eChapters

Enzymes are biocatalysts that play a crucial role in accelerating chemical reactions without undergoing any changes themselves. Enzymes enhance the rate of a reaction by reducing its activation energy without affecting the equilibrium position of the reaction. These catalysts specifically target molecules called substrates and convert them into different molecules known as products. Most enzymes have a proteinous nature, and some have a ribonucleic acid nature (like ribozymes and peptidyl-transferase), showing high substrate and stereo-specificity. Enzyme catalysis is indispensable for the majority of metabolic processes in cells, as it ensures that these processes occur at a rate that is sufficient to sustain life. The progression of metabolic pathways heavily relies on the catalytic activity of enzymes to facilitate individual steps.

The proteinous enzymes are classified into simple and conjugated enzymes.

1. Simple enzymes: Enzymes are made up of only protein components.

2. Conjugated enzymes: Enzymes comprise both protein (apoenzyme) and non-protein components (cofactor).

Vitamins are natural and indispensable nutrients that are essential for our bodies in small quantities. Vitamins are indispensable for a multitude of biological functions, including growth, development, wound healing, maintaining healthy bones and tissues, supporting the immune system, and more. These vital organic compounds exhibit diverse biochemical functions and are essential for metabolic processes. The discovery of vitamins dates back to 1912 when Casimir Funk, a Polish American biochemist, initiated research on their sources, functions, and deficiency disorders. Because of his groundbreaking work, Funk is widely recognized as the pioneer of vitamins.

Types of vitamins

The classification of vitamins is determined by their solubility, resulting in two distinct groups: fat-soluble vitamins and water-soluble vitamins.

Fat-soluble vitamins

Fat-soluble vitamins, as their name implies, need fat for absorption and are stored in the fat cells. These vitamins include Vitamin A, D, E, and K.

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed.

Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials.

Cytotoxicity

Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison.

In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death).

Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome.

Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic.

Why we Need to Evaluate Genotoxicity?

Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells.

Isolation of Genomic DNA

Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below:

1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test.

2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration.

3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs.

4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2).

5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K).

6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA.

7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight.

8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell.

Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Spontaneous contractions of the uterus are controlled and coordinated for various reproductive functions. The non-pregnant uterus facilitates ova and sperm transport, provides adequate embryo placement for implantation, and contributes to the expulsion of menstrual debris through increased contractility, or periods of relative quiescence (Hutchings et al., 2009). Uterine dysmotility has been implicated in the pathogenesis of certain infertilities, implantation failure, endometriosis, dysmenorrhea, and abnormal menstrual events (Kunz and Leyendecker, 2002; Aguilar and Mitchell, 2010). Although these conditions may not contribute to mortality but can significantly affect patient’s reproductive health, and considerably alter their quality of life (Jones et al., 2004; Weissman et al., 2004; Chachamovich et al., 2010). Dysmenorrhoea refers to the occurrence of painful cramps in the lower abdominal region during menstruation and is one of the unresolved gynecological disorders in adolescent girls and women of reproductive age (Wang et al., 2004; Harel, 2008). This condition is mainly characterized by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work (Davis and Westhoff, 2001).

Introduction

Acute lung injury (ALI) is the manifestation of an inflammatory response of lung resulting from severe sepsis, bacterial or viral pneumonia, trauma, and burns in animals (Ali et al., 2020).It is distinguwashed by disruption to the alveolar capillary membranes, atelectasis of the pulmonary airspaces, and neutrophil and protein-rich fluid infiltration into the alveolar space (Li et al., 2019). Although advances in treatment, the disability and fatality rates of ALI remain high, that is around 30-40% (Ali et al., 2020). At the moment, new therapeutic alternatives are urgently required to treat ALI. As a result, this is a very active area of research to understand the molecular principles underlying pulmonary inflammation and ALI (Reiss et al., 2012). The use of animal models of inflammatory lung has allowed for the probing of pathways of inflammation responsible for injury. Specially, rodent models may serve a link between in vitro research to large animals and clinical studies by allowing for high quantities of samples under physiological circumstances at low expenses (Domscheit et al., 2020).LPS, found in the outer membrane of gram-negative bacteria, consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides (Gao et al., 2023). LPS induced lung injury model, closely mimic sepsis and inflammatory lung diseases, is a well-establwashed model in this field of research (Reiss et al., 2012). To mimic acute lung injury, LPS can be administered either directly through intratracheal or intranasal route (direct pulmonary insult) or indirectly through intraperitoneally and intravenous routes (indirect extra pulmonary insult) (Perl et al., 2011). This chapter focuses mostly on rodent model of acute lung injury caused by lipopolysaccharide (LPS), both direct and indirect. Additionally, it also includes the characteristics and underlying molecular mechanism of LPS induced lung injury along with applications in drug development.

Mesenchymal Stem Cells

Recent advances in the field of regenerative medicine have opened up newer therapeutic modalities using cell-based and cell-free therapies, particularly stem cells, as potential candidates. As per the definition, stem cells are the undifferentiated progenitor cells that can regenerate and can also differentiate into various specialized cells (Hoang et al., 2022). They can be classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and postnatal adult stem cells (Locke et al., 2009). Among postnatal adult stem cells, mesenchymal stem cells (MSCs) have emerged as a popular choice of candidate for therapeutics due to the relatively easy availability, simple isolation techniques, low risk of teratogenicity, minimal ethical and legal concerns unlike embryonic stem cells, hence, are easy to apply in clinical research and practice (Voga et al., 2020). Among MSCs of different origin, bone marrow and particularly adipose tissue-derived stem cells (ADMSCs) received more attention and are one of the most common and promising stem cell types. These cells can be simply obtained from subcutaneous and omental fat or liposuction aspirates for the treatment of diverse clinical applications (Kim &Heo, 2014). These cells can also be expanded vigorously in vitro and differentiated into specific cell lineages such as adipocytes, osteoblasts, chondrocytes, and neurocytes and are even genetically stable in long-term cultures (Miana& González, 2018). Mesenchymal stem cells (MSCs) are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the (ISCT) International Society for Cell and Gene Therapy (Dominici et al., 2006).

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Flow Cytometry is a tool used to detect and measure chemicophysical characteristics of a population of cellular particles. Modern day biomedical research needs the molecular assembly of tools to proceed in the advent of biomedical research with real time diagnostic, prognostic and experimental detection of markers. For the same, a naïve tool is flow cytometry. In this technique, a sample having cells or particles is suspended into a fluid and injected into instrument. The ability to perform these measurements in a very rapid time span is one of the key advantages of the flow cytometry process. A single-cell suspension must first be prepared in order to analyse solid Tissues. A flow cytometer is look alike to microscope, except that, instead of producing an image of the cell, it generally offers a cell-by-cell basis high-throughput, automated quantification of specified optical parameters.

They can quantify up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute. Sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. These cells are tagged with fluorescent markers in order to absorb the light and then emitted them in the form of band of wavelengths. Average ten thousand cells can be quickly examined and the data gathered are processed by a computer.

High-performance liquid chromatography or high-pressure liquid chromato graphy (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture. It involves the injection of a small volume of liquid sample into a tube packed with tiny particles (1.8- to 5-micron (μm) particle size called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

Aflatoxins are toxic and carcinogenic compounds produced as metabolites by many Aspergillus species. This fungus invades habitats such as soil, grain, nuts, or decaying vegetation when- ever suitable conditions such as high moisture and temperature are met. B1 is the most toxic of at least 14 different types of naturally occurring aflatoxins. Therefore, many countries and regulatory agencies impose strict limits on B1 as well as other common aflatoxins such as B2, G1, and G2 despite exhibiting lower toxicity. The U.S. FDA set the action level to20 μg/kg (ppb) for human food and to 300 μg/kg (ppb) for animal feed. Aflatoxin B1 is extremely widespread and is present in food and feed products such as cottonseed, corn, and peanuts. It is highly toxic and has been classified as a group 1 carcinogen by the WHO. The aflatoxins B2 , G1 , and G2 are usually found accompanying B1 , in lower concentrations in the contaminated samples. The order of toxicity is AFB1 > AFG1 > AFB2> AFG2.

Introduction

Minerals are present in all body tissues and fluids to carry out the physicochemical processes of life; both in animals and human. It is broadly classified into two categories: (1) macro and (ii) micro or trace elements. The macro elements include calcium, phosphorus, potassium, magnesium, sulfur, sodium and chloride, while elements such as iron, copper, cobalt, iodine, zinc, manganese, molybdenum, selenium and chromium come under micro elements. Animals need large amounts of calcium for construction and maintenance of bone and normal function of nerves and muscles. Phosphorus is a vital constituent of adenosine triphosphate (ATP) and nucleic acid and is also essential for acid-base balance, bone and tooth formation. Iron is an important component of the cytochromes for carrying out cellular respiration in addition to its vital role in red blood cells. Magnesium, copper, selenium, zinc, iron, manganese and molybdenum are important co-factors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways. Animals need iodine to make thyroid hormones. Sodium, potassium and chlorine are important in the maintenance of osmotic balance between cells and the interstitial fluid. Excessive intake of some minerals can upset homeostatic balance and cause toxic side effects.

Isolated tissue preparations are widely used in physiological or pharmaco logical research to delineate the mechanism of action of drugs or any active biomolecules. Traditionally it is used as a tool for bioassay of drugs. The tissue used for such pharmacodynamics studies may be isolated from different animal models viz. frog, rodents, rabbits, sheep, goat, buffalo, chicken etc. Evaluation of drug-receptor interaction and underlying mechanism of action is then performed using isolated organ bath or myograph where the isolated tissues are mounted under optimum resting tension and changes in the tension following exposure of drug is recorded with the help of recording device. With the advancement of science and technology, the recording devices are also gradually upgraded from smoked drum to student’s physiograph to the most advanced Data Acquisition System (DAS) based polyphysiograph.

The isolated tissue preparations (uterine strips, arterial rings, tracheal chains etc.) need essential and appropriate nutrients and environment during experimental which is generally provided by using suitable salt solutions (Physiological Salt Solutions) which provide ionic balance, energy supply, maintain pH and tissue tonicity. The composition of physiological salt solutions is such that it provides an artificial media simulating with the inorganic composition of blood plasma together with a buffer mechanism to maintain an optimum pH of 7.0 to 7.4 and glucose to facilitate tissue metabolism. Commonly used PSS are Frog ringers, tyrode, Kreb’s, de Jalon, Ringer-Locke, modified Kreb’s Henseleit solution etc.

The cytological insult due to exposure of different environmental pollutant viz. pesticides, herbicides etc. impose great loss to the vitality and health of the individuals. The basic pathophysiological distrubances following the expo sure of these deterrents to the different visceral organs leads to dysfunction of these organs. Imbalance between body’s antioxidant defense and peroxidation of the lipid layer of cells’ causing free radical generation is defined as Oxidative stress. Free radicals are the molecules or entity that contains one or more than one unpaired electron. Pathophysiology of several diseases are associated with the generation of oxidative insult and many xenobiotics associated toxicity are also a result of oxidative damage and generation of reactive oxygen species and reactive nitrogen species. Alzheimer’s Disease is one of the fine example of oxidative damage associated neuronal degeneration.

Oxidative stress can be estimated using following parameters:

1. Lipid peroxidation (Paula et al., 2005)

2. Reduced glutathione (Sedlak and Lindsay, 1968)

3. Superoxide dismutase (Madesh and Balasubramanian, 1998)

4. Catalase (Aebi, 1983)

Tissue Collection

Rats are most commonly employed as test subjects to conduct the experiments related to drug metabolizing enzyme assays involving various enzymes i.e. microsomal and non microsomal enzymes. Rats are humanely sacrificed at the end of the exposure period of 28 days of any therapeutic agents or drug and the liver of all the animals has to excise and immediately wash with ice-cold normal saline, weigh and perfuse with ice-cold 1.15% KCl solution containing 0.05 mM EDTA. Livers are processed further for preparation of microsomes for determination of microsomal drug metabolizing enzymes.

Preparation of Microsomes

Livers tissues are homogenized in 3 volumes (1:3) of ice-cold (1-4o C) homogenization buffer containing 1.15% potassium chloride in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates are centrifuged at 15000 g for 30 min (Hitachi cs120GX II). The mitochondrial supernatant (8 ml) is transferred to ultracentrifuge tubes and spun at 105000 g for 1 hour in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions are collected from the middle separating the lipid layer on top in separate micro centrifuge tubes. The microsomal pellet is then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet so obtained is resuspended in 4 ml of resuspension buffer and 1 ml aliquots are stored immediately at -80o C until assayed for drug metabolizing enzymes.

Acetylcholinesterase activity in brain is measured by the method described by Ellman’s et al. (1961).

Reagents

1. Phosphate buffer (0.1M) (pH 8.0) Solution A: 5.22 g of K2 HPO4 and 4.68 g of NaH2 PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2 g of NaOH is dissolved in 150 ml of distilled water. Solution B is added to solution A to get desire pH 8.0 and then finally the volume is made up to 300 ml with distilled water.

2. DTNB reagent 39.6 mg of DTNB with 15 mg of NaHCO3 is dissolved in 10 ml of 0.1 M phosphate buffer.

3. Acetylthiocholine (ATC) 21.67 mg of Acetylthiocholine is dissolved in 1 ml of distilled water.

Procedure

The brain tissue is weighed and 10% homogenate is made in 0.1M Phosphate buffer (pH 8.0). Take 0.4 ml aliquot of homogenate in a cuvette containing 2.6 ml phosphate buffer (0.1M) and 100 µl DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reached a stable value, it is recorded as the basal reading .To this 20 µl of acetylthiocholine is added and change in absorbance is recorded for a period of 10 min at intervals of 2 min. Change in the absorbance per minute determined.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using a large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of the drug discovery process can save time and money both by eliminating least beneficial NCEs.

Preparation of Reagents

Culture media

For the culture of hepatocytes medium M199 (GibcoBRL, USA) is used. One pouch of the dry media is dissolved in 1 liter of filtered distil water. The pH of the media is adjusted by adding 0.4% sodium bicarbonate to be around 7.4. The media is than sterilized by filtration through seitz filter (0.22 mm) and stored in 50 ml sterilized conical flasks.

Isolation of Peripheral Blood Lymphocytes

Chemicals

1. Heparin

2. Sterile PBS (pH 7.2)

3. Histopaque-1077

4. RPMI-1640 Media

Protocol

Collect 5 ml of blood from the animal in 1 ml (20 IU/ml of blood) of diluted heparin and add 2 ml of PBS. Layer over the diluted blood on 5 ml of histopaque-1077 in 15 ml centrifuge tube and centrifuge at 1500 rpm for 40 min. Collect the buffy coat at the interface of plasma and histopaque in separate centrifuge tubes. Wash the cells thrice with PBS and suspended in 2 ml of RPMI-1640 media supplemented with 10% fetal calf serum. Determine the percentage cell viability by 0.1% trypan blue exclusion test, using hemocytometer. Finally dilute the cell suspension to 106 cells/ml in RPMI 1640 medium.

LD 50- It is defined as the concentration/dose that kills 50 % of the experimental population.

Acute toxic studies have been conducted in order to calculate LD50. There are several methods to calculate LD50, some are as follows:

Methods to Determine LD 50

• Karber’s method.

• Miller & Tainter method

• Lorke method

• Fixed dose method

• Acute Toxic Class method

• Up & down procedure

Karber’s Method

This method involves the administration of different doses of test substance to various groups, which has five animals each. The first group of animals is administered with the vehicle in which the test substance was dissolved or diluted

Introduction

The immense use of chemicals and pharmacological entities has put the public health at brisk of potential health hazards. These substances in modern days are presented in the form of common constituents of food, medicines, beverages and junk foods etc. However, long term consumption of these substances might be responsible for chronic toxicity, but the accidental ingestion of large quantities of these substances may result into adverse acute toxic reactions. Hence an experimenter should have the acumen to develop and perform the acute toxic tests in order to find out the lethal dose 50 i.e. LD50 of the substances.

Acute Toxicity Studies

Acute toxicity studies helps to establish the dose dependent adverse effect including mortalities. The LD50 has been used as a major parameter in screening of pharmacological agents for acute toxic potential. Apart from mortality, other biological effects and the time of onset, duration and degree of recovery on survived animals, are also important in evaluation of acute toxicity. Hence, acute toxicity study solely gives information about LD50, therapeutic index and the degree of safety of a pharmacological agent.

Acute toxicity is defined as adverse effects occur following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. Oral administration is the most common form of acute systemic toxicity testing.

Epididymal Total Sperm Count

The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003).

Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor)

Liveability and abnormal sperm

Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa.

The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain.

• A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide.

• 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop.

• Epididymal semen and stain are mixed gently using a blunt fine glass rod.

• After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide.

• The smear is examined under oil immersion objective.

1.1 History and Evolution of Aquaculture

Aquaculture, the cultivation of aquatic organisms such as fish, crustaceans, mollusks and aquatic plants, has a rich history dating back thousands of years. Its evolution has been shaped by diverse cultural practices, technological advancements, and growing demands for sustainable food production. This essay explores the history and evolution of aquaculture, highlighting key developments from ancient times to the modern era.

Ancient Origins of Aquaculture

The earliest records of aquaculture date back to ancient China around 2500 BCE. The Chinese practiced fish farming, particularly with carp, in ponds. Carp were favored due to their resilience and adaptability. This period marked the beginning of organized aquaculture, where fish were bred and raised in controlled environments. The Chinese recognized the benefits of aquaculture for food security and sustainability, laying the foundation for future practices.

In Egypt, evidence suggests that tilapia were farmed as early as 1500 BCE. Ancient Egyptians built ponds along the Nile River to cultivate fish, which were an essential part of their diet. These early aquaculture practices were driven by the need for a reliable food source, especially during periods of low f ish availability in natural waters.

Roman and Greek Contributions

The Romans and Greeks also contributed to the development of aquaculture. In ancient Rome, mullet and oysters were cultivated in man-made ponds called “vivaria.” These ponds were designed to mimic natural habitats, providing ideal conditions for the growth and reproduction of fish. The Romans were known for their engineering prowess, and their aquaculture systems reflected this, with intricate designs to regulate water flow and quality.

2.1 Definition and Key Concepts of Sustainable Aquaculture

Aquaculture, the practice of farming aquatic organisms, has become an essential component of global food production. As the demand for seafood increases, the sustainability of aquaculture practices is critical to ensuring long-term ecological, economic, and social benefits. This essay explores the definition and key concepts of sustainable aquaculture, emphasizing its importance, guiding principles, and the strategies employed to achieve sustainability.

Definition of Sustainable Aquaculture

Sustainable aquaculture refers to the development and management of aquaculture systems that meet the needs of the present without compromising the ability of future generations to meet their own needs. It involves balancing the economic viability, environmental health, and social equity of aquaculture practices. Sustainable aquaculture aims to:

1. Protect and Restore Ecosystems: Minimize environmental impacts and enhance ecosystem services.

2. Support Economic Viability: Ensure the profitability and resilience of aquaculture operations.

3. Promote Social Responsibility: Foster equitable access to resources, benefits, and opportunities for all stakeholders.

3.1 Applications

Nanotechnology, with its ability to manipulate materials at the atomic and molecular scale, offers diverse applications across various sectors, including aquaculture. In this extensive exploration, we delve into the innovative applications of nanotechnology in aquaculture, focusing specifically on areas beyond water parameter control and disease prevention. This comprehensive review highlights how nanotechnology enhances feed efficiency, improves growth and health monitoring, develops novel materials, and addresses environmental and sustainability challenges within the aquaculture industry.

1. Introduction to Nanotechnology in Aquaculture

1.1 Understanding Nanotechnology

Nanotechnology involves the manipulation and engineering of materials at the nanoscale, typically ranging from 1 to 100 nanometers. At this scale, materials exhibit unique physical, chemical, and biological properties that differ significantly from their bulk counterparts. These properties make nanomaterials highly versatile for applications across various industries, including agriculture and aquaculture.

4.1 Genetic Improvement of Aquaculture Species

1. Introduction

1.1 Importance of Genetic Improvement in Aquaculture

Aquaculture serves as a critical component of global food production, supplying a substantial portion of seafood consumed worldwide. However, the industry faces challenges such as disease outbreaks, environmental changes, and increasing demand for sustainable practices. Genetic improvement plays a crucial role in addressing these challenges by enhancing the traits that contribute to productivity, disease resistance, and environmental adaptability in aquaculture species.

1.2 Role of Genomics and Biotechnology

Advancements in genomics and biotechnology have revolutionized the way aquaculture species are bred and managed. Genomics enables the detailed study of an organism’s genetic makeup, allowing researchers to identify specific genes associated with desirable traits. Biotechnological tools, including genome editing and molecular markers, facilitate precise manipulation of these genetic traits, accelerating the breeding process and improving the efficiency of trait selection.

5.1 Internet of Things (IoT) and Smart Aquaculture Practices

1. Introduction to IoT in Aquaculture

1.1 Definition of IoT in the Context of Aquaculture

The Internet of Things (IoT) refers to a network of interconnected devices, sensors, actuators, and software applications that collect, exchange, and analyze data to automate processes and provide actionable insights. In aquaculture, IoT integrates digital technologies to monitor, manage, and optimize farming practices in real-time, enhancing efficiency, sustainability, and productivity.

1.2 Importance and Benefits of IoT in Aquaculture

IoT offers numerous benefits to aquaculture operations:

• Real-time Monitoring: IoT sensors continuously monitor critical parameters such as water quality (e.g., pH, dissolved oxygen, ammonia levels), temperature, and salinity. This real-time data allows farmers to maintain optimal conditions for aquatic organisms, ensuring their health and growth.

6.1 Alternative Protein Sources

Introduction

The rapid expansion of aquaculture as a significant contributor to global food security has highlighted the need for sustainable feed and nutrition practices. Traditional aquaculture feeds rely heavily on fishmeal and fish oil, derived from wild-caught fish, which poses ecological and economic challenges. The overreliance on these marine resources can lead to overfishing, increased production costs, and environmental degradation. To address these challenges, the aquaculture industry is increasingly exploring alternative protein sources that are sustainable, cost-effective, and nutritionally adequate. This essay discusses various alternative protein sources, their benefits, and challenges in the context of sustainable aquaculture feed and nutrition.

Plant-Based Protein Sources

Plant-based proteins are among the most promising alternatives to fishmeal in aquaculture diets. These proteins are derived from crops such as soybeans, peas, and canola. They offer several advantages, including wide availability, cost-effectiveness, and relatively low environmental impact.

7.1 Water Quality and Waste Management

Introduction

Water quality and waste management are critical components of environmental management and conservation efforts, particularly in the context of aquatic ecosystems and freshwater resources. Effective management of water quality ensures the health and productivity of aquatic habitats, supports biodiversity, and sustains ecosystem services essential for human well-being. Similarly, proper waste management practices mitigate pollution, reduce environmental impact, and promote sustainable use of natural resources. This article explores key concepts, challenges, strategies, and innovations related to water quality management and waste management in environmental conservation.

Water Quality Management

Water quality management involves monitoring, assessing, and regulating the physical, chemical, and biological characteristics of water bodies to maintain or improve their suitability for aquatic life, human use, and ecosystem health.

8.1 Disease Prevention and Control in Aquaculture

Aquaculture, the farming of aquatic organisms such as fish, shellfish, and plants, faces significant challenges related to disease management. Diseases in aquaculture can result from viral, bacterial, fungal, or parasitic pathogens, and their outbreaks can lead to substantial economic losses and environmental impacts. Effective disease prevention and control strategies are crucial to sustainably manage aquaculture operations and ensure the health and welfare of cultured species.

Key Strategies for Disease Prevention and Control

1. Biosecurity Measures

Biosecurity is foundational in preventing the introduction and spread of diseases within aquaculture facilities. It involves a set of management practices aimed at minimizing the risk of pathogen transmission through various pathways:

• Quarantine Protocols: New stocks should undergo quarantine periods before introduction to production systems. This allows for health screening and observation to detect and isolate potentially diseased individuals.

9.1 Success Stories from Around the World

Sustainable aquaculture practices are essential for ensuring the long-term viability of the aquaculture industry while protecting the environment, supporting economic development, and enhancing social welfare. This article highlights several success stories from around the world that exemplify innovative and sustainable aquaculture practices. These case studies demonstrate the diverse approaches taken to achieve sustainability in different regions and contexts.

1. Integrated Multi-Trophic Aquaculture (IMTA) in Canada

Location: Bay of Fundy, Canada

Species: Atlantic salmon, blue mussels, kelp

Approach: Integrated Multi-Trophic Aquaculture (IMTA) is a practice where different species are farmed together in a way that mimics natural ecosystems. In the Bay of Fundy, Canada, salmon, mussels, and kelp are cultivated in proximity, creating a balanced system. The salmon are fed traditional aquaculture diets, and their waste provides nutrients for the mussels and kelp.

10.1 Emerging Technologies and Innovations in Aquaculture

Aquaculture, the farming of aquatic organisms, is a rapidly growing sector within the global food production system. As the demand for seafood continues to rise, the industry is turning to innovative technologies and practices to ensure sustainable growth, improve productivity, and address environmental challenges. This article explores some of the emerging technologies and innovations that are shaping the future of aquaculture.

1. Recirculating Aquaculture Systems (RAS)

Definition and Concept: Recirculating Aquaculture Systems (RAS) are land based fish farming systems that reuse water by filtering and treating it before returning it to the tanks. This closed-loop system allows for precise control of water quality and environmental conditions.

Innovations and Benefits

• Water Conservation: RAS significantly reduces water usage compared to traditional open-net pen farming, making it suitable for areas with limited water resources.

• Environmental Impact: By treating and reusing water, RAS minimizes the release of pollutants and nutrients into natural water bodies, reducing the risk of environmental contamination.

• Environmental Impact: By treating and reusing water, RAS minimizes the release of pollutants and nutrients into natural water bodies, reducing the risk of environmental contamination.

Human beings came into existence 1 million years ago. Insects which constitute 70-90% of all animals present in this world came into existence 250- 500 million years ago.Insects are highly specialized group of invertebrates belonging to phyla arthropoda characterized by having a bilateral symmetrical body covered by Exoskeleton chitin; with jointed appendages provided with independently movable muscle and inserted in to special pockets of exoskeleton; with a haemocoelic body cavity containing the circulating blood in which are bathed the internal organs; with a dorsal heart and decentralized ventral nerve cord; and with tracheal system for respiration; metamorphosis direct or indirect.

The insects are bilaterally symmetrical tracheate Arthopods is which the body is divided into head, thorax and abdomen. A single pair of antennae is present and head also bears a pair of mandibles, a pair of maxillae,. The thorax possess three pairs of legs and usually two pairs of wings. The abdomen is devoid of any ambulatory appendages and the genital opening is situated near the posterior end of the body. Post embryonic development is rarely direct and metamorphosis usually occurs.

Insect definition

Insects (from Latin insectum, a calque of Greek ?ντομον [éntomon], “cut into sections”) are a class of invertebrates within the arthropod phylum that have a chitinous exoskeleton, a three-part body (head, thorax and abdomen), three pairs of jointed legs, compound eyes and one pair of antennae.

American Ecologist Whittaker (1969) proposed the five kingdom system of classification. In his ‘Five Kingdom System’, he added three more kingdoms in to Linnean model of two kingdom system. These were: Monera, Protista and Fungi. Whittaker succeeded to overcome the difficulties as well as demerits of two, three and four kingdom systems and to represent the living organisms according to the evolutionary relationships among themselves. He also defined the kingdoms by a number of special characteristics such as whether the organisms possessed a true nucleus or not. Whittaker’s five kingdom system of classification is based on (a) mode of nutrition (b) cell structure and complexity (c) phylogenetic relationship (d) body organization and (e) reproduction.

The large branches within the Animal Kingdom evolutionary tree are ‘phyla’ (the singular being ‘phylum’). ‘Animal phyla’ outlines thirty three phyla, although there are still disputes and disagreements concerning how many phyla are needed to classify the less well-known species. The most easily recognised are Porifera, Cnidaria, Arthropoda, Nematoda, Annelida, Mollusca, Platyhelminthes, Echinodermata, and Chordata. Animal phyla each comprise a branch of the evolutionary tree and these branches are always connected to other branches.

Body Segmentation

In general, insect body is divided in to a series of segments, which in primitive arthropods are known as “somites” or “metameres”. During the process of evolution, these somites gets fused with each other in different ways forming the body parts of the existing arthropods. The type of arrangement of these body segments in embryonic stage is known as primary segmentation while in adult insects is known as the secondary segmentation which differ from primary in having a sclerotized membranous intersegmental region.

In adult and nymphal insects, and hexapods in general, one of the most striking external features is the amalgamation of segments into functional units. This process of tagmosis has given rise to the familiar tagmata (regions) of head, thorax, and abdomen. In this process the 20 original segments have been divided into an embryologically detectable six-segmented head, three-segmented thorax, and 11-segmented abdomen (plus primitively the telson), although varying degrees of fusion mean that the full complement is never visible.

Types of body segmentation.

(a) Primary segmentation, as seen in soft-bodied larvae of some insects. (b) Simple secondary segmentation. (c) More derived secondary segmentation. (d) Longitudinal section of dorsum of the thorax of winged insects, in which the acrotergites of the second and third segments have enlarged to become the postnota. (After Snodgrass 1935)

Endoskeleton of the Head (Tentorium)

• The tentorium forms during development when 2-4 pairs of apophysis/ apodemes (finger-like invaginations of exoskeleton) fuse internally to create a “bridge”.

• The major components of the tentorium are the anterior tentorial arms, the posterior tentorial arms, the dorsal tentorial arms, and the tentorial bridge.

• The anterior tentorial arms originate as invaginations at the lateral limits of the frontoclypeal suture. The coalescence of the anterior tentorial arms forms a more or less solid structure, the tentorial bridge.

• The posterior tentorial arms arise from pits at the ventral ends of the post occipital suture and they unite to form a bridge running across the head from one side to the other.

• In many insects, e.g. Orhtoptera, the central part of the composite tentorium enlarged to form a broad plate referred to as the Corporotentorium.

• The tentorium serves as an internal “truss” that reinforces the head capsule, cradles the brain, provides attachment for muscles and provides a rigid origin for muscles of the mandibles and other mouthparts. The antennal muscles also arise from the tentorium.

Antennae are paired, highly mobile, segmented appendages present between or below or behind the compound eyes. The antennae arise from the 2nd or antennal segment of the head possessing nerves coming from deutocerebrum of the brain. Antennae are absent in order protura and class Arachnida whereas 2 pairs of antenna (antennules) are present in class Crustacea.

Antennae are set in a socket of the cranium called antennal socket (anetnnifer) and connected by an articulatory membrane which allows free movement of antenna. Antennal socket is provided with an antennal suture. Although antennae vary widely in shape and function, all of them can be divided into three basic parts:

• Scape

• Basal segment, larger than other segments and articulates with the head capsule. It is provided with intrinsic muscles.

• Pedicel

• Second segment of antennae. In this, mass of cells called Johnston organ (auditory) is present (eg. houseflies). The above two segments have intrinsic muscles ie., their own muscles. In honey bee, wasps pedicel forms the pivot between scape and flagellum.

• Flagellum/Clavola

• The remaining segments or annuli or flagellomeres are called flagellum which lack individual muscles. Flagellum is supplied with many sensory receptors that are connected by nerves to brain. Flagellum is further divided into three parts

Light spectrum holds rich information about the world. The spectrum of natural illumination changes with daily cycle and objects differ in spectral reflection or emission properties. Like many other animals, insects use chromatic information to f ind favorable habitat, to efficiently locate food sources and to identify conspecific mates using the spectral information. Pollinating insects such as many species of bees and butterflies use their color vision to maximize success in foraging. They detect flowers, memorize the colors and patterns of rewarding flowers, and preferentially collect nectars from the flowers in their later visits. Visual guidance of behaviour is challenging when photons, the elementary particles of light, are scarce. To produce a reliable representation of the surroundings, the visual system must (i) ensure absorption of a sufficient number of photons into a photoreceptor, (ii) house photoreceptors that efficiently convert each photon absorption into a neural signal, and (iii) process these signals appropriately. Insects are a numerous and diverse class of arthropods that have evolved to occupy ecological niches from the brightest to the darkest.

• The thorax is the organ of locomotion due to articulation of wings and legs and is connected with the by neck or cervix.

• Thoracic segments are made up of three sclerites namely, dorsal body plate tergum or nota, ventral body plate sternum and lateral plate pleuron.

• Second and middle tagma which is three segmented, namely prothorax, mesothorax and metathorax. Meso and metathorax which bear wings are called as Pterothorax.

• In the apterygote insects the three thoracic segments are similar in size and structure. In the pterygote insects the thorax is highly modified.

• The prothorax is quite large and is somewhat independent of the other segments. The large shield that covers the dorsal and lateral sides of the prothorax is the pronotum.

• The pronotum is the dorsal sclerite of the prothorax, which can be highly modified in various orders such as the Hemiptera, Blattaria, and Coleoptera. E.g undivided and Saddle shaped in grass hopper, Shield like in cockroach.

• Cockroaches have pronotums that extend forward over the head.

• Scarab beetles and other beetles may also have unusual pronotums.

• Treehoppers have some of the most bizarre pronotums of all insects.

• It is the posterior most and largest region of the insect-body. It is The abdomen is the metabolic and reproductive centre, where digestion, excretion, and the sexual functions take place.

• All the definitive number of segments differentiate in embryo (Epimorphic condition) except protura (Anamorphosis)

• In general more segments are visible in more generalised hemimetabolus insects than in more specialised holometabolus insects. For example in Acridids all 11 segments are visible whereas in muscidae only 2-5 segments are visible

• The basic number of abdominal segments in insect is eleven plus a telson/ tail (Protura) which bears anus. Abdominal segments are called uromeres.

• The undivided tergel plates overlap the sternal ones and the membranous pleura are enclosed in between them.

• The posterior part of each segment overlaps the anterior part of the segment behind it and the two segments are joined by the inter-segmental membrane.

• There are eight pairs of abdominal spiracles. The first pair lies just in front of each tympanum and the other lie dorso-laterally on each of the tergites of the segment to eighth abdominal segments.

• The terga and sterna bear antecostal ridges. The internal dorsal and ventral longitudinal muscles run one antecostal ridge to the other and act as retractors. The contraction of these muscles helps the segments to be retracted. The external muscles originate from the posterior part of the segment and attach to the anterior part of the acrotergite of the next segment. Thus, protraction of these muscles help in the extension of abdomen. Hence, musculature running longitudinally between helps in the contraction and extension of the abdomen.

• The abdominal segments are conveniently divided into three subregions i.e. pregenital, genital and postgenital segments.

Insects development

Once an insect hatches from the egg it is usually able to survive on its own, but it is small, wingless, and sexually immature. Its primary role in life is to eat and grow. If it survives, it will periodically outgrow and replace its exoskeleton (a process known as molting). In many species, there are other physical changes that also occur as the insect gets older (growth of wings and development of external genitalia, for example). Collectively, all changes that involve growth, molting, and maturation are known as morphogenesis.

Metamorphosis

• Metamorphosis actually means change in form Metamorphosis is derived from Greek word ‘Meta’ = Change, ‘morph’ = form or structure.

• The changes in the development from egg to adult, collectively called Metamorphosis (life cycle).

• Post-embryonic changes in form are collectively called as metamorphosis.

• Metamorphosis refers to the way that insects develop, grow, and change form. Metamorphosis include three developmental processes namely growth, differentiation and reproduction which takes place in larval, pupal and adult stages, respectively.

• Majority of insects pass through profound metamorphosis. The growth of an immature insect is accompanied by a series of molts or ecdysis, in which the cuticle is shed off and renewed. The number of molts varies in most insects from 4-8. However some undergo as many as 20 or more molts. A few insects like Bristle tail silver fish (apterygota) may continue molting even after reaching the adult stage. But most of the insects neither molt nor increase in size once then adult stage is reached. The intervals between the ecdysis are called Stadia. The term instar is applied to form of an insect during stadium. In numbering the instar, the form assumed by the insect between hatching from the egg and the first post embryonic molt is termed “First Instar”.

• In most insects, life begins as an independent egg (ovipary).

• Majority of insects are oviparous. Egg stage is inconspicuous, inexpensive and inactive. Yolk contained in the egg supports the embryonic development.

• Eggs are laid under conditions where the food is available for feeding of the future young ones.

• Production of eggs by the female’s body is called oogenesis and the egg laying process is known as oviposition.

• The outer protective shell of the egg is called chorion. Near the anterior end of the egg, there is a small opening called micropyle which allows the sperm entry for fertilization. Chorion may have a variety of textures.

• Size and shape of the insect eggs vary widely.

Alimentary canal of an insect is a tubular structure passing through the body that provides the special internal environment for the food to be mechanically and chemically disintegrated and brought to the vicinity of absorbent cells. Hence, it is involved in the transport of nutrients to the individual cells. It is concerned with ingestion, trituration (chewing), digestion, absorption of nutrient material into haemolymph and egestion of unused food materials. It is tubular structure, which extends from mouth to the anus. It is divided into three distinct regions, namely fore-gut (stomodaeum), mid-gut (mesenteron) and hind-gut (proctodaeum). The fore- and hind-guts are ectodermal in origin and the mid-gut is endodermal.

Longitudinal and circular (intrinsic) muscles are usually associated with each of these three regions and by means peristaltic contractions move the food along the alimentary canal. The functional stomach is the mesenteron usually a circular valve-like fold separates the cavities between stomodaeum and mesenteron is known as stomodael or cardiac valve, and the one closing the entrance to the proctodaeum as the proctodaeum or pyloric valve.

Fore-gut

An insect’s mouth, located centrally at the base of the mouthparts, is a muscular valve (sphincter) that marks the “front” of the foregut. Food in the buccal cavity is sucked through the mouth opening and into the pharynx by contractile action of cibarial muscles. These muscles, located between the head capsule and the anterior wall of the pharynx, create suction by enlarging the volume of the pharynx (like opening a bellows). This “suction pump” mechanism is called the cibarial pump. It is especially well-developed in insects with piercing/sucking mouthparts.

The sense organs in an insect body are distributed on different parts and respond to a given stimulus such as light, sound, touch, chemicals etc. The sense organs may be classified as • Visual organs (or) photoreceptors • Auditory organs (or) organs of hearing

• Chemoreceptors which respond to chemicals

• Tacticle receptors which respond to touch

• Gustatory receptors which respond to taste.

Photoreceptors

Light is perceived by insects through a number of different sense organs and these are the organs that react with light and useful for vision. These are of different types, but most important are the compound eyes.

Compound eyes

History

• Aristotle (384-322 BC): A good beginning of taxonomy was made by Aristotle (Historia Animalium). He erected entomon. He could distinguish mandibulates and haustellates; winged and wingless forms. The insect orders like coleopteran, dipteral, psyche (Lepidoptera) were created by him. Hence, he was called as Father of Biological Classification.

• Linnaeus (1707-1778): Father of Taxonomy and Binomial Nomenclature. He coined the term Insecta. He used the term Systematics. His greatest contribution is Systema Naturae.

• Brawer (1885): Laid foundations for modern system of classification and responsible to divide class Insecta into Apterygota and Pterygota.

• Michael Anderson (1727-1806): Contemporary and rival of Linnaeus. Introduced Numerical Taxonomy and hence is called Father of Numerical Taxonomy. 

• Johan Christian Fabricius (1745–1808), the first systematist considered to specialize on insects, published in 1778 his Philosophia Entomologica (40), the first textbook of entomology.

• Fabricius had already set forth his system of insects in 1775 (Systema Entomologiae), his system of genera in 1776 (Genera Insectorum), and then numerous works on species, culminating in individual accounts of the species of each order

Apterygotes

The Apterygota, which formerly included the other primitively wingless insects currently placed in the class Entognatha, are now restricted to the two orders Archaeognatha and Zygentoma, which in turn were formerly united as the Thysanura. Despite the superficial similarity of the two groups, it is now clear that they are not closely related, mainly because of fundamental differences in the mouthparts. The Archaeognathous mandibles are monocondylic, having a single articulating point with the head so that the mandible can rotate. The Zygentomous mandibles are dicondylic, with two articulating points that restrict the motion to a single plane yet enable the development of a much stronger biting action; this is the type found in all the higher insects, and the Zygentoma may well be the sister group of the Pterygota, or winged insects. Clearly the ‘Apterygota’ is not a monophyletic group and is simply retained for convenience in grouping these two orders that superficially resemble each other and have similar life histories.

• The name Zoraptera, derived from the Greek “zor” meaning pure and “aptera” meaning wingless was given to the order before winged forms were discopvered.

• Members of the order Zoraptera are small (less than 4 mm) and usually found in rotting wood, under bark or in piles of old sawdust.

• They live in small aggregations (gregarious) and appear to scavenge on spores and mycelium of fungi, or occasionally, on mites and other small arthropods.

• polymorphism in populations; with blind, apterous individuals dominating during colony life, but populations produce eyed alates (individuals with wings) for dispersal and founding new colonies.

• males with a distinctive “mating hook” and unique mating behaviors.

• unique grooming behaviors

Adults

• Antennae 9-segmented (moniliform).

• Mouthparts mandibulate, hypognathous.

• Soft bodied, small (usually less than 3 mm).

• Wings often absent with reduced venation when present.

• Tarsi 2-segmented

• Cerci 1-segmented

• ovipositor absent

• incomplete development (egg, nymph, adult)