Chemistry
&
Metabolism
&
Metabolism
- can't be washed off easily by sweat/water
- low mammalian toxicity (doesn't penetrate skin)
- long duration
- targets insects
- know the mode of action
- delivery of molecules
- cost effective
Leps vs Beetles dsRNA uptake
RNAi has to be taken up by insect tissues and differs based on target organism.
Both: Clathrin-dependent endocytosis, transported through endosome
Beetles: dsRNA released by endosome then processed by core RNAi machinery -> silencing mRNA
Less efficient, gut nucleases can degrade dsRNA before it is taken up by RNAi
Leps: dsRNA is not released by endosome, avoiding dsRNA contacting RNAi machinery, systemic spread of dsRNA or siRNAs?
Pyridine carboxamides, Fatty acids, Lipid Biosynthesis Inhibitors, and Inorganic insecticides
Pyridine Carboxamides:
Flonicamid: inhibits feeding behavior by inhibiting the activity of the feeding stimulant receptors in the aphid's stylet, affecting salivation/ingestion
Pymetrozine: also inhibits aphid feeding behavior but targets the nervous system by inhibiting feeding stimulant receptors and inhibits growth and development, affects chordotonal organs and interacts with TRPV channels
Fatty Acids:
Disrupt cell membrane of insects, killing cells and disrupting the nervous system, growth, development, and metabolism
Act rapidly to penetrate cuticle and dissolve in cell's lipid membranes, causing death
Saturated: octanoic acid
Unsaturated: oleic acid (=)
Lipid Biosynthesis Inhibitors:
Target the synthesis of fatty acids in insects, inhibiting the activity of enzymes involved in fatty acid synthesis, causing a depletion of lipids and death (do not disrupt cell membranes)
Inorganic Insecticides:
Boric acid: protoplasmic poison that precipitates proteins within the cytoplasm of cells - disrupts cuticle, causing water loss
Cryolite: naturally occurring mineral that acts as a stomach poison and inhibits and enzymes
How Photorhabdus luminescens and Heterorhabditis attack insects
1) infective juvenile nematodes search for insect hosts in the soil and penetrate the cuticle, releasing bacteria from their gut into the hemocoel
2) bacteria multiply and produce toxins/enzymes including proteases that break down insect tissues, liquifying the insect (loses muscle tone)
3) insect dies in 24-48 hours, nematodes feed on insect and bacteria, bacteria continue to multiple and release toxins
4) nematodes multiply then exit insect into the soil, bacteria look for new hosts
Cellular Respiration
A metabolic pathway that generates ATP, which can be used as energy by the cell
Results in oxygen consumption
Practical considerations for fumigants
- highly toxic and can harm humans, animals, and beneficials
- must be heavier than the air and non-flammable
- should have self-warning properties like smell
- penetration to insect, such as through grains
- concentration, duration, type of pest
- harmful residues can be left behind, residual activity
- enter organism through respiratory tract
- gas-tight area, ventilation
Bt on mammals
Bt toxins are safe for humans due to high specificity
Bt toxin MoA involves binding to specific insect gut receptors in an alkaline pH environment
Mammals lack these receptors and have acidic guts that degrade Bt toxins
Chlordimeform, Methylxanthenizes, and caffeine MoA and synergism
Chlordimeform:
Broad spectrum insecticide, targets nAch receptors and binds to them, preventing transmission of nerve impulses, leading to paralysis and death
Methylxanthenizes:
Bind and block activity of adenosine receptors, disrupting neurotransmission and leading to paralysis and death
Caffeine:
chemical plant defense that interferes with insect feeding behavior and reduces reproduction & kills
similar effect to octopaminergics (overproduction or inhibition of cAMP)
Hydrophilic
Methylxanthines like IBMX synergize with formamidine insecticides and enhance their antifeedant activity by inhibiting the breakdown of cAMP through inhibition of phosphodiesterase enzymes that normally break cAMP into AMP
Cuticular Molting
1. Cuticle matures
2. Pulse of insect brain releases PTTH, driving an increase in the concentration of 20-OH-Ecdysone, initiating apolysis (a separation of the endocuticle from epidermal layer)
3. New cuticle is slowly deposited in that gap between the old endocuticle and the epidermal layer
4. Old cuticle is digested and reabsorbed, allowing the formation of more new cuticle
5. A titer drop in 20-OH-Ecdysone signals eclosion hormone (EH) to be released and ecdysis is initiated (insect loses the old cuticle)
6. Tanning occurs: crosslinking of proteins and small molecules in the exo- and epi- cuticle cause sclerotization (hardening and darkening of the outermost layers), drive by the hormone bursicon
Chitin Synthesis Inhibitors
Disrupt molting by interfering with chitin production.
Timing their application during the the molting process is important.
Disadvantages: broad-spectrum, can develop resistance, persist in environment, expensive
Effects: deformities, reduced feeding/anorexia, weak exoskeleton, reduced fitness/reproduction
Metabolism of different Pyrethroid insecticides
1) secondary alcohols:
terminal alkene is epoxidized and then becomes a diol with the addition of water (rat)
P450 oxidizes alcohol multiple times (alcohol --> hydroxyl --> aldehyde --> acid) (insect)
2) primary alcohols:
Ring hydroxylation: OHs added to ring
Oxidation: CH2OH added to =O with P450
Ester Hydrolysis: B esterase and water hydrolyze molecule in two
DDT, Pyrethroid I, and Pyrethroid II intoxication in Insects and Mammals
Insects:
DDT - excitability, tremors, paralysis, death
Pyrethroid I - hyperexcitation, twitching, kick off legs, erratic flying, paralysis, death
Pyrethroid II - leg coordination failure, extension of limbs, sluggishness, paralysis, death
Mammals:
DDT - motor unrest, increase in spontaneous movement, abnormal susceptibility to fear, hyper-sensitivity to light, touch, and sound, tremors, and convulsions
Pyrethroid I - hypersensitivity, self isolation, agitation, aggression, hair on end, sparring, fine tremors -> whole body tremors
Pyrethroid II - decrease in body temp, lack of coordination, respiratory stress, CS syndrome, squirming, salivation, coarse tremors -> seizures
Site I, II, and III Inhibitors and Uncouplers and ATPase Inhibitors
Site I: electron transport inhibitors
Interfere with ETC in Complex I so no ATP is produced
Ex: Rotenone, Fenazaquin, Pyridaben
Oxygen level has a sharp drop
Site II: respiration disrupters
Inhibit electron transfer to Complex II by FADH2 and oxygen is not consumed (respiration is disrupted)
Ex: Cyanopyrafen, Cyflumetofen
Oxygen slowly declines (gentle slope)
Site III: respiration disrupters
Flow of electrons is disrupted and no oxygen is consumed (respiration is disrupted)
Ex: Hydramethylnon
Oxygen slowly declines (gentle slope)
Uncouplers - slow, disrupt membrane, causing flow of protons, which disrupts proton gradient so ATP-synthase no longer makes ATP
Ex: Chlorfenapyr
Oxygen consumption is unaffected
ATP-ase Inhibitors - function at ATP-synthase and inhibit formation of ATP, no oxygen is consumed
Ex: Diafenthiuron, Tetradifon, Cyhexatin
Oxygen slowly declines (gentle slope)
Steps of the electron transport chain
Stereochemistry and SAR of Pyrethroids
Stereochemistry affects toxicity, potency, selectivity, and metabolic fate. Each enantiomer has different chemical/biological properties. A stereo/chiral center is a C with 4 different atoms attached.
Priority is based on atomic number/weight. Clockwise = R, Counterclockwise = S. Cis = both groups on the same side of the double bond (or both towards or away), Trans =groups on different sides of the double bond or away + towards.
Pyrethroids:
Type I: Cyclopropane ring, single chiral center. Cis is more toxic than trans. Prolong opening of voltage-gated Na+ channels.
Type II: Alpha cyano group. More prolonged and intesnse depolarization of the cell membrane, penetrate cuticle better. Trans is more toxic than cis, S are generally nontoxic except for 1-S-Fenvalerate due to its constituents.
Signal Transduction Pathways performed by GPCRs
G Protein composed of a heterotrimeric complex (a B y)
Alpha subunits bind guanine nts (inactive = GDP, activated = GTP) and dislocates from B and y subunits (form a dimer - second messenger cascades or transcriptional factors)
Alpha subunit produces a variety of second messengers, which activate signaling proteins, ion channels, transcription factors, etc. that influence insect behaviors. These are signal transduction pathways:
alpha s - stimulates enzyme adenylate cyclase to produce cAMP from ATP, resulting in stimulation
alpha i - inhibition of adenylate cyclase and decreased production of cAMP (not an octopamine receptor, tyramine receptor)
alpha q - (inositol pathway) biochemical formation of IP3, a signaling molecule that transverses to calcium stores within the cell (ER or SR) and releases calcium, resulting in an increase in intracellular calcium concentration
Stacked vs Pyramid Bt hybrids
Stacked: express 2 or more Bt toxins that are each effective against different insect pests in order to target multiple specific pests at once (one below ground and one above ground)
Pyramid: express 2 or more Bt toxins that are both effective against the same insect pest in order to protect against resistance (below ground)
DDT and Pyrethroids
Bind to Na+ channel and prevent it from closing, causing prolonged depolarization of the cell membrane, leading to excessive nerve impulses and overstimulation
Pyrethroids have a global effect on central and peripheral nerves, skeletal muscles, and more sensitive sensory neurons, causing potent knockdown and nerve block
Py I binds to open state of channel and keeps it open, many spike train, repetitive firing
Py II stabilizes channel in inactive state and prolongs depolarization and depolarization block, never reaches threshold, inactivated
siRNA Mechanism
1) Initiation: introduction of dsRNA into the cell, which is recognized and cleaved by the enzyme Dicer, making 21-24 nt long siRNAs
2) Loading: siRNAs are loaded into RNA-induced silencing complex (RISC), a multi-protein complex containing Ago protein. siRNA binds to Ago, forming RISC-siRNA complex
3) Unwinding: siRNA is unwound in the RISC complex, resulting in the separation of the sense and antisense strands and the sense strand is degraded, while antisense remains bound to Ago
4) Targeting: RISC-siRNA complex binds to complementary mRNA target through base pairing between the antisense and the target mRNA
5) Cleavage: Ago protein catalyzes cleavage of the mRNA, resulting in degradation of target mRNA and gene expression silencing
Tail Currents (w/ Pyrethroids)
An electrical current that continues to flow after repolarization, returning the membrane potential to the resting state. It is caused by the slow closing of voltage-gated ion channels opened during depolarization.
Type I Py causes a delay in inactivation of Na+ channel and it stays open longer, causing a tail current
Type II Py causes a very delayed inactivation and closure and an even longer tail current
Lower Temperature will extend the opening of the Na+ channel and cause even more pronounced effects
Chemical properties of Pyrethrins and their different groups.
Pyrethrins are made up of an acid and an alcohol. They are unstable at alkaline or acidic conditions and are lipophilic. They are influenced by environmental conditions and readily hydrolyze or oxidize in light and degrade, so not the best insecticides.
Chrysanthemic Acid or Pyrethric Acid and...
Pyrethrolone alcohol (Natural)
Cinerolone alcohol (Cinerines)
Jasmolone alcohol (Jasmolines)
Production of cAMP and its effects
GPCR binds to its ligand and activates G Protein, which activates adenylyl cyclase, which converts ATP into cAMP:
Adenylate cyclase takes ATP and removes 2 Ps, creating AMP, which is acted upon by adenylate cyclase which "cyclitizes" it and creates cAMP
cAMP is a second messenger that can activate protein kinase A (PKA), which can phosphorylate target proteins and influence gene transcription in the nucleus, cytisol, and/or membrane ion channels
It can be turned off by cyclic nucleotide phospho-diesterases which convert cAMP back to AMP
DCDM and Octopamine enhance the production of cAMP (CDM has to be metabolized first to do it)
Botanicals:
Veratridine, Lipid Amides, Indoxacarb & Metaflumizone, Ryanodine & Anthranilic Diamides
1. Veratridine:
Activates Na+ channels and keeps open, repeated nerve firing
Binds differently than Type I Py
2. Lipid Amides:
Hyperexcitation and nerve block like Py, tail current
3. Indoxacarb:
Metabolized into DCJW which binds to Na+ channels, causing a delay in opening and inhibition of nerve impulses. DCJW metabolized again into DCJW acid, binds to diff. Na+ channel site, causes prolonged inhibition of nerve impulses -> paralysis & death
4. Metaflumizone:
Binds to Na+ channels at a diff. site than Py/DDT, causing prolonged and irreversible inhibition of nerve impulses and paralysis & death.
5. Ryanodine:
Skeletal muscle (SR)
AP -> open Ca+ channel -> Ca influx -> RyRs release Ca2+ from SR -> bind to contractile proteins + enable muscle contractions
Neurons (ER)
AP -> presynaptic terminal -> Ca2+ channels open and Ca2+ enters -> increase in Ca concentration causes synaptic vesicles to release neurotransmitters into the synaptic cleft
6. Anthranilic Diamides:
Bind to specific site on RyRs and cause it to remain open, leading to the influx of Ca2+ into the cell, causing muscle contraction/paralysis & death
Cyt Toxin:
1) toxin binds to receptors on brush border membrane of insect midgut
2) c-terminal of hydrophobic portion of molecule inserts into lipid bilayer of a cell and forms pores in midgut epithelium
3) aggregation of toxin molecules continually lodging into cell membrane lets Na+ and water transverse and creates osmotic ion imbalance
4) causes gut swelling and formation of vacuoles and/or apoptosis due to insect immune response
Cry Toxin:
1) Activated and solubilized by proteases in the insect midgut, creating an active toxin from a protoxin
2) toxin monomer binds to cadherin protein receptor, followed by proteolytic cleavage of helix alpha-1 (further digestion of columnar cells of alpha subunit in the midgut)
3) multiple toxins come together to form oligomer
4) oligomer binds to APN (alkaline phosphatases) receptor
5) APN receptor and oligomeric toxin localize to lipid rafts
6) they insert into the cellular membrane like an umbrella and open up, creating a tetrameric pore in the cell membrane
Resistance:
Kdr and Bt mechanisms
Resistance develops from target site mutations or metabolic resistance, caused by insecticide exposure, genetic mutations and selection, behavior, or physical barriers
Kdr (knockdown resistance) arises due to a mutation in the gene that controls Na+ channels, preventing insecticides from binding effectively (Point mutation of Leu -> Phe, between domain 2 and 3)
Super-Kdr is a more extreme Kdr caused by multiple mutations in Na+ channel and an even greater reduction in pyrethroid binding. Often occurs in combination with metabolic resistance or other mechs. (Point mutation in domain 2, linker protein between S4 and S5)
Bt:
Mutations on receptor proteins that reduce binding
Mutations in proteases that activate Bt toxins in the midgut
Increased expression of detoxification enzymes like P450s or esterases that can break down Bt toxins before they reach their target site in the midgut epithelium
Thicker peritrophic membranes (chitin lining midgut) prevent or reduce midgut penetration
Reduced expression of receptor proteins, decreasing number of available binding sites for Bt toxins