The Physiology of Metamorphosis

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The Physiology of Metamorphosis

• Caterpillar to Butterfly
• Tadpole to Frog

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General Introductory Comments

• Meta (change) morpho (form)
• Apoptosis a major event in metamorphosis
• A developmental program is effected through the
  expression of many genes, causing changes in
  phenotype
• Animals capable of metamorphosis are considered
  biphasic

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Terminology

• Holometabolous insect Orders having complete
  metamorphosis
• Instar the insect between molts
• Nymph immature, wingless stage of an insect
  without complete metamorphosis (naiad aquatic
  nymph, specifically aquatic nymph of
  hemimetabolous insects such as dragonflies,
  mayflies stoneflies)
• Larva immature insect with complete
  metamorphosis
• Pupa inactive intermediate stage of
  holometabolous insects between larva adult

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Types of Insect Metamorphosis

• Ametamorphosis no metamorphosis
• Gradual immatures are nymphs change in form
  is gradual (nymphs resemble adults share the
  same habitat) most obvious change involves
  development of external wing pads differences
  in color or markings e.g. grasshoppers, crickets
• Incomplete immatures are naiads immatures do
  not closely resemble adults naiads have tracheal
  gills transition to adults is gradual external
  wing pads develop in later instars nymphs
  adults dwell in different habitats e.g.
  dragonflies, damselflies, mayflies

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Types of Insect Metamorphosis

• Complete immatures are larva pupa larva do
  not resemble adults adult features develop
  during pupa stage immatures adults may or may
  not share habitats immatures are adapted for
  feeding adults are adapted for breeding
  dispersal e.g. butterflies, moths, beetles,
  flies, wasps, etc.

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Complete Metamorphosis Stages

• Egg larva pup adult
• Larva are dramatically different from adult in
  anatomy, habitat lifestyle they use different
  resources
• Wing development is internal from rudimentary
  integumental cells called imaginal discs
• Metamorphosis itself occurs during the dormant
  pupa stage

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Metamorphosis in Insects

• Although the detailed mechanisms of metamorphosis
  differ between species, the general pattern of
  hormone action is usually very similar
• The process of differentiation at the genetic
  level, different genes are turned on to allow for
  synthesis of proteins leading to different
  structures
• Appears to be regulated by effector hormones
  controlled by neurosecretory peptide hormones in
  the brain

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Molting - Prior to Metamorphosis

• Problem of increasing size with having an
  exoskeleton
• The molting process is initiated in the brain,
  where neurosecretory cells release
  prothoracicotropic hormone (PTTH) in response to
  neural, hormonal or environmental factors
• Starts with a cessation of feeding a clearing
  of gut contents
• Trigger for release may be related to stimulation
  of stretch receptors indicating growth to a
  certain size (?)

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Molting cont

• PTTH stimulates the production of ecdysone by the
  prothorasic gland (generally there are 2 of them
  in the thorax)
• Ecdysone is not an active hormone, but a
  prohormone that must be converted to active form
• Conversion is accomplished by heme-containing
  oxidase in the mitochondria microsomes of
  peripheral tissues
• Active form is 20-hydroxyecdysone

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Molting cont

• Each molt is occasioned by one or more pulses of
  20-hydroxyecdysone
• For a molt from a larva, 1st pulse produces a
  small rise in the hydroxyecdysone concentration
  in the larval hemolymph (blood) elicits a
  change in cellular commitment
• The second, large pulse of hydroxyecdysone
  initiates the differentiation events associated
  with molting

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Molting cont

• The hydroxyecdysone produced by these pulses
  commits stimulates the epidermal cells to
  synthesize enzymes that digest and recycle the
  components of the cuticle
• Acting together, PTTH ecdysone trigger every
  molt larva to larva larva to adult

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Molting cont

• The second major effector hormone in insect
  development is juvenile hormone (JH). JH is
  secreted by the corpora allata (located near the
  insect brain) it counteracts the tendency to
  undergo metamorphosis
• The secretory cells of the corpora allata are
  active during larval molts but are inactive
  during the metamorphic molt i.e. JH is
  responsible for preventing metamorphosis)
• In the last larval instar, 1. the medial nerve
  from the brain to the corpora allata inhibits the
  gland from producing JH 2. there is a
  simultaneous increase in the body's ability to
  degrade existing JH

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Molting cont

• Both these mechanisms cause JH levels to drop
  below a critical threshold value - this triggers
  the release of PTTH from the brain PTTH, in turn,
  stimulates the prothoracic glands to secrete a
  small amount of ecdysone
• The resulting hydroxyecdysone, in the absence of
  JH, commits the cells to pupal development
• Larval-specific mRNAs are not replaced, and new
  mRNAs are synthesized whose protein products
  inhibit the transcription of the larval messages.

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Metamorphosis in Insects

• After the second ecysone pulse, new
  pupal-specific gene products are synthesized
  the subsequent molt shifts the organism from
  larva to pupa
• It appears, then, that the first ecdysone pulse
  during the last larval instar triggers the
  processes that inactivate the larva-specific
  genes prepare the pupa-specific genes to be
  transcribed
• The second ecdysone pulse transcribes the
  pupa-specific genes initiates the molt

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Metamorphosis in Insects cont

• Overall, at each larval instar, there is a period
  where the presence of JH prevents the larval
  epidermis from transforming into pupal epidermis
  if JH is present, the epidermis continues to be
  larval, if JH is absent, it becomes pupal
• During the penultimate instar larva, JH titres
  are able to retain the epidermis in its larval
  condition
• During the last instar, there are two windows of
  JH sensitivity.

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Metamorphosis in Insects cont

• The first is for the epidermis - at this time,
  though, ecdysone levels have dropped
  significantly thus, the epidermis will be
  transformed from larval epidermis to pupal
  epidermis
• The second JH sensitive period concerns the
  imaginal disc tissue (destined to become site for
  wings other adult parts)
• At this time, however, the JH titre has risen
  again, so that the imaginal discs are not
  instructed to evert differentiate. The molt
  transforms the larva into a pupa

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Metamorphosis in Insects cont

• The next time the ecdysone pulses occur, no JH is
  seen during the critical periods - the epidermis
  transforms from pupal to adult the imaginal
  discs are allowed to evert differentiate (into
  wings eyes, antennae, legs
• As long as there is sufficient JH, ecdysone
  promotes larva-to-larva molts with lower amounts
  of JH, ecdysone promotes pupation complete
  absence of JH results in the formation of the
  adult (JH inhibits the genes that promote
  development of adult characteristics e.g. wings,
  reproductive organs, etc.)

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Conclusion

• If only ecdysone in its active form is present,
  the epidermal cells are programmed for death, the
  imaginal disc cells proliferate create adult
  structures the pupa becomes an adult (under
  gene control)
• Larval cells break down the nutrients used by
  imaginal discs to form adult parts
• In butterflies, the adult cuticle arises from the
  lysed tissue of the larval epidermal cells unlike
  other insects in which the adult cuticle arises
  from imaginal discs

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Diapause

• Many species of insects have evolved a strategy
  called diapause. Diapause is a suspension of
  development that can occur at the embryonic,
  larval, pupal, or adult stage, depending on the
  species. In some species, diapause is facultative
  and occurs only when induced by environmental
  conditions in other species the diapause period
  has become an obligatory part of the life cycle.
  The latter is often seen in temperate-zone
  insects, where diapause is induced by changes in
  the photoperiod (the relative lengths of day and
  night).

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And who-o-o-o-o are y-o-o-o-u?Caterpillar
(Larva) Physiology

• Anatomy physiology differs from adult
• Herbivorous larva has typical chewing mouth parts
  (mandibles) seen in other insects (eats
  vegetation) some caterpillars only eat very
  specific plants usually the plant their mother
  laid her eggs on e.g. Monarchs larvae eat only
  milkweed plants
• In many moth pupation occurs in a cocoon but most
  butterflies have no cocoon the pupa is a
  chrysalis ( associated with a few silk fibers)
• Larva has many appendages attached to the body
  over the entire length, (adult has the typical 6
  legs associated with most insects) head, thorax
  abdomen elongated body

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Caterpillar Physiology cont

• Larvas body filled with its long digestive tube
  (also has tracheal system) that quickly digests
  the vast quantities of food it eats
• Grows quickly must store sufficient energy to
  fuel molts its pupa stage
• Its primary function is digestion nourishment
• of cells of the larva does not increase as it
  grows these are not the cells of the adult
  caterpillar has imaginal discs within its body
  (clusters of cells destined to become adult body
  parts e.g. compound eyes)
• Coloration varies with species but generally it
  is suited to blend with its host plant more
  colorful larva are often poisonous

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Caterpillar Physiology cont

• Larva have various types of legs attached to
  their thorax are 3 pairs of jointed legs with
  hooks (use them to hold on to food) 5 pairs of
  stumpy prolegs on the abdomen which have little
  crochets (hooks) to hold onto the leaf/stem
  last pair of prolegs (anal prolegs which
  disappear in adult)
• Locomotion move in a ripple fashion contracting
  the muscles the rear segments pushing blood into
  the forward segments which lengthens the anterior
  part of the body legs hold on to the forward
  position then anterior muscles contract pulling
  the posterior segments forward
• Have 6 pairs of simple eyes (ocelli) that detect
  changes in light intensity but cannot form an
  image (composed of photoreceptors pigments)

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Caterpillar Physiology cont

• Sense touch through tiny hairs (setae) all over
  their body these tactile hairs grow through tiny
  holes in the pinnaculum (dark, flattened plates
  on exoskeleton) are innervated communicate
  with brain
• Spinneret is a tube-like structure on larvas
  lower labia that contains spinning apparatus
  (silk glands) larva draws silk from a tube in
  the spinneret used to support themselves spin
  cocoons
• Life span varies 2 weeks - 2 months for some
  species this may be the longest part of the life
  cycle vs. species like Monarch whose adult forms
  over-winter in Mexico/Texas but return to
  Southern Canada to reproduce in spring

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Butterfly Physiology - Overview

• Highly derived sucking mouthparts and consume a
  liquid diet (nectar)
• Mouthparts consist of large labial palps a
  coiled tubular proboscis derived form the
  maxillary gelae mandibles are absent
• Wings, body appendages covered with pigmented,
  dust-like epidermal scales or hair-like setae

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Butterfly Physiology cont

• Exoskeleton made of chitin provides body
  structure protection
• Spiracles on abdomen thorax orifice of
  tracheal system for gas exchange
• Long, tubular heart (dorsal vessel) hemocoel
  circulation for nourishment not O2
• Proboscis, pharynx, foregut (crop), mid-gut, hind
  gut anus digestion
• Fat body storage of energy

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Butterfly Physiology

• Malpighian tubules (long filaments which clean
  the hemolymph put the wastes into the hind gut
  excretion of wastes
• brain, ventral nerve cord, ganglia PNS
  visceral nerves functions in communication
  between cells with external environment
• Johnsons Organ (base of antennae) sense of
  balance (especially when flying)
• Ovaries testes reproduction

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Butterfly Physiology - Head

• Location of feeding sensory structures
• Nearly spherical in shape (houses its brain, 2
  compound eyes, point of attachment of its 2
  antennae, Johnsons organ the moustache-like
  sensory palps)
• No jaws sips liquid food (nectar or liquid from
  rotting fruits) through proboscis (tube-like
  flexible tongue which uncoils for feeding - coils
  into a spiral when not in use)
• Compound eyes contains many hexagonal
  lens/corneas which focus light from each part of
  the insects field of view onto a rhabdome
  (retina) optic nerve carries info to brain
  can also see UV light

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Butterfly Physiology Head cont

• Antennae sensory appendages used for the senses
  of smell balance butterflies have 2 segmented
  antennae with clubs at the end moths do not have
  the clubs Johnsons organ located at base of
  antennae sense of balance especially in flight
  as mentioned earlier
• Palps moustache-like scaly mouth parts on each
  side of proboscis which are covered with sensory
  hairs to determine if something is food

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Butterfly Physiology - Thorax

• Locus for Locomotion divided into 3 segments
  on each segment is a pair of jointed legs site
  of attachment for the 4 wings
• 6 segmented legs 2 front legs of many species
  are quite short frequently are used to clean
  the antennae each foot ends in a pair of
  grasping claws feet also studded with sensory
  organs are used to taste food
• Wings attached to 2nd 3rd thoracic segments
  (meso- meta-thorax) during flight the wings
  are held together because a lobe on the hind wing
  presses on the forewing

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Butterfly Physiology - Wings

• 4 wings (2 forewings 2 hind wings)
• Upon emerging from chrysalis, wings are crinkled,
  wet un-inflated butterfly hangs upside-down
  pumps hemocoel into wings via veins to inflate
  them wings must dry before flying damaged wings
  can not be repaired
• Made of 2 chitonous layers that are supported
  nourished by tubular veins ( some O2 exchange)
• Are covered by 1000s of colorful scales (which
  are tiny, overlapping pieces of chitin outgrowths
  of the body wall) many setae coloration
  pattern varies between the forewings hind
  scent scales (modified wing scales on forewing)
  release pheromones attracting females of same
  species

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Butterfly Physiology - Abdomen

• Relatively soft divided into 10 segments (7-8
  easily seen but several may be fused)
• Site of heart, Malpighian tubules, reproductive
  organs (claspers or ovipositors), many spiracles
  most of the digestive tract (foregut, hind gut
  rectum)

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Ribbit! Ribbit! Overview

• Amphibian Greek derived word meaning double
  life
• A complex process regulated by a number of
  external (environmental) internal (hormonal)
  processes
• Think of the physiological changes that must
  accompany the anatomical ones associated with the
  transition from tadpole to frog i.e. aquatic
  herbivorous grazer to terrestrial carnivore
  considerable alterations in body morphology,
  central peripheral nervous system structures,
  digestive system behaviors

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Overview cont

• E.g. moving from water to land, from being a
  swimmer with a tail to a hopper with legs
• E.g. central auditory system undergoes functional
  neuroanatomical reorganization in parallel with
  the development of new sound conduction pathways
  adapted for the detection of airborne sounds (it
  appears that just at metamorphic climax, there
  may be a brief deaf period in which no auditory
  activity can be evoked from the midbrain

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Environmental Influences

• Environmental factors include temperature, food
  levels, tadpole densities, pond evaporation rates
  predator presence such that increasing
  temperature, decreasing food, overcrowding, pond
  evaporation increase in predation all
  accelerate metamorphosis

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Environmental Influences cont

• Transition from larva to adult involves precisely
  controlled gene regulatory events that occur at
  specific stages during development

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Hormonal Influences Thyroid Hormone (T3)

• low during early larval development peak at
  metamorphic climax
• regulates a series of orchestrated developmental
  changes which ultimately result in the conversion
  of an aquatic herbivorous tadpole to a
  terrestrial carnivorous frog
• Causes the remodeling of nearly every organ in
  the body with respect to its morphology
  physiology
• stimulates jaw head restructuring, gills tail
  resorption, regeneration of the gut into the
  adult form triggers limb development de novo,
  change in eye position, loss of the oral disk,
  lateral line system degenerates

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Hormonal Influences Thyroid Hormone (T3)

• T3 ( steroids) also function by altering gene
  expression, therefore metamorphosis can be used
  to study gene regulation hormone-regulated gene
  expression
• T3 exerts its effects mainly through its
  receptor, the thyroid hormone receptor TR, which
  can repress or activate genes depending on the
  absence or presence of T3

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Hormonal Influences

• Corticosterone increases throughout larval
  development, peaking just prior to thyroid
  hormones at metamorphosis it may induce the
  degeneration of larval tissues such as the gut
  skin prior to regeneration stimulated by thyroid
  hormones
• Corticosterone is synergistic with thyroid
  hormone stimulates the conversion of thyroxin
  to the more active hormone triiodothyronine
• Prolactin levels are high during earlier larval
  stages, inhibit metamorphosis, decline at
  metamorphic climax

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Other Influences

• Transcellular active transport of Na across the
  skin appears to develop during the climax stages
  of metamorphosis in various species of amphibians
  this may be a marker of the development of
  adult-type features
• The more food the tadpole consumes ( they
  generally are voracious eaters), the faster the
  tadpole will grow metamorphose

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Relevant Life Cycle Points

• 6-21 days after being fertilized, egg hatches
• Shortly after hatching, tadpole still feeds on
  the remaining yolk while attached to a weed
  (prior to its swimming stage)
• 7-10 days later, it begins to swim eat algae
  (considered a algae grazer)
• 4 weeks later, gross morphological changes are
  noted e.g.skin appears to grow over gills,
  teeth begin to emerge helping to grate food a
  long, coiled gut is noticeable
• 6-9 weeks, legs begin to sprout, head
  becomes more distinct body elongates diet may
  include larger items like dead insects begins
  to lose its teeth, its mouth grow larger

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Life Cycle Points cont

• at 9 weeks, forelegs become evident
• By 12 weeks, tadpole has only a stubby tail
  soon it will leave the water only to return to
  lay eggs/reproduce as adults
• lungs have replaced gills,

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A Closer Look at the Changes in the Intestine as
an Example

• Long coiled gut has become shorter to accommodate
  dietary change from a grazing tadpole to a
  meat-eating frog small intestine is remodeled
  whereby tadpole epithelial cells undergo
  programmed cell death (apoptosis) are replaced
  by a layer of newly formed adult epithelium
  (research indicates that 2- thyroid
  hormone-regulated genes participate in this
  intestinal remodeling
• In the tadpole, the intestine is a long,
  tube-like structure composed of a single layer of
  epithelial cells with a single epithelial fold
  (typhlosole) where mesenchymal tissue is abundant
  during metamorphosis several dramatic changes
  occur within this organ

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Intestinal Changes cont

• First the mesenchymal component of the tissue
  begins to proliferate extensively this is soon
  followed by the cellular death of the larval
  epithelium concurrently, groups or islets of
  proliferating cells (unknown origin?) begin to
  form these islets are precursors of the adult
  frog epithelium which will eventually replace all
  of the larval epithelium as the transition
  proceeds, adult epithelial cells form a uniform
  layer which begins to migrate into the folds by
  end of metamorphosis have increased in height
  number to produce the final morphological
  structure of the adult intestine which is quite
  similar to that of higher vertebrates

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Intestinal Changes cont

• These intestinal changes occur as a result of
  several cellular processes
• Programmed cell death (apoptosis)
• Cell proliferation
• Cell differentiation
• And occur by 2 general mechanisms
• Cell autonomous events
• Extracellular events that occur by a non-cell
  autonomous event

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Cellular Processes cont

• It is thought during metamorphosis that the
  immune system is shut down to accommodate the
  changes which are related to the acquisition of
  new adult-specific molecules to which the
  tadpoles immune system must be tolerant the
  tadpole is very susceptible to disease at this
  time it may be that tadpoles avoid potentially
  destructive anti-self responses by largely
  discarding the larval immune system at
  metamorphosis and acquiring a new one

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Tadpole Physiology - Overview

• Respiratory System gills (tadpoles are confined
  to aquatic life)
• Young tadpoles are filter feeders algae other
  small plant material
• Osmoregulation suited for aquatic life
  excrete large amounts of dilute urine to balance
  water that enters their bodies from the external
  environment by osmosis
• Lateral line system (system of sense organs which
  often disappear in adults)
• Eyes on sides of head (move forward during
  metamorphosis)

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Frog Physiology - Overview

• Moist skin 2 layers (epidermis dermis) gas
  exchange especially when underwater (therefore,
  skin is heavily vascularized) ecdysis shed
  outer layer of skin not very permeable to water
• Lungs gas exchange when on land ventilate
  with mouths closed throat pulls air in through
  nostrils to lungs (generally developed before
  complete re-absorption of gills lungs help with
  buoyancy
• Teeth in upper jaw tongue attached at front of
  mouth (can be thrown out with amazing speed
  accuracy to grasp prey) mucous glands in mouth
  assist with prey capture

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Frog Physiology - Overview

• Jacobsons Organ blind sacs connected by ducts
  to nasal cavities NB sensory organ plays a
  role in prey recognition
• Diet insects, spiders, small fish, worms
  other tiny animals
• Tympanum (eardrum) just behind eye conducts
  sound directly to hearing structures
• Ectotherms
• Can detect chemical changes through specific
  molecules collected on their eyes skin

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Frog Physiology - Overview

• Eyes protruding from body near 3600 vision
  (needed as dont have very flexible necks
  usually have eyelids for living on land not
  needed in tadpole as its aquatic)
• Long, powerful hind legs jumping feet are
  webbed
• Osmoregulation adapted to terrestrial life
  strike regulation of water loss excretes urea
• Seasonal breeders spring early summer (many
  males have vocal sacs out-pouchings in mouth
  frog fills them with air for vocalization ears
  well developed for hearing sounds in air)
  return to water to lay eggs in clusters (spawn)

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Frog Physiology Overview

• Over-winter in mud at bottom of ponds or under
  moss where bodies remain damp they stay in a
  dormant state some frogs actually freeze
  (research into this for possible application in
  human cryopreservation at Carlton University)

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Quick Points

• Frogs may not stop growing after they reach
  maturity, thus grow throughout their lives
• Tadpoles Prozac with increasing frequency,
  various types of household, cosmetic
  pharmaceutical products are finding their way
  into the waterways that house hundreds of animals
  one laboratory study to determine potential
  impact of such contaminants in the water, put
  tadpole in water containing Prozac without much
  surprise, the Prozac had similar effects on
  tadpoles as human subjects depressant in this
  case it depressed retarded the timing
  sprouting of legs

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Quick Points cont

• Water temperature (e.g. warm) length of
  daylight (e.g. 12 hours) promotes metamorphosis
  to commence
• Increasing interest in using tadpoles in
  biomonitoring (biological indicator of water
  quality) tadpoles are the most sensitive stage
  of frogs life, thus if living in toxic water
  would be a good indicator of water quality