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Protocol for DNA extraction using the Qiagen DNeasy Blood and Tissue Kit (Catalogue No. 69504) presented here

Contents of the kit: DNeasy mini spin columns (2 ml), collecting tubes (2 ml), tissue lysis buffer (ATL), lysis buffer (AL), wash buffer 1 (AW1), wash buffer 2 (AW2), elution buffer (AE) and proteinase-K.

Note: Check for the validity of the product before dilution. Dilute the ATL and AL buffer with molecular gradient ethyl alcohol to a specified volume mentioned on the bottle before starting experiments. Keep a tick mark the bottle cap after dilution with ethyl alcohol for easy identification.

Equipments required: Microscope, centrifuge, vortex mixture, water bath, pestle and mortar/homogeniser, micropipettes, micro tips, eppendorf tubes, needle, micro scissor, forceps, camel hair brush, thermometer, tube stand, masks and gloves.

Polymerase Chain Reaction (PCR) is a laboratory technique, developed by Kary Mullis in the 1980s, used in molecular biology to amplify and replicate a specific segment of the DNA (Mullis, 1990). This reaction in performed in a thermal cycler, involving three main steps: denaturation, annealing and extension.

Principle: PCR is based on the principle of selectively amplifying a specific region of DNA through a series of temperature-controlled cycles. The key components involved in PCR include the target DNA sequence, oligonucleotide primers, DNA polymerase enzyme (Taq DNA polymerase), deoxyribonucleotide triphosphate (dNTPs) (A, T, G, and C), Mg2+ and a thermal cycler apparatus. Initially, the double-stranded DNA is split into single strands under high temperature of 90-95 0C known as the denaturation step. It is followed by cooling the solution to a temperature that allows proper attachment of the oligonucleotide primers to its complementary sites in the strands, known as the annealing step.

Pesticide Toxicity

Chemical tactics to manage pests can include many types of compounds. Some merely repel or confuse pests. Some interfere with weed photosynthesis, insect molting processes or development in some way. Others, including some botanical and most conventional insecticides are broadly toxic to living systems. The term pesticide literally means “pest killer". Generally speaking, when using pesticides, we are trying to garner the advantages of using pesticides while minimizing the risk to humans and other non-target organisms. Risk to humans of pesticide use is often described as:

1. Bordeaux Mixture

It was invented in the Bordeaux region of France by PM Millardet (1982).

Also called Bordo Mix.

Mixture of Copper sulfate (CuSO₄), Slaked lime and Water.

It is known locally in France as Bouillie Bordelaise.

The behavior of pesticides is dependent on many factors, so be sure to consider everything from light and temperature to moisture and pH and recognize that all the factors will impact the active ingredient. Here is some information on these topics, and others, which will improve pest management success.

1.Identification: The first step to disease and pest management success is the proper identification of the insect, disease or weed pest. Without identification of the pest, controls may be misused or improperly timed.

2.Time of Application: Time of application of pesticides plays major role in optimizing pesticidal performance. Early morning or late afternoon are the best times to spray. Dust formulations should be apply in early morning hours (6 am).In the case of foliar spray, it should be done in morning hours between 8 am to 11 am. Avoid spraying during the heat of the day.

3.Water Quality and pH: Primary factors important in water quality are pH and alkalinity (bicarbonates). Most of the pesticides show high efficiency in slightly acidic to slightly basic water pH (i.e. 6.5 to 7.5). Many water sources in alkaline pH (<7.0). Alkali pH (7.5>) is always harmful for pesticides. High pH levels are responsible for alkaline hydrolysis, a process that breaks down the active ingredient in many insecticides, fungicides and herbicides.Add a buffer agent to lower spray solution to a pH of 5.5-6.5. This will give better initial knockdown and longer residual action.

1. Introduction

Insects, like many other organisms, rely heavily on their diet for essential nutrients that regulate their growth and development. The quality of food intake significantly impacts various aspects of an insect’s life cycle. Research, such as that conducted by Lee (2015) highlights that consuming a diet lacking in essential nutrients can lead to premature aging, a process known as senescence, in insects. Furthermore, such inadequate nutrition can hasten the rate of mortality as the insect ages. This underscores the critical role that proper nutrition plays in the longevity and overall health of insect populations.

The Effectiveness of a Pesticide in Controlling a Disease and Pest Depends on

Using the right pesticide for the target disease & pest.

Applying the pesticide at the right time.

Applying the pesticide in the right amount.

Applying the pesticide to the right place.

Sprayer is an equipment in which the spray fluid is broken up into fine droplets and ejected under pressure. Actually, the function of the sprayer is to provide the spray fluid with some energy so that it can be broken into fine droplets through the nozzle and propelled to the target. The energy can be produced manually or with the help of any other source of power. There are a variety of sprayers.

Type of Sprayers: There are a variety of sprayers.

Pesticide Dusting

The dusting powders are low concentration ready to use type, dry formulations containing 2 to 10% pesticide. The inert material or dry diluents is talc, soapstone, attapulgite, etc. and it is non toxic. The sulphur dust is not diluted with inert material.

Majority of the pesticides are applied as sprays. The liquid formulations of pesticide either diluted or directly are applied in small drops to the crop by different types of sprayers. Generally the wettable powder, EC formulations are diluted suitably with water which is a common carrier of pesticides. However, in few cases oil is used as carrier of pesticides.

Any sprayer nozzle essentially breaks the liquid into small droplets and throws them. All nozzles in the field produce rangesof droplets of different size called spray spectrum. The hydraulic nozzles have wide range of size of droplets whereas the rotary nozzles have narrow range. Adroplet of 400 µm diameter is 1000 times bigger in volume than a droplet of 40 µm. The volume of spherical droplets can be worked out as 4/3 µr3. So if the droplet size of aspraying system could be reduced to half, then there will be 8 fold increases in the number of droplets and the coverage will also increase.

i) Common Maintenance

Clean outer surface with brush or cotton waste by using plenty of water or kerosene oil.

Oil the moving or rubbing surfaces of parts with lubricating oil or grease.

Filter the chemical solution/ fuel oil mixture while pouring into the tanks. Make the caps or lids leak-proof with gaskets.

Flush the equipment with clean water to wash inside parts of containers, tubes and nozzles to be free from chemicals.

The objective of correct pesticide application cannot be achieved without good quality Plant Protection Equipment. A sound designed machine shall be proficient as far as pesticide distribution and delivery to the target in minimum time with least wastages is concerned. Hence, the machines should be tested to ascertain that they are:

A. Purchase

a)Do not purchase leaking containers, loose, unsealed or torn bags.

b)Do not purchase pesticides without proper/approved labels.

c)Purchase only just required quantity e.g. 100, 250, 500 or 1000 g/ml for single application in specified area.

Insect Control and Insecticides

Control Tips

Practicing Integrated Pest Management can significantly reduce the amount of insecticides needed to control many insect problems.

Broad-spectrum insecticides are effective against all insects, even the good ones. Other insecticides target certain insects. Using a targeted insecticide minimizes the risk to beneficial or non-target insects.

Some insecticides work immediately to kill insects while others may need some time to take effect.

Using more than one insecticide product in the same location can increase or decrease each one’s effectiveness. It may also result in a greater risk to health and/or the environment.

Insect growth regulators like pyriproxyfen and methoprene do not kill insects; they make it impossible for exposed insects to molt (grow) or lay eggs properly.

Insecticidal baits can be used instead of spraying large areas, especially for social insects like ants. This can decrease the risk of exposure, but remember to place baits where children and pets won’t have access.

The integument is composed of the cuticle and under lying epidermal cells that secrete the cuticle. The integument is a complex vulnerable organ system of diverse function. The insect outer body covering is called integument. The integument is a central subject intimately related to a variety of applied - Problems such as the mode of action, insecticides, ecology, cuticle and endocrine glands present in the body of insects. The insect integument consists of 3 parts and plays crucial role in locomotion, breathing and respiration, feeding, excretion, protection from desication behaviour of insect.

1. Epidermis Layers
2. Basement Layers
3. Cuticle Membrane

ORIGIN OF INSECT

Entomology is a branch of zoology which deals with insects. In this branch we study the insect origin and evolution of insect and their diversity and classification and other parts of insect body. Insects belong to the class insecta in the phylum arthropoda, the largest group in the animal kingdom. All arthropods are characterised by having segmented body, claws, chitinous exoskeleton, ventral nervous system and dorsal heart. The class insecta is generally considered to have evolved from myriapod of some sort during the Devonian period. Based on different mandibles and mandibular movement concluded that insects are not direct insect group myriapods the mouth is located between the prostomium and the first body segment and the anus open in the periproct.
The first great step was the development of a pair of ventral appendage. Insect legs on each body segments and locomotion, parallel to this an improvement in the sense organs of the head occured eyes and Antenna were the ultimate result this level of organisation is met in onychophora. A series of diagramms is helpful in a very simplified way in visualising the hypothtical origin of insects.

Objective: To understand the taxonomic characters and classification of the order Coleoptera, which includes beetles and weevils.

Order: Coleoptera (Coleos = sheath, Pteron = wing) Beetles and Weevils

Taxonomic Characteristics

1. Two pairs of wings, with the forewings thickened and hardened, forming protective covers called elytra, while the hind wings are membranous and protected by the elytra.

2. Both larvae and adults possess biting and chewing mouthparts.

3. Metamorphosis is complete.

4. Beetle larvae are commonly known as grubs. Snout beetle (weevil) grubs are legless (apodous).

Objective: To comprehend the fundamental taxonomic characteristics of the order Hymenoptera and its agriculturally significant families.

The order Hymenoptera displays remarkable diversity in its insect inhabitants. The term “Hymenoptera” originates from “Hymen” meaning membrane or the God of marriage, and “ptera” meaning wings.

Taxonomic Characters

1. Hymenopterans possess mandibulate mouthparts, where the labium and maxilla’s basal sections lie closely side by side, forming a sharp transverse fold near the basal third. In bees, these structures integrate to form a lapping tongue, representing a chewing and lapping type of mouthpart.

2. The thorax is specialized for efficient flight, featuring a collar-like pronotum, an enlarged mesothorax, and a small metathorax. The prothorax and metathorax are fused with the mesothorax.

Excretory system of an organism maintains its homeostasis i.e., a constant internal environment. Here in insects, the blood is haemolymph therefore it determines the excretion of an insect’s body. Excretion also eliminates the nitrogenous wastes and maintains the salt- water balance and also regulates the uniformily of haemolymph. The characteristic feature of insects is the presence of malpighian tubules to eliminate the metabolic wastes, whereas rectum walls carry out the function of reabsorption of salt and water. A minimum amount of water is excreted with nitrogen waste in order to conserve the water in body.