Insect Metamorphosis

~26 Orders with the biggest species diversity

Oldest fossil found is 400my old (Devonian)

Inhabit all terrestrial habitats (even in Antarctica); some FW, few marine and lots of flying ones

Extraordinary successful taxa due to:

• Occupation of many niches
• Coexistence with plants - mutualistic pollinators etc. = coevolution

Some Orders e.g. Lepidoptera,Orthopoda, Diptera, Hymenoptera have a large impact on humans due to being disease carriers, agricultural pests and pollinators

Insect = 3 pairs of legs, 2 pairs of wings, antennae and a compound eye

Open circulatory system = tracheal system

Insulin is an important regulator of metabolism in insects

Corpus cardiacum and corpus allatum:

• Gland-like structures which are connected to the brain
• Important for storing and distributing hormones to the brain, especially during metamorphosis

Evolution of wings/flight

• Escape predators
• Dispersal to new habitats

Diversification of mouthparts for feeding on plants

Plants and insects adaptive radiation - coevolution

• Parallel for ~100 mya until ~50mya explosion of diversity
• Pollinators for angiosperms

Metamorphosis purpose:

• Reproduction
• Alternation of life states for reproduction
• Advantages at each stage for design of the individual
• Differences in diet - caterpillar is designed to gain nutrients by feeding while butterfly is designed to disperse for reproduction 

Ametabolous Development

• Pre-adult is a smaller adult
• Often in primitive insects

Hemimetabolous Development

• Pre-adult is similar to an adult with some structural changes e.g. winds
• Immature infertile nymph stages

Holometabolous Development

• Pre-adult very different
• Complete structural changes during pupa stage
• Only evolved once in all numerous lineages
• Dramatic, time-consuming change between larvae to adult
• Metamorphosis is often costly as larval stage likely to be eaten by predators etc
  • Often the production of loads of eggs/hiding eggs to compensate for this
• Examples - silkworm, tobacco hornworm, Drosophila

Know a lot about the molecular level of metamorphosis in Drosophila

Polyploid organism with the exception of the brain

Imaginal disks = areas of larvae that will be transformed into particular areas of the adult

• Diploid tissues
• Usually no function in the larva except to be adult building blocks

Part of the larval brain is used in the adult but still undergoes a change

• Set of neuroblasts in the larval brain will rapidly proliferate to create a larger brain

Origin occurred in the Carboniferous 

4 Orders have it = Coleoptera, Hymenoptera, Diptera and Lepidoptera

Holometabolous insects more closely related to each other than hemimetabolous insects = diverged once

Ligate a caterpillar during last instar = separate head from the abdomen

The time of ligation effects communication between the different areas of the body

Posterior area of caterpillar would remain larval if the signal is prevented

= there is a critical period for metamorphosis

5th instar = last instar before adulthood

'Adult moult' is produced in a fused 1st instar/5th instar individual = gets signals from the body

If you transplant a 4th instar corpus allatum into a 5th instar abdomen = a '6th instar' is produced

• This is a novel type of moult
• Juvenile but bigger
• Means that there was an important signal produced from the corpus allatum which prevented metamorphosis
• Signal was dubbed 'Juvenile Hormone' = secreted from the corpus allatum
• Juvenile hormone is an inhibitor of Metamorphosis

Corpus allatum:

• All insects undergoing metamorphosis have this 
• Coming off the brain - brain stem?

Prothoracic Gland:

• Required for metamorphosis
• Mainly in the thorax but in Drosophila (highly evolved) there is a ring gland = the corpus allatum, corpus cardiacum and prothoracic gland are all fused together into one gland
  • Ring gland located in the head on top of the brain

Connected a series of pupae = only the anterior one had a brain and prothoracic gland

Anterior pupa triggers metamorphosis in the posterior direction in a chain of connected pupae lacking these organs

• The degree of metamorphosis seen in the posterior pupae correlated to the distance away from the head 
• = less metamorphosis at the far posterior end

1) PTTH from the brain is secreted by the CC 

2) PTTH stimulates the PG to produce E

3) E is released periodically during moulting

4) JH from the CA determines the type of moult

= three endocrine signals (PTTH, E and JH) trigger the process

Acts on the prothoracic glands (PGs) to regulate the synthesis of ecdysteroids

Release of PTTH is controlled by:

• Environmental stimuli e.g. photoperiod, temperature (season)
• Nervous stimuli e.g. stretch receptors (acquired enough resources for a moult)
• Via haemolymph (processing) in Bombyx, Manduca
• Direct PG innervation from neurons in Drosophila

Mode of action:

• Via Receptor Tyrosine Kinase > Ras > Raf > ERK (in Drosophila)

Lateral ventral clock neurons (LNv) signal to PTTH-producing neurons

Light and PTTH signals entrain the PG cells for ecdysone secretion, resulting in the rhythmic release of the hormone

The ecdysone receptor, EcR anticipates these daily rhythms by undergoing cyclic nucleocytoplasmic oscillations in target cells

An overview of proposed mechanisms underlying the regulation of ecdysone biosynthesis in the Drosophila prothoracic gland:

• A range of developmental factors exert effects on the prothoracic gland to control the production of ecdysone pulses, including PTTH, TGFb/Activins, ILPs and circadian signals
• PTTH neurones directly innovate the prothoracic portion of the ring gland = direct release of peptide E
• Similar to other insects = timing of PTTH release is affected by photoperiod and temperature
  • Neurones controlling time directly connect to PTTH neurones
• Pigment dispersing factor (PDF) - set of neurones expressing neuropeptides
  • A subset of these neurones also express sNPF (short neuropeptide F)
• sNPF is received by the PTTH neurones and activates them
• act-B and ILPs come from the neurosecretory cells which activate a portion of the ring gland
  • Doesn't related to time or temperature
  • Relates to growth = only commit to metamorphosis when the individual has stored enough resources

An overview of the proposed mechanisms underlying the regulation of the ecdysone biosynthesis in Drosophila prothoracic gland:

• Multiple signalling cascades that regulate ecdysone biosynthesis in the prothoracic gland
  • There are three signalling pathways - MAPK (Kinase), TGF and Insulin pathways
  • These signalling pathways not unique to insects - similar pathways throughout animals
  • Inactivation of these pathways could compromise ecdysone production and results in developmental defects
• Light, temperature and time influence these pathways
• Not just important to insects but to other cell processes in eukaryotic cells
• Output from prothoracic gland is ECDYSONE, generated by cholesterol with lots of enzymes
  • Enzymes are required for further processing of cholesterol to ecdysone
  • Cholesterol ---> 5B-Ketodiol ----> Ecdysone
• Halloween genes influence the above ^^ pathway - therefore, if they don't exist then the insect is stuck in development as a dead pupa
• BR (broad complex) is a transcription factor which is important for regulating metamorphosis at levels e.g. processing and generation of ecdysone 
  • Requirements for completing metamorphosis is for the inhibition of the BR complex

• Coordinated via the BR complex
• Cholesterol is converted into protohormone ecdysone via a series of reactions that occur in the ER, cytosol (suggested for other arthropod species) and mitochondria
• Ecdysone is exported possibly by secretory vesicles into the haemolymph (inner circulatory system of the fly)
• Protohormone finds its' way to target tissues where it's further processed via the ER using enzyme Shade
• This generates 20-hydroxyecdysone = active form
• 20-hydroxyecdysone binds to proteins which are transcription factors
  • Ecdysone is a steroid hormone = can move through lipid bilayers but needs to bind to protein receptors in the nucleus of cells to coordinate transcription of genes

The precursors for ecdysteroid synthesis are sterols such as cholesterol (campesterol; sitosterol; stigmasterol)

Insects cannot synthesize cholesterols and require cholesterol in their diets to make ecdysone

The primary site of ecdysteroid synthesis is the prothoracic gland; the major product is ecdysone

Ecdysone (inactive form) is converted to 20-hydroxyecdysone (active form) by target tissues

There are some differences in the types of cholesterol in different insects

In Drosophila:

• Ecdysteroids are produced in the prothoracic gland cells of the ring gland, which is attached to the anterior side of the brain
• Hormones are then released into the haemolymph and subsequently converted to the biologically active form 20-hydroxyecdysone (20E) in peripheral tissues

Peaks in 20E correlate to different developmental changes:

• Critical weight reached = forms pupa
• Glue genes allow wandering larvae to attach to substrates using specific products
• Wandering behaviour = larvae crawl out of food and starts to find a place to pupate
• Pupariation = formation of pupa
• Head eversion = bubble of gas pushes head outwards

Ashburner model:

• The hormone-bound and thus active ecdysone receptor directly induces the expression of early puff genes and represses late puff genes
• A small set of early puff genes repress their own expression and are required for the induction of a large set of late puff genes
  • Early puff genes = negative feedback on themselves
• The steps ensure that there's an ordered process in waves of gene expression

Puff genes = genes expressed in polyploid tissues inherited from the larva

• Individual chromosomes line up and form visible structures
• Puffing patterns of chromosomes are correlated with the developmental stage
  • Early way of studying the impact of ecdysone on gene expression - showing puffing in chromosomes 

Early puff genes = hormone directly

Late puff genes = secondary regulation

Early ecdysone response genes (puff genes) negatively feedback on themselves

BR-C complex is important

Later puff genes only stimulated by later gene expression

Complex regulators and regulation = organising different waves of gene expression by early genes promoting late genes and being inhibited by themselves

Different waves of expression may lead to different cascades

At the end of metamorphosis there is a degeneration of the prothoracic gland = means that no other round of metamorphosis occurs after the first one

Triggered by PG exposure to ecdysteroids in the absence of JH

Ecdysteroids acting along trigger apoptosis by the PG cells

Apterygote ametabolous insects retain active PG throughout their lives

First described by Wigglesworth as an inhibitory hormone that prevented the metamorphosis of Rhodnius prolixus

JH is synthesized in and released from the corpus allatum (CA)

JHs belong to sesquiterpenes (lipid hormone) - have long carbohydrate chains

• Variation in species like with ecdysones

JHs have multiple effects during the life on an insect, especially involvement in:

• Inhibition of adult metamorphosis
• Diapause (long-term dormant state)
• Reproduction
• Metabolism

Produced in the corpus allatum of locusts and cockroaches

Different from normal ones

Hydroxylation of the chain may result in molecules with a greater biological affinity, just as 20-hydroxyecdysone is more active than ecdysone

Retinoic acid, methoprene, hydroprene, kinoprene

Used as insecticides

• Insect will do its' final moult but will form a sterile larval form (6th instar) 
• Therefore can't reproduce and compete with reproducing pests for resources

Retinoic acid, methoprene, hydroprene, kinoprene

Used as insecticides

• Insect will do its' final moult but will form a sterile larval form (6th instar) 
• Therefore can't reproduce and compete with reproducing pests for resources

Haemolymph JH titers (concentrations) are regulated by both biosynthesis and degradation

JH synthesis is rapidly controlled along several avenues

• Environmental stimuli e.g. photoperiod
• Endogenous factors e.g. mating, nutritional state

Neurohormones that affect CA affinity:

• Allotropins - stimulate JH production
• Allatostatins - inhibit JH synthesis by the CA
• Allatoinhibin - inhibits the Manduca CA nonreversibly

Because of its' lipophilic nature, JH must be bound to other molecules (JHBPs) in order to move through the aqueous haemolymph

Perhaps more importantly, binding can also protect the hormone from degradation by nonspecific tissue-bound esterases

Juvenile hormone binding proteins (JHBPs):

• Low molecular weight binding proteins with a single JH binding site
• High molecular weight binding proteins (i.e. lipophorins) with multiple JH binding sites
• A 566kDa hexameric protein with 6 JH binding sites

• Drosophila JH is NOT required to prevent precocious development
• Inhibition of downstream TF BROAD (BR complex) is needed to achieve and adult moult

• JH titres decline in 3rd instar larvae, prior to the large E peak that triggers puparium formation
• Taking away JH but not inhibiting the BR complex means that metamorphosis to an adult still occurs

The major role of JH in insects is to modify the action of ecdysteroids and prevent the switch in the commitment of epidermal cells

JH influences the stage-specific expression of the genome that is initiated by ecdysteroids

JH acts via basic-helix-loop-helix PAS domain transcription factors METHOPRENE-TOLERANT (MET) and STEROID RECEPTOR COACTIVATOR (SRC)

Diagram:

a) Model for MET as a JH receptor in insects and JH signalling pathway during larval-pupal commitment

b) JHRE, JH response element

• MET/SRC stimulate juvenile hormones
• In final moult Broad needs to be released from inhibition