ZAHEERKHAN

1. Techniques used in Entomology Research
1. Field sampling: This involves collecting insect specimens from different habitats, such as
forests, fields, or streams, to study their diversity, behavior, and ecological interactions.
2. Morphological analysis: This involves studying the physical characteristics of insects,
including their size, shape, color, and anatomy. Techniques used in this type of analysis include
microscopy, dissection, and staining.
3. Molecular techniques: These involve studying the genetic makeup of insects using various
methods, including DNA sequencing, polymerase chain reaction (PCR), and restriction
fragment length polymorphism (RFLP) analysis.

2. 4. Chemical analysis: This involves analyzing the chemicals produced by insects, such as
pheromones and defensive compounds, to understand their behavior and ecological
interactions.
5. Behavioral studies: This involves observing and quantifying the behavior of insects in
different environments, such as their feeding, mating, and social interactions.
6. Electrophysiology: This involves measuring the electrical activity of insect neurons to
understand how insects process sensory information, such as vision or smell.

3. 7. Imaging techniques: This involves using various imaging techniques, such as scanning electron
microscopy (SEM) and confocal microscopy, to study the structure and function of insect organs
and tissues.
8. Bioassays: This involves testing the effectiveness of insecticides or other chemicals on insect
behavior or physiology.
9. Mathematical modeling: This involves using mathematical models to study insect population
dynamics, disease transmission, or other ecological interactions.

4. 10. Biogeography: This involves studying the distribution and diversity of insects across different
geographical regions and over time.
11. Transcriptomics: This involves studying the entire set of RNA transcripts in an insect, which
can provide insights into gene expression, regulation, and function.
12. Proteomics: This involves studying the entire set of proteins in an insect, which can provide
insights into its metabolic pathways, physiology, and interactions with other organisms.

5. 13. Microbiome analysis: This involves studying the microbial communities that live on or
inside insects, which can provide insights into their symbiotic relationships, disease resistance,
and nutrition.
14. Immunology: This involves studying the immune system of insects, including how they
respond to pathogens, parasites, and other threats.
15. Remote sensing: This involves using satellite imagery or other remote sensing technologies
to study the distribution and abundance of insects in large or inaccessible areas.
16. Network analysis: This involves using network theory and analysis to study the ecological
interactions between insects and other organisms in their environment.

6. Field sampling
2 Major Collecting Approaches
1: Active Collecting
Sweep Netting, Foliage Beating, Searching The Ground, Under Rocks, Sifting Leaf
Litter, Peeling Tree Bark.
2. Passive Collecting: Where Traps Are Set To Either Attract Or Intercept Insects.
Including
1. Pitfall Traps Or Baited Pitfall Traps, 2. Malaise And Flight Intercept Traps, 3. Light
Traps, And 4. Collecting At A Light Sheet, 5. Sticky Traps, 6. Pheromone Traps And
Yellow Pans

7. 1. Sweep netting

8. Foliage Beating or Beat Sampling: A beating sheet or beating net is held
under vegetation and the foliage firmly tapped with a beating stick to dislodge
insects from the branches falling into the net.

9. Sifting Leaf Litter:
A typical leaf litter sieve consists of a gauze with holes of approximately 5 to 10 mm
width. The entomologist places handfuls of leaf litter into the sieve, which is placed
above a white sheet or tray. The sieve is shaken, and insects are separated from the
leaf litter and fall out for inspection.

10. Peeling Tree Bark

11. 2. Passive Collecting
1. Pitfall traps or baited pitfall traps: A pitfall trap is a trapping pit for
small animals, such as insects, amphibians and reptiles. Pitfall traps are a
sampling technique, mainly used for ecology studies and ecologic pest control.

12. Malaise traps are 'tent-like' passive intercept traps that primarily catch flying
insects.

13. Light traps are widely used to survey nocturnal moths. Total species richness and
abundance of trapped moths may be influenced by several factors such as night
temperature, humidity and lamp type. Grasshoppers and some beetles are attracted to
lights at a long range but are repelled by it at short range.

14. Sticky Traps: These traps help with pest control by making use of insects' stimuli
to control or drive them away. Sticky traps trap certain insects by exploiting the fact
that they are attracted to certain colours. These insects then stick to the glue.

15. Pheromone Traps:
Pheromones are chemicals used by insects and other animals to communicate with
each other. Insects send these chemical signals to help attract mates, warn others of
predators, or find food. Using specific pheromones, traps can be used to monitor
target pests in agriculture or in residential areas.

17. Yellow pan traps

18. Packing list for field sampling
1. Beating net and stick – a shallow calico or nylon bag on a metal hoop with a
short wooden handle.
2. Aspirator.
3. Prefilled ethanol vials – to collect some specimens straight to ethanol in field.
4. Collection transfer tubes with strips of tissue or toilet paper inside – for dry
collecting.
5. Notebook.
6. Pen/Pencil.
7. Label card – small squares pre-cut is a good idea.
8. GPS (if you have access to one, not essential).

19.  A beating sheet or beating net is held under vegetation and the foliage firmly tapped
with a beating stick to dislodge insects from the branches falling into the net.

20. An aspirator is a tool used to collect small specimens through the use of
suction. The specimen is then deposited in some type of collecting vessel, where it
can be confined

21. Prefilled ethanol vials – to collect some specimens straight to ethanol in the field

22. Collection transfer tubes with strips of tissue or toilet paper inside – for dry collecting.

23. Note taking in the field
• Always carry a dedicated field notebook with you when out collecting and write up notes
and label specimens once finished collecting, recording all field data at a site before moving
on.
• The baseline information that is required when collecting insects in the field is the location,
date, and collector.
• This combination of information for collected specimens may be referred to as a
“collection event”.

24. • Collecting further supplementary information, is also important, such as habitat, collection
method, and host plant adds more value to the specimen data and may also assist in
identifying that insect.
• For broad-scale surveys, it may serve to develop coding systems by which you can
temporarily label and then refer back to the notebook later when preparing permanent labels
for the specimens
• For host plant-specific insect sampling, for example, we have created a system of codes
that comprise 3 components that make up a unique collection event: a trip code, locality
number, and host number. Note that on any one trip, the localities and hosts are labeled
individually and successively from 1 onwards.

25. Example of a field notebook, recording insects collected from different host plants at a site
using this coded system (with a trip code, locality, numbers, and host numbers):

30. Research on insect morphology involves studying the structure and form of
insects, including their anatomy, physiology, and behavior. Here are some steps you
can follow to conduct research on insect morphology:
1.Identify your research question: What aspect of insect morphology do you want to
study? Is there a specific insect species you are interested in? Knowing your research
question will guide you in selecting appropriate methods and resources.
2.Conduct a literature review: Before conducting your research, it is important to
review existing literature on insect morphology. This will help you identify what is
already known about your research question and provide a foundation for your
research. You can search for scientific articles, books, and other resources online or at
a library.

31. 3. Select your research methods: Depending on your research question, you may use
a variety of methods to study insect morphology. These can include dissection,
microscopy, imaging techniques, behavioral observation, and genetic analysis. Choose
the methods that are most appropriate for your research question and available
resources.
4. Collect and analyze your data: Once you have collected your data, you will need to
analyze it to answer your research question. This may involve statistical analysis,
comparative analysis, or other methods depending on your research question.
5. Communicate your findings: Finally, you will need to communicate your research
findings. This can include publishing scientific articles, giving presentations, or creating
educational materials. It is important to clearly and accurately communicate your
findings so that others can build on your research.

32. Overall, research on insect morphology requires careful planning, attention to detail, and a solid understanding of the
existing literature. By following these steps, you can conduct high-quality research and contribute to our
understanding of the fascinating world of insects.

33. Molecular techniques in Entomology
1.DNA Barcoding: DNA barcoding is a technique used to identify species using a short
DNA sequence from a standard region of the genome. This technique can be used to
identify unknown specimens, verify identifications, and discover new species.
2.Transcriptomics: Transcriptomics is the study of the transcriptome, which is the
complete set of RNA transcripts produced by the genome at a given time. This technique
involves the use of microarray or RNA sequencing technology to identify and quantify the
expression levels of genes in different tissues or under different conditions.
3.Proteomics: Proteomics is the large-scale study of proteins, particularly their
structures and functions. This technique involves the use of mass spectrometry and other
methods to identify and quantify proteins in different tissues or under different conditions.

34. 4. Metabolomics: Metabolomics is the study of small molecule metabolites, such as
amino acids, sugars, and lipids, in biological systems. This technique involves the use of
mass spectrometry and other methods to identify and quantify metabolites in different
tissues or under different conditions.
5. RNA interference (RNAi): RNA interference is a technique used to study gene
function by reducing the expression of specific genes. This technique involves the use of
small interfering RNA (siRNA) or short hairpin RNA (shRNA) to target and silence
specific genes.
6. CRISPR/Cas9: CRISPR/Cas9 is a gene editing technology that can be used to create
specific mutations or knockouts in genes of interest. This technique involves the use of a
guide RNA that directs the Cas9 enzyme to cut specific DNA sequences, allowing for
precise editing of the genome.

35. DNA Barcoding https://www.youtube.com/watch?v=zcd3QYngHX0

36. 3. 1. Proteomics is the large-scale study of proteins, including their structure, function,
and interactions within a biological system. Insect research can benefit greatly from
proteomics methods, as they can help researchers to understand the molecular
mechanisms that underlie various insect biological processes.
https://onlinelibrary.wiley.com/doi/full/10.1002/ps.7077
Here are some examples of proteomics methods that can be used in insect research:

37. 3.1.1 Mass spectrometry: Mass spectrometry is a powerful technique that can be
used to identify and quantify proteins in complex mixtures. This method has been used
to identify proteins in a variety of insect tissues, including the cuticle, gut, and
hemolymph.
3.1.2. Two-dimensional gel electrophoresis: Two-dimensional gel electrophoresis
(2D-GE) is a technique that separates proteins based on their isoelectric point and
molecular weight. This method has been used to identify differentially expressed
proteins in various insect species, including the honey bee, silk moth, and mosquito.
https://www.sciencedirect.com/science/article/pii/S2352340915001870
3.1.3. Protein microarrays: Protein microarrays are a high-throughput method for
studying protein-protein interactions. This method has been used to study protein
interactions in the nervous system of the fruit fly, Drosophila melanogaster.

38. 3.1.4. Shotgun proteomics: Shotgun proteomics is a technique that uses mass
spectrometry to identify proteins in complex mixtures without prior fractionation. This
method has been used to identify proteins in the salivary glands of mosquitoes and other
insects.
3.1.5 Label-free quantification: Label-free quantification is a method for quantifying
proteins without the use of isotopic labels. This method has been used to study the
proteomes of various insect tissues, including the fat body and ovaries of the honey bee.

39. 3.1.1.1 Mass spectrometry is a powerful technique that can be used to quantify
proteins in complex mixtures. Here are some common mass spectrometry methods for
protein quantification:
3.1.1.1.1 Label-based quantification: Label-based quantification involves introducing
a stable isotope label, such as SILAC or iTRAQ, to the protein samples prior to mass
spectrometry analysis. This allows for the comparison of protein abundance between
different samples. The labeled peptides are then separated and quantified using mass
spectrometry.
3.1.1.1.2 Label-free quantification: Label-free quantification is a method for
quantifying proteins without the use of isotopic labels. This method relies on the
detection and quantification of peptide ions in a mass spectrometer. The abundance of
each peptide ion is used to infer the abundance of the corresponding protein.

40. 3.1.1.1.3. Selected reaction monitoring (SRM): SRM is a targeted mass spectrometry
technique that can be used for protein quantification. This method involves selecting
specific peptides for analysis and monitoring their abundance across different samples.
SRM is often used for the quantification of low-abundance proteins.
3.1.1.1.4 Data-independent acquisition (DIA): DIA is a label-free mass spectrometry
method that uses precursor ion selection to fragment peptides into smaller fragments.
These fragments are then detected and quantified by the mass spectrometer. DIA can be
used for the quantification of large numbers of proteins in a single analysis.
https://www.sciencedirect.com/science/article/abs/pii/S1874391921000543

41. Metabolomics: Metabolomics is the study of small molecule metabolites, such as
amino acids, sugars, and lipids, in biological systems. This technique involves the use
of mass spectrometry and other methods to identify and quantify metabolites in
different tissues or under different conditions.

42. 3.1.1.1.5 Multiple reaction monitoring (MRM): MRM is a targeted mass spectrometry
method that can be used for the quantification of specific peptides in a complex mixture.
This method involves selecting specific precursor ions and monitoring their
fragmentation into specific product ions. MRM is often used for the quantification of low-
abundance proteins.

43. 1. Mass spectrometry (MS): MS is a powerful analytical tool that can identify and
quantify a wide range of metabolites in complex biological samples. In entomology, MS is
commonly used for the analysis of insect hemolymph, which contains a wide range of
metabolites.
2.Nuclear magnetic resonance (NMR): NMR is a non-destructive technique that can
provide detailed information on the chemical structure of metabolites. In entomology,
NMR is often used for the analysis of insect tissues and excreta.
3.Gas chromatography-mass spectrometry (GC-MS): GC-MS is a powerful analytical
tool that can identify and quantify volatile and semi-volatile metabolites in complex
biological samples. In entomology, GC-MS is commonly used for the analysis of insect
pheromones and other semiochemicals.

44. 4. High-performance liquid chromatography (HPLC): HPLC is a powerful separation
technique that can be coupled with MS or NMR to identify and quantify metabolites in
complex biological samples. In entomology, HPLC is often used for the analysis of insect
excreta and hemolymph.

45. CRISPR/Cas9 is a powerful genetic tool that enables targeted genome editing in a wide
range of organisms, including insects. In entomology, CRISPR/Cas9 techniques have
been used to investigate the function of genes involved in insect development, behavior,
and physiology, as well as to develop new insect control strategies.
Some of the applications of CRISPR/Cas9 techniques in entomology include:
1.Gene knockout: CRISPR/Cas9 can be used to create targeted gene knockouts in
insects, which can help to determine the function of specific genes in insect
development, behavior, and physiology.
2.Gene editing: CRISPR/Cas9 can also be used to introduce specific mutations or
modifications into insect genes, which can help to study the effects of these changes on
insect traits.

46. 3. Gene drive: CRISPR/Cas9 can be used to create gene drives in insects, which can
spread beneficial traits or suppress harmful traits through a population. This approach
has potential for the development of new insect control strategies.
4. Insect sterilization: CRISPR/Cas9 can be used to create genetic modifications that
result in insect sterilization, which can help to control insect populations without the use
of pesticides.
Overall, CRISPR/Cas9 techniques are revolutionizing the field of entomology by enabling
targeted genetic manipulation of insects. This has the potential to improve our
understanding of insect biology and develop new insect control strategies that are more
effective and environmentally friendly.

47. III. Chemical analysis is an important tool in entomology for identifying and quantifying
the various chemicals that play important roles in insect behavior, physiology, and
ecology. Chemical analysis techniques are used to identify and measure the amounts of
insect pheromones, defensive chemicals, and other secondary metabolites that are
important for insect interactions with their environment and other organisms.
Some of the commonly used chemical analysis techniques in entomology include:
1.Gas chromatography (GC): GC is a powerful separation technique that can be used
to separate and identify different volatile and semi-volatile compounds in complex
mixtures. In entomology, GC is often used to analyze insect pheromones and other
volatile compounds.

48. 2. High-performance liquid chromatography (HPLC): HPLC is a powerful
separation technique that can be used to separate and identify different non-volatile
compounds in complex mixtures. In entomology, HPLC is often used to analyze non-
volatile insect secondary metabolites.
3. Mass spectrometry (MS): MS is a powerful analytical tool that can be used to
identify and quantify specific chemicals in complex biological samples. In entomology,
MS is often used in conjunction with GC or HPLC to identify specific insect
pheromones or secondary metabolites.
4. Nuclear magnetic resonance (NMR): NMR is a non-destructive technique that can
provide detailed information on the chemical structure of compounds. In entomology,
NMR is often used to identify the chemical structure of new insect pheromones or
secondary metabolites.

49. Overall, chemical analysis techniques are helping entomologists to better understand the
chemical ecology of insects and their interactions with the environment and other
organisms. This information can be used to develop new insect control strategies or to
improve our understanding of the natural world.

50. 3. Pesticide research techniques

51. Insecticides are a type of pesticide that are specifically designed to control or eliminate
insect pests. Insecticides can be classified into several different categories based on
their chemical composition, mode of action, and target insects. Here are some of the
commonly used insecticide classifications:
1.Organochlorines: Organochlorine insecticides were widely used in the past but have
been largely phased out due to their persistence in the environment and potential for
bioaccumulation in food chains. They act by disrupting the nervous system of insects,
causing paralysis and death.
2.Organophosphates: Organophosphate insecticides are still used today but are
highly toxic to humans and other mammals. They act by inhibiting the activity of the
enzyme acetylcholinesterase, which is necessary for proper nervous system function
in insects and mammals.

52. 3. Carbamates: Carbamate insecticides are less toxic than organophosphates but can
still be harmful to humans and other mammals. They act by inhibiting the activity of the
same enzyme as organophosphates, acetylcholinesterase.
4. Pyrethroids: Pyrethroid insecticides are synthetic versions of pyrethrins, which are
naturally occurring insecticides found in chrysanthemum flowers. They act by causing
paralysis and death in insects by disrupting their nervous system.
5. Neonicotinoids: Neonicotinoid insecticides are relatively new and highly effective
insecticides that act by targeting the nervous system of insects. They have been
implicated in the decline of pollinator populations, including bees.

53. Biological insecticides: Biological insecticides are made from living organisms, such as
Plants, bacteria, viruses, and fungi, and are used to control specific insect pests. They
act by infecting, parasitizing, or otherwise causing harm to the target insect.
Overall, the classification of insecticides can be helpful in understanding their properties,
mode of action, and potential risks and benefits. It is important to use insecticides
responsibly and follow all safety precautions to minimize their impact on the environment
and human health.

54. Biopesticides are certain types of pesticides derived from such natural materials as
animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda
have pesticidal applications and are considered biopesticides. As of August 31, 2020,
there were 390 registered biopesticide active ingredients.

55. Biopesticides are derived from natural sources and can be used for pest management in
agriculture, horticulture, and forestry. Here are some research techniques for biopesticide
research: Plant/ other organisms selection and identification:
1.Isolation and characterization of biopesticides: The first step in biopesticide research is the
isolation and characterization of potential biopesticides. This involves the identification and
isolation of microbial or plant-based compounds that have pesticidal properties.
Isolation of plant extracts and compounds involves the separation of desired compounds from
other plant constituents such as lipids, carbohydrates, and proteins. Here are some common
techniques used for the isolation of plant extracts and compounds:
Solvent extraction: Solvent extraction is one of the most widely used techniques for the isolation
of plant extracts and compounds. It involves soaking the plant material in a solvent such as
ethanol, methanol, or water to extract the desired compounds.

56. Solid-phase extraction: Solid-phase extraction (SPE) is a technique used to isolate and purify
compounds from complex matrices. The plant extract is passed through a solid-phase extraction
column, which selectively retains the desired compound based on its physical and chemical
properties.
Steam distillation: Steam distillation is used to isolate volatile compounds such as essential oils
from plant material. The plant material is subjected to steam, which extracts the volatile
compounds, and the resulting mixture is then separated by condensation.
Soxhlet extraction: Soxhlet extraction is a technique used to extract compounds that are less
soluble in solvents. The plant material is placed in a thimble and extracted repeatedly with a
solvent using a Soxhlet extractor.

57. Chromatography: Chromatography is a powerful technique used for the isolation and
purification of plant extracts and compounds. It involves separating the mixture of compounds
based on their physical and chemical properties such as size, charge, and polarity. Common types
of chromatography used in plant extract isolation include column chromatography, thin-layer
chromatography (TLC), and high-performance liquid chromatography (HPLC).
Precipitation: Precipitation involves adding a reagent to the plant extract to cause the desired
compound to precipitate out of solution. Common reagents used for precipitation include acid,
base, and salt.

58. 2. Bioassays: Once potential biopesticides have been identified, bioassays can be used
to evaluate their efficacy against target pests. Bioassays involve testing the biopesticide
against the target pest under controlled laboratory conditions.
3. Field trials: Field trials are conducted to determine the effectiveness of bio-pesticides
in real-world conditions. This involves applying the biopesticide to crops in the field and
monitoring its impact on target pests.
4. Formulation development: Biopesticides are often formulated to improve their
efficacy and stability. Formulation development involves optimizing the formulation to
improve its delivery and efficacy.
5. Genetic engineering: Genetic engineering can be used to enhance the production of
biopesticides. This involves introducing genes into microbial or plant-based organisms to
increase the production of pesticidal compounds.

59. 6. Transcriptomics and proteomics: Transcriptomics and proteomics can be used to
study the molecular mechanisms underlying the pesticidal activity of biopesticides. This
involves analyzing the gene expression and protein levels of organisms treated with
biopesticides.
7. Metabolomics: Metabolomics can be used to study the metabolic changes that
occur in organisms treated with biopesticides. This involves analyzing the metabolic
profiles of organisms treated with biopesticides using techniques such as mass
spectrometry.
Overall, a combination of these techniques can be used to develop and optimize
biopesticides for pest managem

60. 5. Behavioral studies techniques in entomology: Entomology is the study of insects,
and behavioral studies in entomology involve observing and analyzing the behaviors of
insects in their natural habitats or in controlled laboratory settings. There are several
techniques used in behavioral studies in entomology, some of which are:
1.Observation: The simplest technique in entomology is to observe the insects in their
natural habitats or in the laboratory setting. Observing their feeding patterns, mating
behaviors, movement patterns, and other behaviors can provide valuable insights into
their behavior.
2.Choice experiments: In these experiments, insects are given a choice between
different options, such as different types of food, pheromones, or other stimuli. By
observing which option the insect chooses, researchers can gain insights into their
preferences and behavior.

61. 3. Y-maze and T-maze experiments: In these experiments, insects are placed in a
maze with two or three arms. The insect is then allowed to choose which arm to follow,
and the researchers can observe which arm they choose and how long they spend in
each arm.
4. Wind tunnel experiments: Wind tunnels are used to study the flight behavior of
insects. The wind tunnel creates a controlled environment where researchers can
observe how insects respond to different airflows and stimuli.
5. Electrophysiology: This technique involves recording the electrical activity in the
insect's nervous system in response to different stimuli. This can help researchers
understand how insects perceive and respond to different stimuli.
6. Genetic manipulation: By genetically manipulating insects, researchers can study how specific
genes affect their behavior. For example, by turning off or overexpressing a specific gene, researchers
can observe how it affects the insect's behavior.

62. Choice experiments

63. Y-maze and T-maze experiments

64. Wind tunnel experiments

65. Electrophysiology techniques are commonly used in entomology to study the
nervous system and sensory organs of insects. These techniques allow researchers to
measure electrical signals in individual neurons and sensory organs, which can provide
valuable insights into how insects perceive their environment and how their nervous
systems function.
Some of the most commonly used electrophysiology techniques in entomology include:
1.Extracellular Recording: This technique involves placing a small electrode near a
neuron or sensory organ and measuring the electrical activity generated by the neuron
in response to a stimulus. Extracellular recording is used to study sensory perception,
neural coding, and information processing in insects.

66. 1.Intracellular Recording: This technique involves inserting a small electrode directly
into a neuron and measuring the electrical activity inside the neuron. Intracellular
recording is used to study the membrane properties, synaptic interactions, and action
potentials of individual neurons.
2.Electroantennography: This technique involves measuring the electrical activity
generated by the antennae in response to odors. Electroantennography is used to
study the olfactory system of insects and can provide insights into how insects detect
and discriminate between different odors.
3.Electroretinography: This technique involves measuring the electrical activity
generated by the retina in response to light. Electroretinography is used to study the
visual system of insects and can provide insights into how insects detect and process
visual information.

68. Imaging techniques are essential tools in entomology, providing researchers
with a way to visualize and analyze the internal and external anatomy of insects, as well
as their behavior and interactions with the environment. There are several imaging
techniques used in entomology, including:
1.Light Microscopy: Light microscopy uses visible light to produce images of insects
and their structures. This technique is used to visualize the external and internal
anatomy of insects, as well as their behavior and interactions with the environment.
2.Scanning Electron Microscopy (SEM): SEM uses an electron beam to produce
highly detailed images of the surface structure of insects. SEM is used to visualize the
fine details of insect morphology, such as the structure of insect cuticle, antennae, and
legs.

69. 4.Transmission Electron Microscopy (TEM): TEM uses an electron beam to produce
highly detailed images of the internal structure of insects. TEM is used to visualize the
ultrastructure of insect cells, such as the structure of the insect nervous system and
internal organs.
5. X-ray Microscopy: X-ray microscopy uses X-rays to produce high-resolution, three-
dimensional images of the internal structure of insects. X-ray microscopy is used to
visualize the internal structure of insect specimens without having to physically dissect
them.
5. Confocal Microscopy: Confocal microscopy uses laser light to produce highly
detailed, three-dimensional images of insect structures. Confocal microscopy is used to
study the internal structure of insect tissues, such as the structure of the insect nervous
system and internal organs.

70. Overall, imaging techniques are essential tools for entomologists, providing a way to visualize and analyze the
intricate anatomy and behavior of insects. By using a combination of imaging techniques, researchers can gain a
better understanding of how insects function and interact with their environment.

71. 8. Bioassays are an essential tool in entomology for assessing the toxicity of various
chemicals, including insecticides, to insects. In entomology, bioassays can be
performed on individual insects, groups of insects, or whole colonies. There are
several types of bioassays used in entomology, including the following:
1.Contact bioassays: These are performed by placing insects on a treated surface or
in contact with a treated substance. The amount of time the insect remains in contact
with the treated surface or substance, and the number of insects that are killed by the
chemical, are used to determine the toxicity of the chemical.

72. Contact bioassays are commonly used in entomology to evaluate the toxicity of a chemical to insects
through direct contact. In a contact bioassay, a known amount of the chemical is applied to a surface,
such as filter paper or plastic, and allowed to dry. The surface is then exposed to a known number of
insects for a specific amount of time.
The mortality rate of the insects is then recorded after a set period, usually 24 or 48 hours. The mortality
rate is calculated as the percentage of insects that have died after exposure to the chemical.
Contact bioassays can be used to determine the lethal concentration of a chemical, which is the amount
of the chemical required to kill a specific percentage of the insect population. The lethal concentration can
be used to evaluate the effectiveness of different chemicals and to compare the toxicity of different
formulations.
Contact bioassays can also be used to assess the residual activity of a chemical on different surfaces. For example,
a contact bioassay can be performed on different types of surfaces, such as glass, plastic, or wood, to evaluate the
efficacy of a chemical in controlling insect populations on different surfaces.
Overall, contact bioassays are a useful tool in entomology for evaluating the effectiveness of different chemicals in
controlling insect populations, as well as assessing the residual activity of chemicals on different surfaces.

74. 2. Topical bioassays: These are performed by applying the chemical directly to the
insect's cuticle, usually on the thorax or abdomen. The mortality rate of the insects is
used to determine the toxicity of the chemical.
Topical bioassays are a type of bioassay used in entomology to determine the toxicity of a
chemical to insects through direct application to the insect's cuticle. In a topical bioassay, a
known amount of the chemical is applied directly to the insect, usually on the thorax or
abdomen, using a micropipette or a brush.
The insect is then observed for a set period, usually 24 or 48 hours, to record the mortality rate.
The mortality rate is calculated as the percentage of insects that have died after exposure to the
chemical.

75. Topical bioassays can be used to determine the LD50, which is the amount of the chemical
required to kill 50% of the insect population. The LD50 can be used to compare the toxicity of
different chemicals and formulations.
Topical bioassays can also be used to assess the efficacy of insecticides against different life
stages of insects, such as eggs, larvae, pupae, or adults. This information can be useful in
developing targeted pest management strategies.

76. 3. Residual bioassays: These are performed by applying the chemical to a surface
and allowing it to dry before exposing insects to the treated surface. The mortality rate
of the insects is used to determine the toxicity of the chemical.
4. Fumigation bioassays: These are performed by exposing insects to a gas or vapor
of the chemical. The mortality rate of the insects is used to determine the toxicity of the
chemical.

77. 5. Feeding bioassays: These are performed by offering insects a treated food source.
The mortality rate of the insects is used to determine the toxicity of the chemical.
Bioassays are useful in entomology for determining the effectiveness of insecticides and
other chemicals in controlling insect populations. They can also be used to assess the
resistance of insects to certain chemicals and to screen new chemicals for potential
insecticidal prope

78. 9. Mathematical modeling techniques have a wide range of applications in entomology,
which is the scientific study of insects. These techniques allow researchers to understand the
behavior, ecology, and population dynamics of insect populations. Here are some examples of
mathematical modeling techniques and their applications in entomology:
1. Population dynamics models: These models describe how the size of insect populations
changes over time. They are used to study factors such as birth rates, death rates, migration, and
environmental factors that affect the growth of insect populations.
2. Spatial models: These models describe the spatial distribution of insect populations and how
they interact with their environment. They are used to study how insect populations spread and
how they are affected by factors such as habitat fragmentation, climate change, and human
activities.

79. 3. Epidemiological models: These models describe the spread of insect-borne diseases, such as
malaria and dengue fever. They are used to study how the diseases are transmitted, how they
spread through populations, and how they can be controlled.
4. Behavioral models: These models describe the behavior of insects, such as their mating
behavior and foraging behavior. They are used to study how insects interact with their
environment and how they respond to changes in their environment.

80. Biogeography is the study of the distribution patterns of species and ecosystems across
geographical space and through geological time. It is a field of study that has become
increasingly important in entomology, as insects are highly diverse and have complex
distributions across the globe. There are several techniques that can be used in
biogeography to study the distribution patterns of insects. Some of these techniques are:
1.Historical biogeography: This involves reconstructing the historical events that have
shaped the current distribution patterns of insects. This can be done by examining the
geological history of a region, as well as the evolutionary history of the insect taxa in
question. Historical biogeography can provide insights into how insects have evolved
and dispersed across different regions.

81. 2. Phylogenetic biogeography: This involves using molecular data to construct
phylogenetic trees that can be used to infer the historical biogeographic events that
have shaped the distribution patterns of insects. This can provide more precise
information on the relationships between different insect taxa and their distributions.
3. Ecological biogeography: This involves studying the ecological factors that
influence the distribution patterns of insects, such as climate, habitat, and other
environmental factors. This can help to identify the ecological factors that limit the
distribution of certain insect taxa and can provide insights into how insects are adapted
to different environments.

82. 5. Biogeographical mapping: This involves creating maps that show the
distribution patterns of different insect taxa across different regions. This can help to
identify areas of high insect diversity and can be used to prioritize conservation
efforts for threatened or endangered insect species.
Overall, biogeography techniques can provide valuable insights into the distribution
patterns and evolutionary history of insects, which can be used to inform
conservation efforts and improve our understanding of insect biodiversity.

83. Transcriptomics is the study of the complete set of RNA transcripts produced by an
organism. It has become an important field of study in entomology, as it allows
researchers to better understand the molecular processes underlying insect
development, behavior, and adaptation. There are several transcriptomics techniques
that can be used in entomology, some of which are:
1.RNA sequencing (RNA-seq): This involves sequencing the entire transcriptome of an
insect, which provides information on the genes that are expressed in different tissues
or under different conditions. RNA-seq can also be used to identify novel genes that are
specific to certain insect taxa.

84. 2. Microarray analysis: This involves using microarrays to compare the expression
levels of thousands of genes simultaneously in different insect tissues or under
different conditions. Microarrays can also be used to identify genes that are
differentially expressed between different insect species.
3. Reverse transcription polymerase chain reaction (RT-PCR): This involves
amplifying specific RNA transcripts from an insect and quantifying their expression
levels. RT-PCR can be used to validate gene expression data obtained from RNA-seq
or microarray analysis.
4. Single-cell RNA sequencing (scRNA-seq): This involves sequencing the
transcriptome of individual cells, which allows researchers to identify cell types and
study gene expression patterns at a single-cell resolution. scRNA-seq can also be
used to identify rare cell types that are difficult to detect using other techniques.

86. The microbiome of insects is a complex community of microorganisms that play important roles
in the ecology and physiology of the host insect. Entomologists use various techniques to analyze
the microbiome of insects, which include:
1. Culture-based techniques: These techniques involve isolating and culturing bacteria in a
laboratory setting. This approach is limited to detecting bacteria that can be grown under
laboratory conditions and may not accurately reflect the diversity of the microbiome.
2. DNA sequencing: This technique involves analyzing the DNA of the microorganisms present in
the insect's microbiome. Two common methods for DNA sequencing are 16S rRNA sequencing
and metagenomics. 16S rRNA sequencing targets a specific gene present in bacterial DNA, while
metagenomics sequences all DNA present in a sample, allowing for the identification of bacteria,
viruses, and other microorganisms.
13. Microbiome analysis

87. 3. Fluorescence in situ hybridization (FISH): This technique involves using fluorescent probes to
visualize specific bacteria in a sample. FISH can be used to identify specific bacterial taxa and
their spatial distribution within the insect.
4. Shotgun proteomics: This technique involves analyzing the proteins produced by the
microorganisms present in the microbiome. This approach can provide information about the
metabolic activities of the microbiome.
5. Metabolomics: This technique involves analyzing the metabolites produced by the
microorganisms in the microbiome. Metabolomics can provide insights into the functions of the
microbiome in the insect.
These techniques can be used alone or in combination to provide a comprehensive analysis of
the microbiome of insects. Understanding the microbiome of insects can have important
implications for pest management, disease control, and conservation efforts.

88. 13.1.1 Culture-based techniques in entomology involve isolating and culturing bacteria
from the insect's microbiome in a laboratory setting. The general procedure for culture-
based techniques in entomology is as follows:
13.1.1.1 Sample collection: The first step is to collect samples from the insect.
Depending on the type of insect and the location of the microbiome, samples can be
collected by swabbing, dissection, or other techniques.
13.1.1.2 Sample processing: The collected samples are then processed in the
laboratory to remove any non-bacterial components and to dilute the bacterial cells.
13.1.1.2 Inoculation: The processed sample is then inoculated onto an appropriate
growth medium. Different types of media can be used to promote the growth of different
types of bacteria.

89. 13.1.1.4 Incubation: The inoculated plates are then incubated at an appropriate
temperature and for an appropriate length of time. The temperature and time will
depend on the type of bacteria being cultured.
13.1.1.5 Colony isolation: Once the bacteria have grown on the plate, individual
colonies can be isolated and purified by streaking the colonies onto a fresh plate.
13.1.1.6 Identification: The isolated bacteria can then be identified based on their
morphological and biochemical characteristics. Various tests can be performed to
determine the type of bacteria, such as Gram staining, catalase test, oxidase test, and
others.
13.1.1.7 Characterization: Further characterization of the isolated bacteria can be
done by DNA sequencing or other molecular techniques to determine the phylogenetic
relationship of the bacteria.

90. Culture-based techniques have limitations, as they only detect bacteria that can be
grown in the laboratory setting and may not accurately represent the diversity of the
microbiome. Nevertheless, they can provide valuable information about the
composition of the microbiome of insects and their potential functions.

91. DNA sequencing techniques are increasingly being used in entomology to identify and study
insect species. Here are some of the common techniques used:
Sanger sequencing: This is a traditional method of DNA sequencing that involves the use of
chain-terminating nucleotides to sequence DNA. Sanger sequencing can be used to sequence
short stretches of DNA, such as single genes or regions of genes, and is often used in barcoding
studies to identify insect species.
Next-generation sequencing (NGS): NGS technologies, such as Illumina and Ion Torrent, have
revolutionized DNA sequencing by allowing researchers to sequence large amounts of DNA at
once. This has made it possible to sequence entire insect genomes and transcriptomes, which
has led to a better understanding of insect genetics and evolution.

92. Polymerase chain reaction (PCR) sequencing: PCR sequencing is a technique that involves
amplifying a specific region of DNA using PCR, and then sequencing the amplified DNA using
Sanger sequencing or NGS. This method is often used in insect population genetics studies to
examine genetic variation within and among populations.
Metagenomics: Metagenomics is a technique that involves sequencing all the DNA in a sample,
including DNA from the insect itself as well as any microorganisms that may be associated with
the insect. This technique can be used to study the insect's gut microbiome, which can play an
important role in its physiology and ecology.
Overall, DNA sequencing techniques have greatly expanded the scope of entomology research,
allowing researchers to study insect genetics, evolution, and ecology in greater detail than ever
before.

93. Immunology is the study of the immune system and how it protects the body from
foreign invaders such as viruses, bacteria, and parasites. Insect research techniques
involve the study of insects and their biology, behavior, and interactions with other
organisms. There are several techniques used in both immunology and insect research,
including:
Microscopy: This involves using a microscope to view and analyze cells, tissues, and
other structures. In immunology, microscopy is used to study the immune system's cells
and their interactions with pathogens. In insect research, microscopy is used to study
insect morphology, behavior, and interactions with their environment.
Flow cytometry: This is a technique that uses lasers and specialized instruments to
analyze cells in a fluid sample. In immunology, flow cytometry is used to identify and
quantify different types of immune cells. In insect research, flow cytometry can be used
to analyze the cells of insects and their interactions with pathogens.

94. Behavior assays: These are tests used to study insect behavior, such as response to
stimuli, feeding behavior, and mating behavior. Insect behavior assays can help
researchers understand how insects interact with their environment and with other
organisms.
ELISA: This stands for enzyme-linked immunosorbent assay and is a common
laboratory technique used to detect and measure proteins or other substances in a fluid
sample. In immunology, ELISA is used to detect antibodies and other immune system
proteins in blood and other fluids. In insect research, ELISA can be used to detect and
quantify insect proteins, such as those involved in insecticide resistance.
PCR: This stands for polymerase chain reaction and is a technique used to amplify DNA
sequences. In immunology, PCR is used to detect and identify pathogens in blood and
other fluids. In insect research, PCR can be used to identify and quantify insect DNA,

95. Hemocyte isolation and analysis: Hemocytes are immune cells found in the hemolymph (insect
blood) and are responsible for phagocytosis, encapsulation, and melanization of invading
pathogens. Hemocytes can be isolated from the hemolymph using techniques such as
centrifugation and fluorescence-activated cell sorting (FACS) and analyzed using microscopy,
flow cytometry, and molecular techniques.
RNA interference (RNAi): RNAi is a technique used to silence the expression of specific genes in
insects. RNAi can be used to study the function of genes involved in the immune response and
to identify potential targets for pest control.

96. Infection assays: Infection assays involve infecting insects with pathogens and monitoring the
immune response. These assays can be used to study the molecular and cellular mechanisms of
insect immunity and to identify genes and proteins involved in the immune response.
Microbial culture and identification: Microbial culture and identification techniques can be used
to isolate and identify pathogens from infected insects. This information can be used to study the
interaction between insects and their microbial environment and to develop strategies for
controlling insect pests.
Proteomics and transcriptomics: Proteomics and transcriptomics are powerful tools for studying
the molecular mechanisms of insect immunity. These techniques can be used to identify proteins
and genes involved in the immune response and to investigate changes in gene and protein
expression in response to infection.

97. Proteomics refers to the study of proteins in a biological sample. Proteomics techniques have
been applied in various fields of biology, including entomology. Here are some of the
proteomics techniques that have been used in entomology:
Gel electrophoresis: This technique is used to separate proteins based on their size and charge.
It involves running a sample of proteins on a gel matrix and applying an electric field to
separate the proteins.
Mass spectrometry: This technique is used to identify and quantify proteins in a sample. It
involves ionizing the proteins and analyzing the resulting ions based on their mass-to-charge
ratio.
Two-dimensional gel electrophoresis: This technique combines two forms of gel
electrophoresis to separate proteins based on their size and charge. It allows for the separation
of thousands of proteins in a single sample.

98. Liquid chromatography: This technique is used to separate proteins based on their chemical
properties. It involves passing a sample through a column filled with a stationary phase, which
separates the proteins based on their interactions with the stationary phase.
Isotope labeling: This technique is used to quantify protein expression levels. It involves labeling
proteins in one sample with a heavy isotope and proteins in another sample with a light isotope.
The two samples are then mixed, and the relative abundance of each protein can be determined
based on the ratio of heavy to light isotope.
Overall, proteomics techniques have been used in entomology to study various aspects of insect
biology, including the identification of insect venom proteins, the characterization of insect
immune responses, and the discovery of insecticide resistance mechanisms.

99. DNA barcoding protocol
The DNA barcoding protocol for insect research follows the same general steps as for other
organisms, but with some modifications to account for the specific characteristics of insects.
Here is a detailed overview of the DNA barcoding protocol for insect research:
Specimen collection: Insects can be collected using a variety of methods, depending on the type
of insect and its habitat. Common collection methods include sweep netting, pitfall traps, and
light traps. Insects should be properly labeled and stored in a way that preserves their DNA. For
DNA barcoding, it is important to collect multiple specimens of each species to ensure genetic
diversity and avoid misidentification.

100. DNA extraction: DNA extraction from insects can be challenging due to their small
size and tough exoskeletons. There are several methods available for insect DNA
extraction, including CTAB-based protocols, commercial kits, and non-destructive
methods such as leg clipping. Some insects, such as beetles, require additional
tissue preparation steps to remove pigments and other interfering compounds.
1. Collect the insect: The first step in DNA extraction is to collect the insect and
store it properly. The insect should be stored in a tube with a label indicating the
species and collection date. Insects can be stored at -20°C or in 70% ethanol until
ready for DNA extraction.
2. Preparation of the tissue: Insects can be processed as a whole or by selecting
specific tissues such as legs, wings, or antennae. The tissue should be cut into
small pieces with a scalpel or razor blade, and any visible debris or pigments
should be removed. For some insects, such as beetles, the tissue may require
additional preparation to remove pigments and other interfering compounds.

101. 3. Disruption of the tissue: The tissue needs to be disrupted to release the DNA. This can be
done by mechanical or chemical methods. Mechanical methods include grinding the tissue
with a pestle and mortar or by using bead-beating or sonication. Chemical methods include
using detergents such as SDS, CTAB or Triton-X to lyse the cells.
4. DNA extraction: DNA extraction can be performed using a variety of commercial kits, or by
following a homemade protocol such as the CTAB extraction method. In general, the DNA
extraction protocol should include the following steps:
I. Cell lysis: The tissue homogenate is treated with a lysis buffer to release the DNA.
Protein precipitation: Proteins are precipitated using an organic solvent such as phenol-
chloroform, or by using magnetic beads that selectively bind the DNA.
II. DNA precipitation: The DNA is precipitated with ethanol or isopropanol, washed with 70%
ethanol, and air-dried.

104. III. DNA re-suspension: The DNA is dissolved in a suitable buffer such as TE buffer or water.
IV. Quality control: The quality and quantity of the extracted DNA should be assessed using a
spectrophotometer or fluorometer. The DNA should be checked for purity, yield, and integrity.
The DNA should be stored at -20°C or -80°C until ready for downstream applications.
In summary, the DNA extraction protocol for insects involves collecting the insect, preparing the
tissue, disrupting the tissue, DNA extraction using commercial kits or homemade protocols, and
quality control of the extracted DNA. The extracted DNA can be used for a variety of downstream
applications such as PCR, sequencing, and genotyping.

105. IV. PCR amplification: The PCR amplification step for insect DNA barcoding typically targets the
mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which has a variable region that
allows for species identification. PCR primers specific to this gene region are used to amplify the
target DNA fragment. Insect DNA can be challenging to amplify due to the presence of inhibitors
and low DNA quantity, so multiple PCR reactions may be necessary.
V. Sequencing: The amplified DNA fragments are then sequenced using high-throughput
sequencing technologies, such as Illumina or PacBio. Insect DNA barcoding typically uses short-
read sequencing platforms, as the COI gene fragment is only ~600 base pairs long. The resulting
sequence data is analyzed to ensure high quality reads are obtained for downstream analysis.

106. Data analysis: The final step in insect DNA barcoding is to analyze the sequence data to identify
the species of the insect. This is typically done by comparing the sequence data to a reference
database of known COI sequences, such as the Barcode of Life Data System (BOLD) or the
National Center for Biotechnology Information (NCBI) GenBank. Species identification is based on
a similarity threshold between the query sequence and the reference database, with a high
similarity indicating a high probability of a match. The reference database can also be used to
assess genetic diversity within a species and to detect potential cryptic species.
In summary, the DNA barcoding protocol for insect research involves specimen collection, DNA
extraction, PCR amplification, sequencing, and data analysis. This protocol is a valuable tool for
insect identification, biodiversity research, and conservation efforts.

107. Insect pests of stored grain
1. Lose of grains: 1/3 or ¼ of total store grains
 Much of them due to insect attack.
 Many grain pests preferentially eat out grain embryos, thereby reducing the protein
content of feed grain and lowering the percentage of seeds which germinate.
 Overseas customers demand insect-free grain.