Process of detoxification in insects is mediated by certain enzymes. Insects use enzymes associated with labial salivary glands or guts to detoxify plant defensive compounds or suppress plant induced defenses. Ex.- Esterases, mixed function oxidases, glutathione s-transferases

2.  Plant nutritional quality affects preference and performance of
insect
 Optimal diet is important for growth, reproduction and/or fitness
 The ratio of dietary protein-to-digestible carbohydrate (P:C) is one
component that can be selected by insect herbivores, such as
caterpillars and locusts (Simpson and Abisgold, 1985; Lee et al., 2002
and 2003).
For example- caterpillars of the beet armyworm, S. exigua, prefer a
protein-biased diet of 22P:20C (Merkx-Jacques et al., 2008).
 Dietary proteins provides nitrogen needed by insects for growth
and reproduction (Lee, 2007).
For example- Spodoptera exigua caterpillars do not grow well on a diet
containing less than 0.6% casein (Broadway and Duffey, 1986b).

3. –
• Process of conversion of a toxic
substances to a non-toxic substance and its
removal from the body
Detoxification
• Are macromolecular biological catalysts,
• It accelerates or catalyze chemical reactions,
• Enzymes act upon substrate molecules and convert
them into products.
Enzyme
• Process of detoxification mediated by enzymes
• Insects use enzymes associated with labial salivary
glands or guts to detoxify plant defensive compounds
or suppress plant induced defenses
• E.G., Esterases, mixed function oxidases, glutathione
s-transferases
Enzymatic
detoxification

4. • Insects protect themselves from deleterious effects of the plant defenses
(physical or chemical defenses).
• Chemical defensive strategies, plant nutritional quality and the presence of
constitutive or induced secondary metabolites are of key importance
• Secondary metabolites affect insect behavior or their growth,
development and reproduction (Harborne, 1993).
• Insects have evolved strategies to overcome these plant defenses.
• Biochemical resistance, includes the increased metabolic capability of
detoxifying enzymes, such as carboxylesterases and glutathione S-
transferases (GSTs) (Brattsten, 1988; Li et al., 2007).
• These enzymes can make plant chemicals more hydrophilic to increase their
excretion or convert them into nontoxic forms (Ahmad and Hopkins, 1993).
plant defenses
direct defense,
indirect defense

5.  In addition to these gut-associated enzymes, insects
produce effectors in the oral secretions (OS) to suppress
the induction of plant defenses.
 The activity of these can be affected by the diet that the
insect feeds on. This may reflect two dietary factors:
• Nutritional quality or
• Secondary metabolite
 Diet nutritional composition, such as the protein-to-
digestible carbohydrate (P:C) ratio, is a major factor
influencing the labial salivary GOX activity of caterpillars
of the cotton bollworm, Helicoverpa armigera and the
beet armyworm, Spodoptera exigua (Babic et al., 2008;
Hu et al., 2008).

6. • Based on enzymatic systems
• Protects the insect by detoxifying/sequestering insecticide molecules.
• The enzymes are developed as protection against naturally occurring
plant toxins (allelochemicals) such as alkaloids, terpenes and phenols, in
order to overcome the potential toxicity of the plants they feed on
(Gatehouse, 2002; Despres et al., 2007; War et al., 2012; Heidel-Fischer
and Vogel, 2015; Rane et al., 2016).
• Enzymes can detoxify xenobiotics into a non- toxic compound and/or
into a form more suitable for rapid elimination from the body.
• Resistant insects metabolize the insecticide faster because they possess
forms of the enzyme with a higher catalytic rate, or higher quantities
of the enzymes as a consequence of increased transcription or gene
amplification.

7. Insects use enzymes associated with labial salivary glands or guts to detoxify plant
defensive compounds or suppress plant induced defenses. Current studies suggest that the
activity of these enzymes can be affected by diet due to two main factors:
• plant secondary metabolites or
• nutritional quality.

8. Metabolism in insects
Activation Detoxification
2 methods –
inactive
compound
active
compound
metabolic reaction
that converts
active
compound
another active
compound or
compounds
Toxic
compound
non-toxic
compounds
metabolic reaction
that converts

10. (Li et al., 2007; Hollingworth and Dong, 2008; Yu, 2008; Berenbaum and Johnson, 2015)
Metabolic pathways
Phase I reactions
primary processes
Oxidation
Reduction
Hydrolytic
process
Glutathione
mediated reaction
Phase II reactions
Secondary processes
Glutathione
conjugation
Glucoside
conjugation
Amino acid
conjugation

11. Phase I Reactions
• This class includes all those enzymes in which
• One atom of an oxygen molecule is reduced to
water
• While other atom is used to oxidize the substrate
• This takes place by mixed function oxidase
Oxidation
• In this reaction halogen is replaced by hydrogen
atom
Reduction
• Positivity of phosphorous atom of insecticide is
reduced
• Takes place by estrases.
Hydrolytic
process
• Glutathione is utilized
• Eigther in a purely catalytic manner or
• Consumed by the direct binding to substrate
Glutathione
mediated reaction

12. Phase II Reactions
• harmful electrophilic compounds are
conjugated with GSH(reduced
glutathione)and with this the other
nucleophilic centers such as proteins and
nucleic acids are protected
Glutathione
conjugation
• harmful xenobiotics or their metabolites
combine with glucose to form
conjugates
Glucoside
conjugation
• activation of xenobiotic acid through
enzyme requiring ATP and followed by
condensation with endogenous amino
acid
Amino acid
conjugation

13. Enzymes
Phase I (functionalization)
reactions: oxidations and
reductions
Cytochrome P450s, flavin-
containing monooxygenases
(FMOs), hydroxylases,
lipooxygenases,
cyclooxygenases, peroxidases,
mononamine oxidases
(MAOs)and various other
oxidases, dioxygenases,
quinone reductases, dihydrodiol
reductases, and various other
reductases, aldoketoreductases,
NAD-and NADP-dependent
alcohol dehydrogenases,
aldehyde dehydrogenases,
steroid dehydrogenases,
dehalogenases.
Phase II (conjugation)
reactions: transfer
chemical moieties to
water-soluble derivatives
UDP
glucuronosyltransferases,
GSH S transferases,
sulfotransferases,
acyltransferases,glycosylt
ransferases,
glucosyltransferases,
transaminases,
acetyltransferases,
methyltransferase
Hydrolytic enzymes
Glycosylases,
glycosidases,
amidases,glucuronidases,
paraoxonases,
carboxylesterases,
epoxide hydrolase
and various other
hydrolases,
acetylcholinesterases and
various other esterase

14. Detoxification Enzymes
 Also known as Drug Metabolizing Enzymes (DME) or
Effector-Metabolizing Enzymes
 Involved in detoxification of plant metabolites, dietary
products, drugs, toxins, pesticides
 Selection result from variation in diet, climate, geography, toxins
(pesticides)

15.  Exogenous compounds (toxins, pesticides) compete with
endogenous ligands (estrogen, other hormones) acting
as agonists or antagonists.
• for binding to receptors (estrogen, glucocorticoid)
• channels (ion or other ligand)
 Such binding to receptors could result in toxicitiy,
abnormal development, or cancer
 Detoxification enzymes act to break down these
chemicals before they bind to receptors or channels

16.  GOX is an oxidoreductase that catalyzes the oxidation of
 β-D-glucose D- glucono-β-lactone + H2O2
 GOX has been detected in the salivary secretions and/or glands of-
 S. exigua, the corn bollworm,
 Helicoverpa armigera, the tobacco budworm,
 Heliothis assulta and the corn earworm,
 Heliothis zea
 Among these insects, generalists seem to have relatively high GOX
activity compared to specialists (Eichenseer et al., 2010).
 Thus, GOX may be also involved in expanding the host plant range by
insects (Eichenseer et al., 2010).

17.  Converts glucose gluconate, a carbohydrate
that cannot be utilized by the insect,
 Allow caterpillars to cope with the detrimental
effects of excess carbohydrate consumption since
plants often contain sufficient or excess
carbohydrates, such as sucrose, with limited quantity
and/or quality of proteins
ex.- The GOX activity of H. armigera is significantly increased
by higher content of sugar in the diets (Hu et al., 2008).
 Plant secondary metabolites did not affect GOX
activity. (Hu et al., 2008).

18. • A large group of phase 1 metabolic enzymes
• Metabolise a variety of exogenous and endogenous substrates.
Act against a broad range of chemical classes, including pyrethroids,
organophosphates and carbamates (Hollingworth and Dong, 2008).
• Catalyse the hydrolysis of ester insecticides into their corresponding acid and
alcohol compounds; this increases the polarity of the insecticidal metabolites
which can then be excreted more readily from the insect body.
• Sequestered toxic molecules are no longer available for interactions with
target proteins
• Associated with insecticide resistance in many insect species as a consequence
of quantitative and/or qualitative changes, resulting in the overproduction of
the enzymes or in modifications of their structures (Li et al., 2007).

19. • Catalyse the transfer of one atom of molecular oxygen to a substrate and the
reduction of the second atom of oxygen to form water; the process requires the
transfer of two electrons provided by NADPH cytochrome P450 reductase
(Feyereisen, 2005; Guengerich, 2008).
• The reaction is commonly described as:
RH + O2 + NADPH + H+ ROH + H2O + NADP+
• Are phase 1 enzymes involved in the
detoxification of xenobiotics
• Able to convert lipophilic compounds into
polar metabolites that can be easily
eliminated from the body; (Feyereisen
2005, 2015; Liu et al., 2015).

20. • Specifically, catalyze conjugations by facilitating nucleophilic attack of
the sulphhydryl group of endogenous reduced glutathione (GSH) on
electrophilic centres of a range of xenobiotic compounds, including
insecticides or acaricides (Konanz and Nauen, 2004) and various plant
toxins (Despres et al., 2007).
• Thus the xenobiotics have increased solubility and are excreted from the
insect system by the formation of mercapturic acid derivatives (Habig et
al., 1974; Enayati et al., 2005).
• Detoxifies both endogenous and xenobiotic
compounds
• Involved in intracellular transport, biosynthesis
of hormones & protection against oxidative
stress (Ketterman et al., 2011).

21.  Glutathione S-transferases (GSTs) are a major family of
multifunctional detoxification enzymes
 Since some GSTs can detoxify lipid hydroperoxides,
α,β-unsaturated aldehydes, lipid epoxides and may
involve in the repair of radical-damaged DNA, this
group of enzyme is critical in protecting insects against
oxidative stress
 Therefore, several mechanisms might work together to
detoxify xenobiotics. For example, in the case of
pyrethroid resistance, GSTs protect insects either by
offering a passive protection through binding the
insecticide molecules or by detoxifying lipid
peroxidation products induced by pyrethroids
(Kostaropoulos et al., 2001; Vontas et al., 2001).

22.  GST genes and activity can be induced by plant allelochemicals or
insecticides. For example, expression of glutathione S-transferase cDNA of
the Oriental leafworm moth, Spodoptera litura, was up-regulated by some
insecticides, such carbaryl, DDT, deltamethrin, and Bacillus thuringiensis (Bt).
Deng et al. (2009)
 GSTs activity increased when the English grain aphid, Sitobion avenae larvae
were fed on resistant wheat cultivar, which had high concentration of
phenolic compounds (Leszczynski et al., 1994).
 Caterpillars of the gypsy moth, Lymantria dispar and forest tent caterpillar,
Malacosoma disstria fed on leaves supplemented with phenolic glycosides had
increased GSTs activity compared to the control group (Hemming and
Lindroth, 2000).
 GST activity of the Oriental tobacco budworm, Helicoverpa assulta larvae fed
on chilli pepper was lower than those fed on tobacco or artificial diet (Wang
et al., 2009).

23.  The fruit fly, Bactrocera tau fed on balsam pear has higher GST
activity compared to those fed on cucumber, pumpkin, towel gourd
and white gourd (Li and Liu, 2007).
 Even fed on different cultivars of the same species, the activity of
GSTs of insects can vary , when three insect species, the
grasshopper, Atractomorpha lata, the green peach aphid, Myzus
persicae and the diamondback moth, Plutella xylostella fed on eight
cultivars of sesame, they had different GST activity. This may be
due to that different cultivars produced diversed quantity and
quality of chemicals in response to these insect herbivores (Sintim
et al., 2012).

24.  Plants often protect themselves against insect herbivores by
proteinase inhibitors (PIs), which act on insect gut-associated
proteinases
 Ingestion of plant-derived proteinase inhibitors (PIs) by insects will
impede protein digestion and lead to a decrease in bioavailability of
essential amino acids required by the insect for growth, development
and
For example, using the artificial diet containing soybean trypsin
inhibitor to rear the sugarcane borer, Diatraea saccharalis, led to a
delay of larval development, increasing the length and number of
instars and decreasing female longevity (Pompermayer et al., 2001).

25.  To overcome trypsin inhibitors in their diet, insects have several
mechanisms such as expressing new proteinases that are insensitive to
the inhibitor
 The corn earworm, Helicoverpa zea larvae express two different trypsin
isozymes depending on if larvae are fed on control or inhibitor-
containing diet (Volpicella et al., 2003).
 The tobacco budworm, Heliothis virescens larvae also vary their
complement of trypsin enzymes when fed on control or inhibitor-
containing diet (Brito et al., 2001).
 Herbivorous insects can also regulate midgut trypsins by differential
regulation of multiple genes encoding different digestive proteinases
For example, in response to dietary inhibitors, like soybean trypsin
inhibitor, there was an initial up-regulation of all proteinases genes in
the caterpillars of the cotton bollworm, Helicoverpa armigera, which is
followed by a down- regulation of genes that encode proteinases
sensitive to the inhibitors but sustained expression of genes encoding
inhibitor-insensitive proteinases (Bown et al., 1997).

26.  Carboxylesterases are hydrolases that use water molecules to cleave ester bonds,
phosphoesters and amides turning target chemicals into corresponding alcohols
and acids
 Important for insecticide resistance. Three different classes of agrochemicals,
pyrethroids, organophosphates (OPs) and carbamates, can be detoxified by
carboxylesterases (Ahmad, 1986; Casida and Quistad, 1998; Shan and Hammock,
2001; Oakeshott et al., 2005).
 Midgut carboxylesterase activity increases when caterpillars of the tobacco
budworm, Heliothis virescens, are exposed to profenofos (Harold and Ottea, 1997).
 Carboxylesterases bind to the substrates and hydrolyze them (Wheelock et al.,
2005). In the Australian sheep blowfly, Lucilia cuprina, substitution of amino acids
at the acyl pocket of the carboxylesterase increased the overall activity compared
to the wild type of enzyme (Devonshire et al., 2007).

27.  In insects, carboxylesterases are regulated in many ways,
 including gene amplification,
 selection for and expression of mutant carboxylesterases and
 enhanced transcription of non-amplified, structural genes (Wheelock et
al., 2005).
For example, overproduction of carboxylesterase E4 or its paralog FE4 protein
enables the green peach aphid, Myzus persicae to degrade diverse insecticides
including OPs, carbamates, and pyrethroids (Field and Devonshire, 1998).
 Plant diet can affect herbivore carboxylesterase activity. For example, in the
Oriental tobacco budworm, Helicoverpa assulta, larvae fed on chili pepper have
lower carboxylesterase activity than those fed on tobacco and artificial diet (Wang
et al., 2009).
 In the beet armyworm, S. exigua, carboxylesterase activity was the highest in
larvae feeding on Chinese cabbage, but decreased by nearly 60% if caterpillars
were fed on maize seedlings (Zhang et al., 2011).
 In the silverleaf whitefly, Bemisia tabaci, populations on cabbage had higher
carboxylesterase activity levels compared with garden egg populations (Avicor et
al., 2013). Insecticides added into diets can also increase carboxylesterase activity
(Gao and Liang, 1993).

28.  The high levels of reactive oxygen species (ROS), such as superoxide anion
radicals, singlet oxygen, hydrogen peroxide and highly reactive hydroxyl radicals
can negatively affect insects by damage the cells by reacting with the membrane
lipids and this will impair the absorption of ingested nutrients in the
 Ascorbate peroxidase (APOX) can reduce H2O2 levels by converting it to H2O
(Asada, 1992).
 Insect gut APOX activity can be affected when plant allelochemicals like o-
dihydroxyphenols and tannic acid are added to the diets (Barbehenn, 2002;
Lukasik et al., 2009).
 Caterpillars of the Egyptian cottonworm, Spodoptera littoralis fed on potato
leaves rich in allelochemicals, such as chlorogenic acid and tannins, have higher
APOX activity in comparison to those reared on semi-artificial diets (Krishnan and
Kodrík, 2006).
 The level of APOX activity was higher in the African maize stalk borer, Busseola
fusca larvae fed on non-transformed maize plants compared to those fed on Bt
maize plants (George and Gatehouse, 2013).

29. . Current studies suggest that the activity of these enzymes can be affected by diet
due to two main factors:
• plant secondary metabolites or
• nutritional quality.
• During the last 50 years,
 increased use,
 overuse and
 even misuse of pesticides
has led to the selection of resistance in more than 500 arthropod pest species.
• Michigan State University developed an online database (APRD)
(http://www.pesticideresistance.com) to list the resistant cases reported.
• Following the first report of resistance at the beginning of the last century
(Melander, 1914), the number of cases continued to grow, with an exponential
increase during the late 1970s and early 1980s (Georghiou and Lagunes-Tejada,
1991).

30.  Today, the order with the highest number of resistant species is
• Diptera (27 %), followed by
• Lepidoptera (25 %),
• Hemiptera (17 %) and
• Coleoptera (10 %) (Whalon et al., 2012).
 Diptera can have severe economic impact as many of them transmit diseases
to humans and domestic animals, while others are pests of agricultural plants.
 In the other orders, many of the resistant species represent a serious threat for
agricultural production and are responsible for important agricultural yield
losses causing problems for future food security.
27%
25%17%
10%
21%
diptera
lepidoptera
hemiptera
coleoptera
other

31.  Insecticide resistance evolves predominantly by two
mechanisms:
1. the enhanced production of metabolic enzymes, which
sequester or detoxify the xenobiotics, and/or
2. mutations of target proteins, which make them less
sensitive to the xenobiotics.
 Subsidiary physiological mechanisms which contribute to
reduce insecticidal effects have also been described, e.g., a
lower penetration of the chemicals or an increased
excretion.

32. The detoxifying enzymes are
important for insects to protect them against
the plant defenses, both nutritional
deficiency and toxic plant secondary
metabolites. The study of dietary effects on
the activity of these enzymes on different
diets will be helpful in prevention and
management of resistance build-up against
plant secondary metabolites and insecticides
for efficient management of insect-pests in
future. Furthermore, the information obtained
from these studies would provide details
essential for the informed synthesis of effective
and environmentally friendly actives.