant. The gut of each insect were dissected in sterile condition and macerated in M9 minimal
bacterial growth medium (Thermo Fisher Scientific). One ml of macerated gut sample was added to 100
ml of growth medium containing 5µg/ml of different insecticides in it. The conical flask was incubated in
shaker 37°C with constant shaking at 150 rpm up to 15 days. Aliquots from each flask were removed at
regular interval of 3, 6, 9, 12 and 15th day considering day 1 as first day of inoculation. These aliquots
were subjected to GC-MS/ LC-MS analysis to study the degradation pattern of original pesticide.
Similarly one part of each aliquots plated on to M9 media containing 5µg/ml of different commercial
insecticides and incubated at 37°C for growth of pesticide degrading bacteria. Individual bacterial
colonies grown on various pesticide containing plated will be isolated and subjected to enzyme
production.

Pesticide hydrolysis assay

Overnight broth culture of both microflora of insect gut and probiotics were plated on to brain heart
infusion medium containing different concentration of pesticides. The radius of halo zone (zone of
clearence) will be measured after over night incubation.

Molecular identification of most potent pesticide metabolizing bacteria in gut of pesticide resistant
insects and their phylogenetic analysis

Molecular characterisation by 16s rDNA sequence analysis will be carried out using universal
primer. Identification will be achieved by comparing the sequence data with the reference data
collection using BLASTN 2.3.1 program (www.ncbi.nlm.nih.gov/BLAST/). The phylogenetic tree
will be generated using clustering algorithm Neighbor-Joining (NJ) method with 1000 bootstrap
replicates, partial deletion (95% site coverage as cut off) using Molecular Evolutionary Genetics
Analysis version 7.0 for big datasets MEGA7.

Study of pesticide tolerance under increasing concentration of pesticides

The best performing organism in term of pesticide degradation isolated from previous
experiment are subjected to dose dependent effect analysis of different pesticides. The bacterial
growth will be assessed at regular interval and presented graphically.

GC-MS and LC-MS/MS analyses of pesticide degradation

The degradation performance of selected microbes will be assessed by culturing each isolates in
a flask containing 50 ml of M9 media with 10µg/ml of different pesticide. Culture will be incubated
on shaker like earlier experiment each with three replicates. At regular interval the sample will be
collected from each flask and centrifuged. The supernatant were seperated and subjected to liquid
extraction using different solvent system. The resulting extract will be subjected to GC/ LC-MS analysis
to confirm the degradation of pesticides.

Purification of Enzyme

All the following procedure will be carried out at 0-4°C .The supernatant from the previous step will be
subjected to (NH4)2SO4 precipitation overnight. The precipitate will be collected by centrifugation at
12000g for 30 minutes and will be dissolved in minimum volume of 50mM Tris- Hcl buffer (pH7.0). The
enzyme solution will be subjected to dialysis against same buffer and concentrated further. The enzyme
solution will now loaded to sephadex-G column washed with Tris- Hcl buffer using Akta stat protein
purification system (GE health care Life sciences). Different fraction will be collected and the molecular
weight of the native enzyme will be determined using mol weight markers. Fraction with high specific
activity will be determined and will be subjected to further purification. The highly purified enzyme will
be used for making formulation of pesticide degradation.

Enzyme assay

Finally the enzyme assay will be carried out using different pesticide as substrate. The protein
concentration will be determined according to Lowry’s method. One unit of enzyme activity will be
defined as the amount of enzyme that catalyzes the degradation of 1 mmol of substrate ( test pesticide)
per min under standard conditions.

Enzyme characterisation

The purified enzyme will be subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and its mol weight will be determined using protein marker. The isoelectric point (pI) will be
estimated by PAGE using isoelectric focusing kit. The pH optimum, temperature optimum,
thermostability of the purified enzyme and effect of metal ions will also be determined.

Experimentation with cell lines

Effect of pesticide residues from fruits and vegetables before and after its degradation were
exposed to human neuronal, glial and melanoma cell lines. Cytotoxicity potential of different pesticide
in term of expression of oxidative stress related enzyme, cell proliferation, viability of cells, apoptosis
will be assed.