Diapause and cold hardiness in insects – biochemical aspects

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. Diapause and cold hardiness in insects – biochemical aspects 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

3. 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

4. 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)

5. 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?

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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 )

12.   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

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

14. Phases of Diapause

15. 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.

16. 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.

17. 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 .

18. 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.

19. 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.

20. 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

21. 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

22. 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

23. 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 .

24. 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.

25. 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.

26. 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 .

27. 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.

28. 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.

29. 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 ca 2+   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).

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

31. 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.

32. 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.

33. 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

34. 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. 

35. 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.

36. 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

37. 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.

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

39. 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.

40. Biosynthesis of Glycerol

41. Thermal relations

42. 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.

43. 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

44. 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.

45. 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.

46. 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

47. 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 .