Diapause is a period of suspended or arrested development during an insect's life cycle. Insect diapause is usually triggered by environmental cues, like changes in daylight, temperature, or food availability. “State of arrested development in which the arrest is enforced by a physiological mechanism rather than by concurrently unfavorable environmental conditions”. (Beck, 1962) Diapause and cold hardiness in insects : Why?

1. Mogili Ramaiah
IARI, Ph.D Scholar
Division of Entomology
New Delhi-110012
Welcome
Term-Paper Presentation

2. Contents
 Introduction
 History
 Basic classification
 Diapause in insects
 Phases of Diapause - Biochemical aspects
 Case studies - Diapause
 Cold hardiness in insects
 Cold hardiness – Biochemical aspects
 Case studies – Cold hardiness
 Important terms
 Summary
 Conclusion
 References

5. Introduction
Survival Strategies
It's not easy being a insect.
There are a lot of "windshields" on the road of life!!
 Diapause
 Cold-hardiness
 Migration
 Parthenogenesis
 Polymorphism

6. Diapause
 Diapause is a period of suspended or arrested development
during an insect's life cycle. Insect diapause is usually
triggered by environmental cues, like changes in daylight,
temperature, or food availability.
 “State of arrested development in which the arrest is
enforced by a physiological mechanism rather than by
concurrently unfavorable environmental conditions”.
(Beck, 1962)

7. History
 Word Diapause was coined by Wheeler to egg stage of Grass
hopper, Conocephalus ensiferum
 Slightly modified terminology, as proposed by Shelford (1929)
and later recommended by Lees (1955) and Danks (1987).
[Dormancy, Diapause and quiescence]
 Phases of diapause – Kostal V (2006)
 Model insects for cryoprotectant metabolism, the larvae of freeze
avoiding gall moth, Epiblema scudderiana (Clemens) and freeze
tolerant gall fly, Eurosta solidaginis (Fitch) – Richard et al. 1987
Diapause and cold hardiness in insects : Why?

8. Basic classification
 Dormancy ; Is a generic term covering any state of suppressed
development (developmental arrest), which is adaptive (that is
ecologically or evolutionarily meaningful and not just
artificially induced), and usually accompanied with metabolic
suppression.
a)Quiescence ; An immediate response (without central
regulation) to a decline of any limiting environmental factor(s)
below the physiological thresholds with immediate resumption
of the processes if the factor(s) rise above them.
b) Diapause ; A more profound, endogenously and centrally
mediated interruption that routes the developmental
programme away from direct morphogenesis into an
alternative diapause programme of succession of physiological
events; the start of diapauses usually precedes the advent of
adverse conditions and the end of diapause need not coincide
with the end of adversity. (Shelford ,1929)

9. DIAPAUSE QUISCENCE
 Highly evolved form of
dormancy and is
maintained irrespective of
environment
 Diapause is not an
immediate response
 Long adaptive form of
dormancy
 Stage is determined
 Irreversible dormancy
 Heterodynamic insects
 Egg of silk worm, Larvae
of PBW
 Temporarily inhibited by
unfavorable environment
 Quiscence is an immediate
response
 Short or long depending on
environmental conditions
 Stage is not determined
 Reversible dormancy
 Homodynamic insects
 Pupa of Helicoverpa
armigera

10. Facultative vs. Obligatory diapause
1. Insects having facultative diapause may go through
several generations without showing diapause –
multivoltine
2. Insects having obligatory diapause have a diapause in
each generation - univoltine
 Influence of environmental factors

11. Aestivation Hibernation
 The period of suspended
activity in individuals
occuring due to seasonal
high temperature
 Summer diapause
 Eg. Red hairy caterpillar
 The period of suspended
activity in individuals
occuring due to seasonal
low temperature
 Winter diapause
 Eg. Pink bollworm
 Seasonal variations

12. Life stage of insect
Egg diapause: Mulberry silkworm, Grass hoppers,
locust
Larval diapause: Pink bollworm
Pre pupal diapause: Plodia interpunctella
Pupal diapause: Pieris brassicae, Red hairy caterpillar
Adult diapause : White grub, Epilachna, Leptinotarsa
Imaginary diapause: Mosquitoes

13. Egg diapause is of two types
1. True egg diapause - embryonic development stopped
due to presence of an egg-inhibiting hormone
2. False egg diapause – embryo develops but fails to
hatch. Really a first-instar larval diapause (no
ecdysone)

14.  Larval and pupal diapause
caused by failure of secretion of ecdysone
Adult diapause caused by failure of reproductive
organs to mature and produce gametes due to absence
of a gonadotrophic hormone
Photoperiod (day length) usually causes facultative
diapause
1. Long-day insects - PBW
2. Short-day insects – silk moth

15. Phases of Diapause
I. Pre diapause
Induction phase
Preparation phase
II. Diapause
Initiation phase
Maintenance phase
Termination phase
III. Post diapause

16. Phases of Diapause

17. Induction phase
 Its occurs at a genetically predetermined stage of life and
occurs well in advance of the environmental stress.
 This sensitive stage may occur within the lifetime of the
diapausing individual, or in preceding generations i.e,
resulting in egg diapause.
 During this phase, insects are responsive to external cues
called token stimuli.
 Token stimuli can may be any change in photoperiod,
thermoperiod, or allelochemicals from food source.
 This triggers the switch from direct development pathways to
diapause pathways.

18. Preparation phase
 The preparation phase usually follows the induction phase.
Though insects may go directly from induction to initiation
without a preparation phase.
 During this phase, insects accumulate and store molecules
such as lipids, proteins and carbohydrates.
 These molecules are used to maintain the insect throughout
diapause and to provide supplement for development
following diapause termination.
 Diapausing puparia of the flesh fly, Sarcophaga crassipalpis
increase the amount of cuticular hydrocarbons lining the
puparium, effectively reducing the ability of water to cross
the cuticle.

19. Initiation phase
 Photoperiod is the most important stimulus initiating
diapause.
 The initiation phase begins when morphological
development ceases.
 In some cases, this change may be very distinct and can
involve moulting into a specific diapause stage, or be
accompanied by colour change, behavioural change,
migration, aggregation or some enzymatic change.
 Adults of the fire bug, Pyrrhocoris apterus, have the
enzymatic complement that allows them to accumulate
polyhydric alcohols, molecules that help to lower their
freezing points and thus avoid freezing during diapausing.

20. Maintenance phase
During the maintenance phase, insects experience
lowered metabolism and developmental arrest is
maintained.
Sensitivity to certain stimuli which act to prevent
termination of diapause, such as photoperiod and
temperature is increased.
 At this stage, insects are unresponsive to changes in
the environment that will eventually trigger the end of
diapause, but they grow more sensitive to these stimuli
as time progress.

21. Termination phase
 In insects that undergo obligate diapause, termination may
occur spontaneously, without any external stimuli.
 In facultative diapausers, token stimuli must occur to
terminate diapause.
 These stimuli may include chilling, freezing or contact with
water, depending on the environmental conditions being
avoided.
 These stimuli are important in preventing the insect from
terminating diapause too soon.
 The effect of diapause slowly decreases until the insect can
resume its developmental process under favourable
condition.

22. According to physiological and ecological mechanisms
of its incidence and termination, diapauses can be
classified into three types
Parapause : an obligatory hereditary arrest of development
or activity arising in every generation at a species specific
instar
Oligopause : an arrest of development or activity with
control of its induction, maintenance and termination,
similar for all these periods
Eudiapause : a facultative arrest of development or activity
with different controlling mechanisms of induction and
termination, e.g., through photoperiod and chilling,
respectively

23. Diapause larvae vs Non diapause larvae
Lipid content is more
Water content lower
Trehalose content more
Low oxygen
consumption
Protein and amino acids
are more
Free fatty acids low
Lower metabolic rate
Lipid content is less
Water content higher
Trehalose content less
High oxygen
consumption
Protein and amino acids
are less
Free fatty acids high
High metabolic rate

24. Egg Diapause and Metabolic Modulations during
Embryonic Development in
the Silkworm, Bombyx mori L. (Lepidoptera:
Bombycidae)
Tribhuwan Singh, Pramod Kumar Singh and Khursheed
Ahmad Sahaf
Annals of Biological Research, 2013, 4 (1):12-21

25. Carbohydrate metabolism
 Amount of glycogen accumulated in diapause eggs is 1.7 times
higher than in non-diapause eggs. More than 90% of
carbohydrate accumulated in the silkworm diapause eggs is
glycogen.
 The glycogen initially present in the diapaused eggs rapidly
broken down into sorbitol and glycerol at the onset of
diapause.
 Glycogen accumulated in the silkworm egg is from the
glycogen stored in the fat body during pupal stage, which is
converted into trehalose and is released into haemolymph and
then absorbed by developing oocyte.

26. The trehalase localizes in plasma membrane of
vitellogenic follicles where haemolymph trehalose
hydrolyses into glucose to be taken up by oocytes.
The glucose is immediately used to synthesize glycogen
as a storage reserve, by which hyperglycogenia is
induced in diapause eggs.

27. Changes in amino acid
 Significant changes in some free amino acids occurred
during the initiation and termination of diapause.
 In particular, a sudden large increase in alanine content
(about 50μ mol / g eggs) occurred at the initiation of
diapauses. Afterwards alanine declines gradually with the
increase of glutamate and especially proline.
 Proline content is low during the initiation and maintenance
of diapause but increased suddenly during the termination
period indicating the conversion of alanine to glutamate and
proline in diapausing eggs. Proline accumulated may be
utilized during embryogenesis.

28.  In diapausing insects, high concentration of free amino acids as
well as of polyols and sugars serves to decrease the super cooling
point.
 The super cooling point of Bombyx mori eggs is lower during
diapause and hibernation may be due to increase of total amino
acids and the accumulation of alanine or proline.
 Proline serves as an energy source for later stages of embryonic
life.
 In diapausing eggs of silkworm, pyruvate produced from
glycogen by the glycolytic pathway appears to be converted first
to alanine and then to praline via glutamate to maintain energy
sources during diapause for resumption of embryogenesis.
Therefore, the diapause eggs can survive for one year or more if
necessary, despite their small reserves .

29. Nucleotide metabolism
 In silkworm eggs, diapause is decided during the
maturation process of the eggs in the ovary of pupal body.
Therefore, there is a close relationship between diapause
occurrence and metabolism of egg cells.
 In insects, nucleic acid is not only related to the expression
of genes but also influence protein synthesis, cell division,
growth and development.
 In univoltine genotypes, sub-oesophageal ganglion if
removed at early pupal stage, the female will lay non-
diapausing eggs, while normal female laid diapause eggs.

30. If the mature eggs inside the ovariole of above two
groups taken out, it is found that DNA content of
diapause eggs is 25.29% lower than that of non-
diapause eggs and RNA content of diapause mature
eggs is 25.48% less but the DNA / RNA ratio of these
two groups were the same.
Hence, it is inferred that DNA content of mitochondria
of diapause eggs is probably lower than non diapause
eggs.

32. A summary of lipid storage and mobilisation in
overwintering insects.
 Generally, diapause induction involves the inhibition of insulin-like
peptide production in insects, which removes the inhibiting effect of
the insulin receptor on FOXO, allowing lipid accumulation to occur.
During diapause maintenance, the adipokinetic hormone (AKH) is
produced in response to amp-activated protein kinase (AMPK)
accumulation as well as other unknown factors, which stimulates the
production of diacylglycerol (DAG) from triacylglycerol (TAG) via a
cyclic adenosine monophosphate (camp) and ca2+ signalling cascade.
DAG is then exported from the lipid droplet and the cell is
transported through the haemolymph by binding to high-density
lipophorin (hdlp) using unknown factors with the help of a lipid
transport particle (LTP), forming low-density lipophorin (ldlp).
Abbreviations: apoplp-iii, apolipophorin 3; DAG-BP, diacylglycerol
binding protein; FFA, free fatty acid; JH, juvenile hormone. Figure
redrawn after denlinger and armbruster (2014) and canavoso et al.
(2001).

34. Phases of pupal diapause mapped onto a metabolic rate
trajectory and physical developmental markers in Rhagoletis
pomonella.

35.  In Rhagoletis pomonella, we are attempting to understand
adaptive changes in pupal diapause regulation over much
shorter evolutionary distances.
 Diapause development proceeds through multiple phases,
separated by important transitions.
 In Rhagoletis diapause we have pinpointed these phases
through metabolic and developmental phenotyping, and
have thoroughly characterized the process of diapause
termination at the transcriptomic level.

36. Regulation of diapause :
 It is regulated at several levels.
 Environmental stimuli interact with pre defined genetic
pathway to effect neural signals, endocrine pathways and
metabolic and enzymatic changes.
 Environmental regulators of diapause generally display a
characteristic seasonal pattern. Depending upon the season,
insect respond to the photoperiodism and thermoperiodism.
 The neuroendocrine system of insects consists primarily of
neurosecretory cells in the brain, the corpora cardiaca,
corpora allata and the prothoracic glands.

37.  There are several key hormones involved in the regulation
of diapause: juvenile hormone (JH), diapause hormone
(DH), and prothoracicotropic hormone(PTTH).
 PTTH stimulates the prothoracic glands to produce
ecdysteroids that are required to promote development.
 Larval and pupal diapauses are often regulated by an
interruption of this connection, either by preventing release
of PTTH from the brain or by failure of the prothoracic
glands to respond to PTTH.
 The corpora allata is responsible for the production of JH,
failure of this leads to adult diapause

38. Endocrine glands involved in diapause
1. Neurosecretory cells in protocerebrum – “brain” hormone
2. Corpora cardiaca – stores and releases brain hormone
3. Prothoracic glands – produces and releases ecdysone
4. Subesophageal ganglion – produces and releases egg diapause
hormone
DH acts to stimulate trehalase activity in developing ovaries to
bring about hyperglycogenism in mature eggs, a prerequisite for
diapause initiation.

39. Cryptobiosis: a phenomenon when insects become
quiescent due to adverse climatic conditions and shows
no visible sign of metabolic activity
Eg ; Polypedalium vanderplanki by complete
dehydration, the insect can survive for many years.
Athermopause: when factors other than temperature
such as hygiene, nutrition are involved in dormancy.

40. Cold hardiness in insects
Depending on the maintenance of body temperature, animal
kingdom is divided into
1) Warm Blooded Animals (Homeothermic): These animals
maintain a constant body temperature within certain narrow
limits irrespective of the temperature variations in the
external environment (‘Endothermic animals’) Eg.
Mammals
2) Cold Blooded Animals (Poikilothermic) : These animals are
not capable of maintaining constant body temperature . These
are also called as ‘Ectothermic animals’ as they depend upon
the environment than the metabolic heat to raise their body
temperature. Eg. Insects
3) Socio-homeothermic Animals: These organisms maintain their
body temperature slightly above the atmospheric temperature
and are able to air condition their nests. They maintain their
own temperature inside their colony irrespective of the
temperature outside. Eg. Honey bees

41.  Cold hardy insects: insects which have a capacity to survive
through quiet prolonged exposure to non freezing low
temperatures. They are killed only if tissue are suddenly
frozen
 Ice nucleating proteins present in cold hardy insects,
absorb the nucleus of water preventing the formation of ice.
 Super cooling: maintenance of liquid state of blood below
the freeze point. Eg ; Cold carpenter ant
 Undercooling means cooling below O 0C, where as super
cooling means cooling below freezing point.

42. Survival at low temperature (Cold
hardiness)
Freeze intolerant insects
1. Freeze – avoiding - no mortality > SCP (Epiblema)
2. Highly chill- tolerant - some mortality > SCP
3. Moderately chill- tolerant - relatively low SCP
4. Chill – susceptible – high mortality low SCP
5. Opportunistic species – high mortality around O 0C
Freeze tolerant insects
1. Partial
2. Moderate
3. Strong
Super cooling point for most freeze tolerant insects is in
the range of -5 to -10 oC

43. Cold hardiness
 Ice formation reduced by emptying the gut before the cold
period as food in the gut may form nuclei for ice formation.
 Reduction in the water content, which increase the osmotic
pressure of the haemolymph.
 Compounds that stabilize the SCP, peptides or glycopeptides
that perhaps adsorb to the surface of newly formed ice
crystals and prevent new water molecules from reaching ice
crystals. There production induced by low temperature and
short periods.
 JH titers increases in haemolymph, which have regulatory
role in the production of the antifreezing agents.
 Crystals of calcium phosphate in the Malphigian tubules.
 Production of polyhydric alchohols, glycerol.

44. Biosynthesis of Glycerol

46. Thermal relations

47. The metabolic reasons for production of alternative
cryoprotectants such as Ribitol, threitol, erithritol and
ethylene glycol alsoneed to be explored in appropriate
species.
Because of CO2 loss in the conversion of glycogen to
these C5, C4 and C2 polyols, their synthesis appears to
be insufficient in terms of carbon conservation but
there may but energetic or redox balance reasons for
production of these compounds.

48. Summary
Diapause is a delay in development evolved in response
to regularly recurring periods of adverse environmental
conditions.
Phases of diapause
Physiological characteristics of a diapausing stage
Regulation of Diapause
Cold hardiness
Biosynthesis of polyols

49. Conclusion
Insects clearly posses a range of coordinated and
integrated mechanisms that have evolved to allow them to
survive and flourish under potentially adverse
environmental conditions.
Diapause an important part of the life-cycle in many
species of invertebrates.
It is considered in ecological studies with the aim to
model and predict population responses to the
environment which changes either seasonally or linearly,
on an evolutionary scale.

50. Increasing precision in the knowledge of how the
responses to environmental factors change at an
individual ontogenetic level.
Studies of insect thermal relations have direct
applications to numerous research fields, including pest
management, cryopreservation and forensic entomology.
Such studies will continue to play a key role in
forecasting the effects of climate change and in the
prediction of potential impacts of agricultural pest
species or disease vectors in the future.

51. It is not the strongest the species that survives, nor the
most intelligent that survives. It is the one that is the
most adaptable to change

53. Questions
 Why we need to study diapause and cold hardiness in
insects?
 Phases of diapause.
 Important terms Diapause, Quiescence and Dormancy etc.
 Differences between Diapause vs Non Diapause, Diapause vs
Quiescence, obligate vs facultative diapause, Aestivation vs
Hibernation etc.
 Regulation of diapause by insect hormones.
 Cold hardiness - Cryoprotectants, Production of polyols .