GENES OF INTEREST IN ENTOMOLOGICAL RESEARCH

Presentation by,

Mr. Guru P. N.Ph. D. 015/39

Cry toxin Bt: Cry1Ab, Cry1Ac, Cry2a, Cry9c, Cry2B, Vip I,

VipII etc.

Plant metabolites: Flavonoids, aklaloids, terpenoids.

Enzyme inhibitors: SbTi, CpTi.

Enzymes: Chitinase, Lipoxigenase.

Plant lectins: GNA.

Toxin from predators: Scorpion, spiders.

Insect hormones: Neuropeptides and peptidic hormones.

Genetic engineering of Plants for Insect Resistance

SEX DETERMINATIONSex determination is the process by which the gender of a bisexual organism becomes fixed, so that the individual progeny develops either as a son or a daughter.

Except diptera, sex determination in insects is cytological.

General aspects of sex determination in insects

Hermoproditism: same genotype producing male and female organs in same individualEg: Icerya

Gyanandromorphs/ intersexes: genetically abnormal individuals

1.XX/XY systems (1:1) Heterogametic sex: produce two gametically different types of

gametes

Homogametic sex: gametes of only one type

S1S1 X S1S2 (parents) 1:1 S1S1 and S1S2 (progeny)

Heterogametic : male in most and female of lepidoptera, trichoptera and diptera few.

When female is heterogametic sex, then named as Z & W i.e., ZW/ZZ

ZW (female) X ZZ (male) 1:1 ZZ and ZW (progeny)

2. XX/XO systems Y chromosome tends to degenerate leads having few or no

alleles functional

This is progressive evolutionary phenomena

XX (female) X XO (male) 1:1 XX and XO

It mainly based on genetic balance

It occurs in all type of insects both primitive and advanced

Ancestral form of sex deermination of orthopteroid insects

Neo-X systems: Orthopteroid, odonata, hemipteroid and coleoptera

3. Multiple sex chromosome system Difference between sexes involves larger number of

chromosomes

Most common has just two X’s

i.e., X1X2 Y or X1X2O and notation is

X1X1X2X2 / X1X2Y or X1X1X2X2 / X1X2O

Generally 1-6 X’s are present (extreme is 12)

Ex: hemiptera, dermaptera, diptera (multiple factor)

4. Multiple factor system

5. Haplodiploidy system Eg: Hymenoptera, Thysanoptera

Alternate alleles at a single locus- segregating in opposition

Females : S1S2, S1S3, S2S3 Males : S1S2S3 – if eggs are unfertilized

: S1S2, S2S2, S3S3 - inbred populations

This is intern also referred as “complementary sex determination mechanism”.

6. Molecular basis of sex determination Mainly restricted to Drosophila melanogaster

Sexlethal (Sxl) located on X-chromosome this is essential for determination of females

If, X:A is 1.0 (diploid with XX), Sxl produces an active product

that causes embryo to develop as female

X:A is 0.5 (diploid eggs with XY or XO) then Sxl is silent and embryo develops as male

OrthopteroidDictyoptera (Blattodea + Mantodea), Phasmida, Orthoptera, Embioptera XX/XO

Isoptera, Dermaptera XX/XYMost Heteroptera XX/XY or XOHomopteraAurenorhyncha XX/XOSternorhyncha Psyllodea : XX/XO

Coccoidea : XX/XOAphoidea : XX/XO

Coleoptera Diversified XX/XO, XX/XY, multiple, neo-X/neo-Y,……

Hymenoptera Haplodiploid

Lepidoptera Female heterogamy XY/XX or ZW/ZZ

Panarpoid XX/XO

Sex determination system in different insect orders

Model of JH III within a surface representation of the substrate-binding pocket of MsJHE.

Hormonal Control of Insect Metamorphosis

BrainTemperature, Light, Stress, etc.

Prothoracic Gland

Ecdysteroid

Corpus Allatum

Prothoracicotropic hormone (PTTH)

Juvenile Hormone (JH)

LarvaPupa

Adult

Development of winged morph

Biosynthesis in the corpora allata glands and release into the hemolymph. The primary mode of JH metabolism in the hemolymph is through the action of JHE. JHE hydrolyzes

JH in the presence or absence of hemolymph

Juvenile hormone esterase

Family : hydrolases (carboxylic ester bonds)

Other names : JH esterase, juvenile hormone esterase and juvenile hormone carboxyesterase.

Important in the regulation of the JH titre

JHE's appear to be produced by the fat body

JH esterase induction

JH I being the most potent inducer

Factors present in the head of the insect are potent inducers of JH activity.

Starvation of lepidopteran larvae also induces appearance of JH esterase

The insect nervous system is the target of the majority of synthetic chemical insecticides that are in agricultural use.

JH analog insecticides, for example, do not target the endocrine system of mammals and can show selectivity within an insect order or even family, a level of selectivity that is not obtained with classical insecticides.

The jhe gene has potential as a highly insecticidal transgene given an appropriate vector for expression.

What we can do with JHE ?

What is the future ?

Insertion of transgenes into the baculovirus genome that express a peptide hormone or

enzyme such as JHE that can alter normal host physiology.

Recombinant protein is the JHE from Tyroglyphus molitor, a member of the order Coleoptera (beetles)

(Hinton and Hammock, 2003b).

A dipteran, Drosophila melanogaster (Campbell et al., 2001)

Four lepidopterans, Heliothis virescens

(Hammock et al., 1990; Bonning et al., 1992), Choristoneura fumiferana

(Feng et al., 1999), Manduca Sexta

(Hinton and Hammock, 2001), and Bombyx mori

(Hirai et al., 2002)

Tried in

Introduction

• More than 50 neuropeptides have been described – Behavior– Pain perception– Memory– Appetite– Thirst– Temperature– Homeostasis– Sleep

Neuropeptides: neurohormones or neurotransmitters?• Neurohormones: when neurons secrete their

peptides into the vascular system to be transported to a relatively distant target

• Neurotransmitter: Many axon terminals of neurosecretory cells secrete their products at the synapse to directly affect a post synaptic cell

• Neuropeptides can do both – depends on nerve terminal

Stages of action

• Synthesis (ER and Golgi apparatus)

• Packaging into large-dense core vesicles (with modifying enzymes)

• Transport (fast-axonal transport)

• Release• Action (prolonged)• Termination by diffusion

and degradation

Synthesis is sequential

Synthesis is controlled by alternative splicing

Neuropeptides

• The endogenous opiates• Neuropeptide Y• Galanin• Pituitary adenylate cyclase–activating peptide (PACAP) • Melanocyte-stimulating hormone (MSH)• Neurokinin A (NKA)• Substance P (SP)• Neurotensin• Calcitonin-gene–related protein (CGRP)• Vasoactive intestinal polypeptide (VIP)

Neuropeptide Y

• A 36 amino acid peptide• Acts via G-protein coupled receptors (decrease in

cAMP, decrease in Ca+2 , and increase in K+ conductance)

• Function: augments the vasoconstrictor effects of norepinephrine

Diet

MembranePL

Synthesis of acetylcholine

• Choline + acetylcoenzyme-A by choline acetyltransferase in cytoplasm

• Transported into and stored in vesicles.

• Removal: hydrolysis by acetylcholinesterase

PROTEASE INHIBITORSROLE IN INSECT CONTROL

Protease inhibitors (PIs)

Are one class of plant defense proteins against insect pest infestation.

Plant derived protease inhibitors inactivate proteases of animals and microbial origin

Inhibiting endogenous enzymes

They are involved in the protection of plants against pests and possibly pathogens.

Protease-inhibitor families in plant tissues.

1. Soybean trypsin inhibitor (Kunitz) family

2. Bowman-Birk inhibitor family

3. Barley Trypsin inhibitor family

4. Potato Inhibitor I family

5. Potato Inhibitor II family

6. Squash Inhibitor family

7. Ragi 1-2/Maize bifunctional inhibitor family

8. Carboxypeptidase A. B inhibitor family

9. Cysteine proteinase inhibitor family (cystatins)

10. Aspartyl proteinase inhibitor family

Important ones are

Serine protease inhibitors

Cysteine proteinase inhibitor

Metallo- and aspartyl proteinase inhibitors

Aspartic and Metallo-Proteinase Inhibitors

Six families of Hemiptera,

pH optima =3-5

Serine Proteinase Inhibitors

Competitive inhibitors

Lepidoptera: (alkaline range of 9-11) : serine proteinases and

metalloexo peptidases are most active. (orders present: Lepidoptera,

Diptera, Orthoptera, Hymenoptera, and Coleoptera)

Antinutritional effects

Cysteine Proteinase Inhibitors (cystatins)

Most have been found in animals, but several isolated from plant

species, pineapple, potato, corn, rice, cowpea, etc.,

Found in midguts of several families of Hemiptera and Coleoptera

(digestion of food proteins).

Mildly acidic (pH optima of cysteine proteinases 5)

Plant PI’s

Mode of action

PI molecules inhibit protein digestive enzymes in insect guts, resulting in amino acid deficiencies and thereby developmental delay, mortality, and/or reduced fecundity

Hyperproduction of proteases to compensate for the loss of activity, causing the depletion of essential amino acids. The imposed nutritional stress would subsequently retard insect development.

Growth delay in a natural setting would also lengthen exposure of phytophagous insects to their predators and pathogens.

Increase in mortality.

Blocking insect midgut proteinases

The presence of inhibitor leads to the loss of nutrients particularly sulphur containing amino acids, and thereby weak and stunted growth and ultimate death

Insect resistant transgenic plants expressing PI’s

The first ever transgenic plants were Produced using cowpea trypsin inhibitor cDNA clone. The transgenic plants were resistant against herbivorous insects such as Collosobrchus maculatus, Heliothis spodoptera and Diabrotica and Tribolium sp.

Future prospects of inhibitory proteins

(i) Gene Combinations/Packaging/ Pyramiding

(ii) Protein Engineering

(iii) Single-chain Antibodies

(iv) Phage Display

Bowman-Birk Inhibitor (BBI) family

Closely related to serine protease inhibitors.

These proteins are classified as double-headed serine protease inhibitors due to the presence of two reactive site domains within the same polypeptide, one each for trypsin (Lys-Ser) and chymotrypsin (Leu-ser) molecules.

The cowpea trypsin inhibitor constitutes a some-what larger gene family of four major isoinhibitors.

Cowpea trypsin Inhibitor Gene (CpTi gene)

Isolated from cowpea plant (Vigna unguiculata).

This is the first plant-originated insect resistance gene to be successfully transferred into other plants species.

Against Lepidoptera, Coleoptera and Orthoptera

Inhibit the insect proteases involved in digestive processes.

Consequently, this will result in a depletion of essential amino acids required for growth, which in turn cause the larvae to die.

The CpTI gene is heritable to the next generation

LECTINS : Roles in Pests Control

“Lectins are a class of proteins of non-immune origin that possess at least one non-catalytic domain that specifically and reversibly bind to mono-or oligosaccharides”.

They are similar to antibodies in their ability to agglutinate red blood cells; however lectnis are not the product of immune system.

They may bind to a soluble carbohydrate or to a carbohydrate moiety that is a part of a glycoprotein or glycolipid.

“Based on the overall domain architecture of plant lectins, four major groups can be distinguished:

Merolectins, Hololectins, Chimerolectins and Superlectins

Entomotoxic lectins

Lectins are found to be effective on the insect orders like, Lepidoptera, Coleoptera, and Homoptera

Harmful effects of lectins

Larval weight decrease,

Mortality,

Feeding inhibition,

Delays in total developmental duration,

Delays in adult emergence and fecundity on the first and second

generation

Plant lectinsLectins are a group of proteins that are found in plants and they inhibit predation bybeing harmful to various types of insects and animals that eat plants.

Lectin (plant source) Insect Mannose specific:

ASA (Allium sativum) Myzus persicaeDysdercus cingulatus

ASA I, II Dysdercus cingulatusD. Koenigii

ASAL (Allium sativum leaf) D. CingulatusLiphaphis erysimi

Mannose/ glucose specificConA (Canavalia ensiformis) Aphids

LCA (Lens culinaris) Acyrthosipum pisum

PSA (Pisum sativum) A. PisumClover leaf weevil

N-acetyl-D-glucosamine specificACA (Amaranthus caudatus) A. Pisum

WGA (Triticum aestivum) Diabroticus undecimpuctataL. Erysimi

The first lectin to be purified on a large scale and was available on a commercial basis was Concanavalin A; which is now the most well- known lectin to control of some pest insects

Canavalia ensiformis, or Jack-bean, is used for animal fodder and human nutrition.

It is also the source of concanavalin A lectin.

Galanthus nivalis or snowdrop is the best-known and most widespread plant. (Fam:

Amaryllidaceae).

Currently, the two major groups of plant derived genes used to confer insect resistance on crops are lectins and inhibitors of digestive enzymes (proteases and amylase inhibitors)

Transgenic tobacco expressing Pinellia ternata agglutinin (pta) gene induced enhance level of resistance to M. persicae.

Transgenic plants with insecticidal lectin gene

Transgenic plants with lectin genes to confer resistance against insects

Transformed plant Lectin Target pest

Maize WGA Ostrinia nubilalisDiabrotica undecimpunctata

Rice GNANilaparvata lugensNephotettix virescensCnaphalocrocis medinalis

Tobacco PSA Heliothis virescens (tobacco bud worm)

Tobacco GNA Helicoverpa zeae (cotton bollworm)

Fungal lectins with insecticidal activity

•Mushrooms contain various potential interesting proteins, including lectins in their organs such as mycelium, spores and fruiting bodies

Some lectins from fungi including Xerocomus chrysenteron (XCL), Arthrobotrys oligospora (AOL) and Agaricus bisprous (ABL)

While, XCL is having higher insecticidal activity on Dipteran (Drosophila melanogaster) Homopteran (Myzus persicae and Acyrthosipon pisum)

reversible antiproliferative effects

Action mechanism of lectin at the tissue level of insects (MoA)Binding of the lectin to the midgut tract causing disruption of the epithelial cells including

Elongation of the striated border microvilli,

Swelling of the epithelial cells into the lumen of the gut lead to complete closure of

the lumen,

Permeability of cell membrane to allow the harmful substances penetrations from

lumen towards haemolymph and

Impaired nutrient assimilation by cells,

Allowing absorption of potentially harmful substances from lumen into circulatory

system, fat bodies, ovarioles and throughout the haemolymph

Fungal lectins with insecticidal activity

Lectin (fungal source) Insect Host

XCL (Xerocomus chrysenteron)

M. persicae, Acyrthosipon siphum

Peach, potato

Drosophila melanogaster Pea

SSA (Sclerotinia sclerotium)

A. Pisum Pea

RSA (Rhizoctonia solanii agglutinin)

Spodoptera littoralolis Cotton

PeCL (Penicillium chrysogenum)

M. persicae, A. pisum

Peach, potato, pea

(A) Xercomus chrysenteron

(B) Penicillums chrysogenum

Indirect effects of lectins

Interaction with virus transmission

Mannose-binding lectins are able to bind to carbohydrate on micro-organisms.

Circulatory viruses contain numerous N-linked glycosylation sites on their surface cells. Many of these sites contain high-mannose glycans which could interact with mannose-binding lectin such as ConA

Synergistic effects on other proteins

The insecticidal activity of protease inhibitor and α-amilase inhibitors were significantly increased when these inhibitors enzymes incorporated with lectin

Rice transgenic plant carrying three insecticidal genes including lectin gene (encoding gene GNA), cry1A and cry 2A, have enhanced levels of resistance to a wide range of different rice pests

THANK YOU

GENES OF INTEREST IN ENTOMOLOGICAL RESEARCH

Presented by, Mr. Guru P. N.Ph. D. 015/39

Dept. of Agril Entomology