References used according to the letters given at the

1

References used according to the letters given at the bottom of the slides

A: Santer, RM, Walker, MG Emerson, L, Witthames, PR (1983) On the morphology of the heart ventricle in marine teleost fish (Teleostei), Comp. Biochem. Physiol. Vol. 76A, No. 3, pp. 453-457. B: Ostrander, GK (2000) The Laboratory Fish. Academic Press. C: Farrell, AP (1991) From Hagfish to Tuna: A Perspective on Cardiac Function. Fish Physiological Zoology, 64, (5), 1137-1164; URL: http://www.jstor.org/stable/30156237

D: Evans, HE, Clairborne, JB (2006) The Physiology of Fishes, Third edition. CRC, Taylor and Francis. Marine Biology Series. E: Root, RW (1931) The respiratory function of the blood of marine fishes. Biological Bulletin of the Marine Biology Laboratory, Woods Hole, 61, 427-457. F: Radull, J (2002) Investigations on the use of metabolic rate measurements to assess the stress response in juvenile spotted grunter, Pomadasys commersonnii (Haemulidae, Pisces). PhD thesis, Rhodes University. G: Kaunahama, K (2008) Effect of salinity on stress response of juvenile white steenbras, Lithognathus lithognathus. MSc thesis, Rhodes University

H: Jobling, M (1998) Fish bioenergetics. Chapman and Hall, London. I: An Overview of the Histological Study of Marine Finfish; http://research.myfwc.com/features J: Hontela and Stacey 1990. General Concepts of Seasonal Reproduction. In: Munro, A.D., Scott, A.P., Lam, T.J. (Eds.), Reproductive Seasonality in Teleosts: Environmental Influences. CRC, Press, Boca Raton, pp. 53–78. K: Arukwe, A, Goksøyr, A (2003) Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology, 2:4, 1-21. L: Hibiya, T (1982) An atlas of fish histology. Normal and Pathological Features. Gustav Fischer Verlag. M: Atlas of fathead minnow normal histology; http://aquaticpath.umd.edu/fhm/resp.html N: Fielder, DS, Allan GL, Pepperall, D, Pankhurst PM (2007) The effects of changes in salinity on osmoregulation and chloride cell morphology of juvenile Australian snapper, Pagrus auratus, Aquaculture 272: 656–666. O: de Jesus, EG, Toledo, JD, Simpas, MS (1998) Thyroid hormones promote early metamorphosis in grouper (Epinephelus coioides). General and Comparative Endocrinology, 112: 10-16.

Fish physiology

Horst Kaiser

Ichthyology II, 2012

2

Respiration

3

Life in water (1/2)

• Water is 840 times more dense and 60 times more viscous than air.

• Oxygen: – Air: 210 ml/L O2 at 21% partial pressure – Water: up 15 mg/L O2 (dep. on temperature) – Sea water holds 18% less O2 than freshwater

• Oxygen consumption in fish – 17 mg/kg/h @10°C – 100-500 mg/kg/h @ 30°C

• More than 40 genera of fishes breath oxygen using other methods than their gills.

4

Life in water (2/2)

• Energy demand to accomplish respiration:

– approx. 50% of total demand but can be up to 90%

• Blood volume: 2-4 mL / 100 g; (nucleated RBC)

• Gill surface area: 150–300 mm2 / g tissue

• Systolic blood pressure: about 44 mm Hg

• Gill irrigation: 5-20 L H2O / kg BM / h

• Opercular beat counts: 40-60 / minute

5

Henry’s law

VO2 = α PO2

VO2 = O2 concentration in ml/L (or mg/L)

α = solubility coefficient: the volume of O2

dissolved in water: ml O2/L/atm

PO2 = PO2 partial pressure (atm)

6

Metabolism of trout vs turtle

Trout Turtle

Oxygen requirement ~ 5 ml / min / kg ~ 5 ml / min / kg

Ventilation volume

600 ml H2O / min / kg

50 ml air / min / kg

Routine costs for ventilation 10 % 2 %

ref. D

Water flow

Blood flow

Blood flow

Water flow

Water flow

8

Gills and kidney: Osmoregulatory organs

9

PAIRED HOLOBRANCHS

Gill rakers

Four paired holobranchs

tongue

10

11

12

Decreasing oxygen level

Increasing oxygen level

Water interface at the lamella

Countercurrent exchange The difference in oxygen partial pressure PO2 between water and plasma strongly influences the uptake of oxygen at the gill lamella / water interface!

13

14

Hagfish gill pouch

Atlantic mackerel Scomber scombrus

Horse mackerel Trachurus trachurus

Oyster toadfish Opsanus tau

Eel, Anguilla japonica

Angler Lophius piscatorius

Skipkack tuna, Katsuwonus pelamis

16

0 200 400 600 800 1000 1200 1400 1600

Scomber scombrus

Trachurus trachurus

Katsuwonus pelamis

Anguilla anguilla

Lophius piscatorius

Opsanus tau

Notothenia rossi

Gobionotothen gibberifrons

Patagonotothen tesselata

Unit gill area: mm2 / g body mass

17

0 5 10 15 20 25 30 35 40 45

Scomber scombrus

Trachurus trachurus

Katsuwonus pelamis

Anguilla anguilla

Lophius piscatorius

Opsanus tau

Notothenia rossi

Gobionotothen gibberifrons

Patagonotothen tesselata

Number of gill lamellae / mm filament length

Model: R=a*Wb

R=0.56*W 0.679

0 2 4 6 8 10 12 14 16 18

Weight (g/fish)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

mg

O2

/ h

Oxygen consumption in grunter

Data are from John Radull’s thesis

Model: R=a*Wb

R=0.67*W -.042

0 2 4 6 8 10 12 14 16 18

Weight (g/fish)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

mg

O2

/g/h

Oxygen consumption in grunter

Data are from John Radull’s thesis

Possible explanations for metabolic rate changes with size

• Developmental changes of relative weights of different organs

– Liver and gills weigh relatively less

– Swimming musculature becomes more developed

• Metabolic intensities of different tissues may decline with increasing size (age)

• Differences between species

• Possible effect of test temperature

• Possible interactions between temperature and species

• Salinity?

Metabolic rate in fish

• Standard metabolic rate (SMR)

• Routine metabolic rate (RMR)

• Active metabolic rate (AMR)

• Metabolic scope (MS): MS = SMR – AMR

21

0.00

0.20

0.40

0.60

0.80

12:00 16:00 20:00 0:00 4:00 8:00

Hour of day

Ox

yg

en

co

ns

um

pti

on

(m

g.g

-1.h

-1)

AMR

SMR

22 ref. F

MS

POS

IS

Oxygen probe

Mechanical stirrer

Syringe

Water flow

Glass flask

Water flow

Intermittent respirometry

23 ref. G

Physiology of the stress response

An interesting and important relationship between the stress

response and respiration

27

The stress response

• Primary stress response: Perception of a stressor and initiation of physiological responses.

• Secondary stress response: Biochemical and

physiological changes (i.e.,: release of stress hormones).

• Tertiary stress response: Changes in metabolic rate. This may be followed by various other responses.

28

29

Endocrinology of the stress response

Sympathetic nerves

Chromaffin cells

Catecholamines

Hypothalamic factors

Pituitary gland

ACTH

(Adrenocorticotropic hormone)

Inter-renal cells

Cortisol

Brain / hypothalamus

Generalised change in adrenaline and cortisol concentration in fish plasma following a stressor

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Time (h)

Pla

sma

con

cen

trat

ion

(n

M)

30

Adrenaline

Cortisol

The main effects of elevated cortisol levels

• Initiates a switch from anabolism to catabolism

• Affects osmoregulation

• Very immunosuppressive

• Important effects on reproductive processes

31

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 50 100 150 200 250

Time after stress application (min)

Oxygen c

onsum

ption (

mg g

-1h

-1)

Ref.: Radull et al. 2000

The effect of handling stress on metabolic rate changes in spotted grunter

32

0

0.2

0.4

0.6

0.8

1

1.2

02468

Dissolved oxygen concentration (mg L-1

)

Ox

yg

en

co

ns

um

pti

on

(m

g g

-1h

-1)

33 ref. F

Respiration

Gas exchange

34

Respiration

Ventilation

O2 diffusion

O2 dissolves into plasma

O2 binds to haemoglobin

in RBCs

O2 diffuses across

capillary walls &

cytoplasm to mitochondria

Once O2 has been

offloaded CO2 diffuses into the RBC

CO2 is transported to gills and

excreted

35

• Fish have nucleated red blood cells! • Some species have more than 1 type of Hb • Each protein molecule has 4 globin subunits each

with one haem group to bind O2

• Hb exists in 2 states, a tense (T) state with low affinity to O2, and a relaxed state (R) with high affinity.

• A shift from T -> R increases O2 binding capacity • The four units cooperate to increase O2-uptake • What changes the state from T to R?

Hemoglobin (Hb)

36

Blood and serum Cap Capillary glass tube filled with blood

Spin tube at 13 g

Plasma / Serum Red blood cells / packed cell volume

Cap

Small volume of lymph

37

Oxygen carrying capacity depends on …

• Number of red blood cells

• Concentration of haemoglobin within the red blood cells

• Oxygen binding properties of haemoglobin

• Partial pressure of oxygen

38

Where do red blood cells come from?

39

Spinal cordSpinal cord

Haemopoietic granular cell precursorsHaemopoietic granular cell precursors

Haemopoietic sinusHaemopoietic sinus

Dorsal aortaDorsal aorta

KidneyKidney

Haemopoietic foramenHaemopoietic foramen

Muscle fibersMuscle fibers

Vertebral BoneVertebral Bone

Bone marrowBone marrow

HaemodinamicHaemodinamic organ in dorsal aortaorgan in dorsal aorta

HAEMOPOIETIC ORGAN IN HAEMOPOIETIC ORGAN IN

RAINBOWRAINBOW TROUTTROUT

Carbon dioxide transport – the chemistry of CO2 in water

CO2 H2O H2CO3 HCO3- H+ + +

Carbon dioxide

Carbonic acid

Bicarbonate ion

44

Extracellular

Carbonic anhydrase speeds up carbon dioxide dissociation in the cell

CO2 H2O H2CO3 HCO3- H+ + +

CO2 H2O H2CO3 HCO3- H+ + +

Carbonic anhydrase

Red blood cell

Slow reaction

Fast reaction

45

At the tissues

CO2 H2O H2CO3 HCO3- H+ + +

Carbonic anhydrase Fast reaction

Drop in pH !

Hemoglobin changes state and releases O2 O2 diffuses to plasma

and tissues

HCO3- transport to plasma

in exchange for Cl-

46

At the gills

CO2 H2O H2CO3 +

Carbonic anhydrase Fast reaction

Release of CO2 increases pH

Hemoglobin changes state and is ready to pick up O2 at the gills.

CO2 diffusion to plasma and then to the water

HCO3- H+ +

HCO3- transport to blood

cell in exchange for Cl-

47

O2 CO2

Gill interface Blood stream

pH

O2 to organs

Excess CO2 through respiration, and in water

Drop in blood pH (lactic acid, stress, excessive swimming)

Reduced opercular movement -> impaired CO2 excretion

Hyper- capnia

Hyper- lacticaemia

Increased Bohr and root effect

O2 CO2

CO2

pH

48

Partial pressure of oxygen (PO2)

Perc

en

t sa

tura

tio

n o

f H

b w

ith

O2

higher pH (gills)

lower pH (tissues)

Bohr effect

Exponential increase due to Hb subunit cooperation Dissociation

curves parallel

Reduced affinity under acidic conditions 49 ref. B

Partial pressure of oxygen (PO2)

Perc

en

t sa

tura

tio

n o

f H

b w

ith

O2

Bohr effect – species differences

Reduced affinity under acidic conditions 50 ref. B

Sessile species with high Hb-affinity to oxygen Can cope with low-oxygen conditions

Active species with low Hb-affinity to oxygen require high oxygen levels to survive.

0

2

4

6

8

10

12

14

16

18

0 500 1000 1500 2000 2500 3000

Pe

rce

nta

ge o

xyge

n in

blo

od

Red blood cells / ml (*1000)

Toadfish

Goosefish Puffer fish

Sculpin

Sea robin

Mackerel

51 ref. E

Root effect

52

Sea robin (Prionotus spp) Toadfish (Opsanus spp)

Mackerel (Scomber scombrus)

Why is the Root effect unique to fish?

• Oxygen supply to the retina

• Gas bladder function

53

Release of gas from the gas bladder

• Physostomous fishes

– Pneumatic duct to the gut in most but not all species

• Physoclistous fishes

– Closed gas bladder

– Release gas into the blood (specialised oval area)

– Excess gas is carried to the gills

54

The rete counter-current system Example: gas bladder rete in eel • Cross section: 5 mm2 • Volume: 21 mm3

• Surface area: 30 cm2

• Capillaries: 20.000 to 40.000 • Artery diameter: 9 - 10 µm • Venous capillary: 11 – 13 µm • Diffusion distance (capillaries): 1 µm • Capillary length: 4 mm • Holes in capillary: 20 – 80 nm • Hole diaphragm: 5 nm

Eye

Gas bladder

55 ref. B

PCO2 / PO2 Lactate pH Lactate pH

Lactate pH Lactate pH

PCO2 / PO2

PCO2 / PO2 PCO2 / PO2

Pre-rete Arterie

Post-rete Vein

Gas gland

epithelium

Secretory bladder

Bladder caps

Post-rete Arterie

Pre-rete Vein

Rete mirabile: Changes of gas content, pH and lactate in the capillaries

Rete mirabile

Three processes: • Bohr / Root shift • Salting out • Counter-current diffusion

O2

CO2

56 ref. D

57

Partial pressure of oxygen (PO2)

Perc

en

t sa

tura

tio

n o

f H

b w

ith

O2

higher pH (gills)

much lower pH (retina / swimbladder)

Root effect

Subunit cooperation Some

subunits fail to load O2

O2 Saturation is not reached even if sufficient O2 available!

Decreased capacity under acidic conditions

Ice fish • White gills • Almost transparent blood • No haemoglobin

Channichthyids 58

Colourless blood

Bulbus arteriosus Ventricle

Liver

An interesting case: respiration in icefishes

• Some species, i.e., Nototheniidae, do not have haemoglobin

• Many fish take up oxygen across the skin (up to 35%) – do icefish employ this strategy?

• Icefish gill morphology does not differ much from that of most other fishes

• Surface are is similar to that of other species

• In this case, which factors determine O2 uptake?

59

We observe

• Gill histology in ice fishes does not contribute to oxygen uptake.

So how do they do it?

60

The Fick principle

• O2-consumption = Vg x [O2] x % extraction

– O2-consumption = mg O2 /kg / h

– Vg = Gill water flow in ml O2 / kg / h

– [O2] = O2 (mg/L) in water pumped over gills

If % extraction is low (= no haemoglobin carrier!), Vg

should increase. Vg in ice fishes is relatively high.

However, this is only part of the explanation.

61

The Fick principle

• O2-consumption = Q x [(A-V) x O2] – O2-consumption = mg O2 /kg / h

– Q = Cardiac output (ml / kg / h)

– A and V = O2 (mg/L) in Arterial and Venal blood

Ice fishes without haemoglobin dissolve O2 in

plasma, thus, they have a low oxygen-carrying

capacity.

Prediction: Q should be relatively high. Data show a 7-

fold increase in Q relative to many other species.

62

63

STEEN and BERG, Comp. Biochem. Physiol., 1966, Vol. 18, pp. 517 to 526

Adaptations in ice fishes

• Relatively high cardiac output / large hearts

• Relatively high blood flow to the gills

• Modification of secondary gill lamellae to increase diameter

• Lower blood pressure

• Ice fishes make use of perfusion in addition to diffusion to pick up O2.

64

The circulatory system

65

Gills Organs

Organs

Organs

Lungs / Skin

Lungs

Fish (teleost)

Amphibian

Mammal

66

Heart

67

68 From: http://accessscience.com

African lungfish, Protopterus. The blood is directed to the dorsal aorta or lungs depending on whether the fish is breathing in air or water. (After D. Randall et al., eds., Eckert Animal Physiology, 4th ed., W. H. Freeman, New York, 1997)

Gills

Fish (teleost)

Liver

Intestine

Kidney

Caudal region

Head, anterior region

Ret

urn

to

hea

rt

69

Organs Gills

Fish (teleost)

70

Heart

Fish (teleost with ABO – generalised) – Colours resemble adaptations

Gills

Organs

Heart

ABO

ABO = air breathing organ

Unique features of the heart in fish • Cells of the heart muscle receive poorly oxygenated venous blood (V). • Single ventricle produces low pressure (<40 mm Hg) • Lack of coronary circulation • Relatively high ventricular stroke volume • No valves at the venous inflow • Pacemaker

V A

71

Bulbus arteriosus

Ventricle

Atrium

ref. A

Bulbus arteriosus • Thick elastic walls, smooth muscle • Stores up to 100% of stroke volume to control pulse pressure

Ventricle • Pumps blood to bulbus arteriosus • Pumped volume = stroke volume (in fish)

Blood circulation in the head of trout Gray: pre-branchial arteries (afferent) Red: post-branchial arteries (efferent)

72

Bulbus arteriosus

Ventricle

Atrium

ref. A, B

Type trabecular heart of an air breathing catfish

• A = Atrium • V = Ventricle • B = Bulbus arteriosus

White arrow = direction of blood flow

Trabeculae Spongy myocardium of the ventricle

Longitudinal trabeculae (mechanical strength)

Entrance from the sinous venosous

1 mm

valved junction

73 ref. B

• Cardiac myocytes * • Fibres (arrow)

• Myocardial fibres in the spongy tissue layer • Organised in fascicles • Fascicles form branching lacunae • Large surface area in fish due to lack of blood supply

Compact myocardium of active fishes

BA = Bulbus arteriosus AV = Atrioventricular valve BV = Bulboventricular ring (a)= outer layer (b)= inner layer

74 ref. B

The mysterious secondary circulatory system in fishes

• A system of very small capillaries branching ( ) off mostly from efferent filamental gill arteries (EF)

• Secondary arterioles (S) are too small to transport red blood cells

• Volume of blood can make up to 20% of total blood volume.

• Blood flow under hormonal control • Function unclear

– Lymph precursor? – Osmoregulation? – Pool of reserve blood? – Some nutrient supply?

40 µm

76 ref. C

Blood flow – the basic definitions

• F = Flow [ml / min]

• Pa = Arterial pressure [mm Hg]

• Pv = Venous pressure [mm Hg]; nb: Pv ≈ 0

• δP = Pa – Pv

• R = Vascular resistance [mm Hg / ml]

• η = Viscosity [Pascal sec]

• L = Length of the blood vessel [mm]

L

PrF

8

4

78

0

20

40

60

80

100

120

140

0 2 4 6 8 10V = ml O2 / kg / min

Q =

ml /

kg

/ m

in

Hagfish (10 – 15 °C)

Lincod (10 °C)

Rainbow trout (11°C)

Leopard shark (20°C)

Dogfish (20°C)

Yellowfin tuna (26°C)

Skipjack tuna (26°C)

Oxygen consumption (V) and cardiac output (Q)

Oxygen consumption V(O2) = Q (Pa O2 – Pv O2) Pa O2 = O2 of arterial blood Pv O2 = O2 of venous blood 79 ref. C

80

Arterial blood pressure

Cardiac output (Q)

Stroke volume

Heart rate Vascular

resistance (R)

•Length of blood vessels • Hormonal control • Vessel radius

• Life history (10 – 300 ml/min/kg) • Arterial O2 level -> high Q in ice fishes • Exercise, size, age

• Temperature • Activity • O2 demand • O2 availability • Species • Metabolic rate

To increase Q most fish species adjust stroke volume more than heart rate (except tunas)

ref. D

81

Stroke volume

Ventricular strength

End-diastolic volume

Arterial blood pressure

(“afterload”)

• Energy of contraction is a function of muscle fibre length. • A fish heart can pump almost its own volume • Greater ventricular filling = greater stroke volume

• Operational pressure (mm Hg) is species specific

• Filling time • Filling pressure

ref. D

82

Arterial blood pressure

Cardiac output (Q)

Stroke volume (SV)

Ventricular strength

End-diastolic volume

Arterial blood pressure

Heart rate (HR)

Vascular resistance (R)

Examples

Q (ml/min/kg) HR (beats/min) SV (ml/kg)

Carp, resting, 6° C 9 7 1.22

Carp, resting, 15° C 15 17 0.77

Trout, resting, 11 °C 17.6 38 0.46

Dogshark, resting, 19 °C (2.8 kg) 52.5 43 1.21

Flounder, resting, 10 °C 16 34 0.50

Sturgeon, resting, 19 °C 36 48 0.83

Tuna, 26 °C; 1.4 kg 115 97 1.3

ref. D

Endocrinology

83

Endocrinology

• Objectives of the following lectures

–A comparative overview of hormones in fishes

–Reproductive endocrinology • The effect of pollutants on fish

endocrinology

–Endocrinology of osmoregulation

84

Why do we study endocrinology?

• To understand fish physiology

• To manage fish health

• To understand and manipulate reproduction

• To evaluate the effect of pollutants on fish reproduction

• And more …

85

Glands

Exocrine Endocrine

Discharge of excretory products through ducts

No ducts

Examples: Intestinal glands Sweat glands

Richly vascularised

Many cytoplasmatic organelles

Interact with receptor cells

86

Endocrine system

• Chemical communication and regulation of ... – Development

• Growth and maintenance

• Larval development

• Energy availability

– Osmoregulation

– Reproduction

– Stress response

– Digestion

– Behaviour

87

Hypo- thalamus Pituitary

Pineal gland

Head kidney chromaffin cells

Kidney Corpuscles of Stannius

Urophysis

Gonads

Pancreatic tissue Gastrointestinal peptides

Heart

Thyroid gland

88

Hormones as messengers • Endocrine

– Affect distant parts in the body – Transported via the circulating blood

• Paracrine – Local effects / diffusion

• Pheromonal – Secretion into the environment

• Autocrine – Work in the cell in which they are produced

89

Receptors • These are large protein or

glycoprotein molecules.

• Located at the target cells.

• They are constantly broken down or synthesised.

• Quantity affected by hormone levels.

• Down or up-regulation by hormones.

90

Classification of hormones

• Steroids / steroid-related hormones

• Tryrosine derivates

• Hormones with a peptide and glycopeptide structure

91

Steroids / steroid-related hormones

• Lipid soluble.

• All steroids are cholesterol derivates. 4-ring structure.

• Producing organs: Mainly gonads, but in fish also adrenal cortex.

92

Steroid hormone

Cell membrane

Receptor protein

Nuclear membrane

Induction of mRNA production

Protein synthesis

Cell response 93 ref. H

Peptide hormone

Receptor AC

Cell membrane

ATP cAMP

Activation Protein Kinase Protein Kinase

Protein Cell response

AC = Adenylate cyclase cAMP = cyclic AMP PD = Phosphodiesterase

PD

94 ref. H

95 ref. B

Endocrinology of reproduction

Maturation and ovulation of oocytes

96

97 ref. B,

An Overview of the Histological Study of Marine Finfish; Fish and Wildlife Research Institute, Florida, USA;

98 ref. I

99 ref. I

100 ref. I

a

Yolk granulesGVBDo

Oil globule

GV

b

c d

GVBDoGV

GVGV

Histological sections of GnRHa-induced wild-caught L. niloticus broodstock under-going final oocyte maturation; a-b) early oocyte maturation (centre GV); c) oocyte with fully formed oil globule with periphery GV; d) oocytes undergoing germinal vesicle breakdown, GV= germinal vesicle, black arrow head = multiple nucleoli, GVBDo = germinal yolk vesicle break-down oocyte

102 ref. I ref. I

Examples for predictive factors

• Changes in day length

• Slow changes in temperature

• Other factors in the environment depending on the stages of gonadal development

103

Modifying factors

• Water quality

• Lunar cycle

• Broodstock nutrition

• Social interactions

104

Synchronising cues

• Water quality changes

• Floods

• Rapid temperature changes

• Atmospheric changes

105

* *

* * *

Go

nad

al m

ass

/ o

ocy

te d

iam

eter

Winter Spring Summer Fall

Patterns of oogenesis

carp

tench

Various barbus spp.

106

Hypothalamus

Endocrine gland

Anterior pituitary

Target tissue

Tropic hormone

End hormone

Metabolite(s) Releasing hormone

Adopted from Jobling 1998 107

108

109

110

Hypothalamus

Pituitary

Liver

Gonads

(Ovaries)

GnRH = Gonadotropin-Releasing Hormone

GnRH Inhibitor

GtRH Inhibitor

Antagonist (Domperidone; Pimozide)

Testosterone

(ie.: 11-α-Keto Testosterone)

Aromatase

GtH = Gonadotropin Hormone

Estrogen (ie., 17-ß Estradiol)

Vitellogenin

GtH 1 GtH 2

GnRH

+

- -

- + Vitellogenesis

Feedback

Secretion

Moderating effect

Conversion

Summary of oocyte maturation / ovulation

111

Case example 1: Goldfish (Carassius auratus)

• Environmental cues

• Behavioural cues

• Endocrinological control

• Pheromonal control

112

Aspects of goldfish reproduction

• Pre-ovulatory waiting phase

• Influence of a diurnal rhythm

• Spawning follows ovulation very tightly

• Three cues for initiation of ovulation:

– Vegetation

– Temperature

– Presence of males

• But scotophase is an overriding factor

113

114 ref. J

12:00 04:00 12:00

12:00 20:00 04:00 12:00

Female

Male

GtH

17,20P Ovul (PG) courtship

17,20P PIP

17,20P milt courtship

spawning

synchronicity

20:00

PG=Prostaglandin

PIP=PG-induced pheromone

115 ref. J

Case example: Masu salmon

• Migrational cues

• Behavioural cues

• Endocrinological control

• Sneaker behaviour

116

Masu salmon: two reproductive strategies in males

117

Fresh water Fresh water Sea water Fresh water

Fresh water Fresh water Fresh water Fresh water

Salmon parr: precocious sneakers

Salmon parr: migratory form

Masu salmon: reproductive behaviour

118

L-kynurenine

1

2

3

4

Testosterone -> upstream migration in females Testosterone -> nest digging behaviour in females

Precocious male (sneaker)

1

4

Testosterone -> induces upstream migration in males, including precocious sneakers

5

Osmoregulation

120

iii CnLOsmol /

• λ = Osmotic coefficient (ranges from 0 -1)

• n = Number of particles (ions) into which a molecule dissociates (example: NaCl = 2; glucose = 1)

• C = the molar concentration of the solute

• i = The index i represents the identity of a particular solute

121

Gills and kidney: Osmoregulatory organs

122

H2O

300 mOsm

1 mOsm

Ingest

Freshwater

Seawater

High volume urine Low Na+, Cl-

400 mOsm

Filtration

Filtration H2O

1000 mOsm

Na+, Cl-, H2O

Na+, Cl-

Na+, Cl-

Na+, Cl-

Na+, Cl-

Na+, Cl- Passive Active

Low volume urine Isotonic Na+, Cl- 123

H2O

Ingest / drink

Seawater

400 mOsm

Filtration

1000 mOsm

Na+, Cl-, H2O

Na+, Cl-

1. Plasma hyposmotic to seawater; approximately 60% lower 2. Constantly dehydrated and loaded with salt 3. Up to 90% of Na+ and Cl- is removed in the intestine 4. Dehydration forces organism to excrete small volume of urine 5. Urine is isosmotic to plasma and seawater; not hyperosmotic 6. There is active extra-renal excretion of salts via the gills!

Low volume urine Isotonic Na+, Cl-

Na+, Cl-

124

300 mOsm

1 mOsm Freshwater

High volume urine Low Na+, Cl-

Filtration H2O

Na+, Cl-

Na+, Cl-

Na+, Cl-

1. Plasma hyperosmotic to freshwater 2. Over-hydrated and salt-depleted 3. Large volumes of dilute urine 4. Take up Cl- from the environment in exchange for HCO3

-

5. Take up Na+ in exchange for ammonia (NH4+)

HCO3-

NH4+

125

0

0.5

1

1.5

2

2.5

DW 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00Hard fresh water

Osmoidentity

Osmoconformer (marine)

Stenohaline (marine)

Euryhaline (teleost, marine type, Fundulus heteroclitus)

Euryhaline (teleost) Freshwater type O. mossambicus

Stenohaline (freshwater)

Euryhaline (elasmobranch) (high urea levels)

Hyper-osmoregulation

Hypo-osmoregulation

Soft fresh water

Blo

od

osm

ola

lity

(Osm

/kg)

Environment osmolality (Osm/kg)

Osmoregulatory capabilities of major fish groups

Seawater

126

Hagfishes

0

100

200

300

400

500

600

Na+

Cl-

K+ x 10

0

200

400

600

800

1000

1200O

smo

lalit

y Osmolality (mOsm)

Seawater Freshwater

mM

/L

127 ref. D

Active transport?

Where and how?

Form and function of the chloride cell

128

1. primary lamella; 2. extracellular cartilaginous matrix; 3. chondrocytes; 4. secondary lamella; 5. epithelial cell; 6. mucous cell; 7. chloride cell; 8. pillar cell; 9. lacuna (capillary lumen); 10. red blood cells within lacuna.

129 ref. M

130 ref. B

Salinity tolerance of Australian snapper - a case study

Background and rationale

• Transport of euryhaline fish from FW -> SW causes proliferation of chloride cells

• Aquaculture of marine species – Often coastal sites / environmental fluctuations

– Lack of information on rapid salinity change on chloride cell morphology

• Australian snapper life history includes estuaries

131

Salinity tolerance of Australian snapper - a case study

Experimental design

• Assess the effect of fast salinity changes

– SW -> 15 ppt

– SW -> 45 ppt

• Measure

– Blood hematocrit

– Blood serum chemistry

– Morphology of chloride cells

132 ref. N

Gill sections 168 h after transfer

A: 30 -> 30 ppt B: 30 -> 15 ppt C: 30 -> 45 ppt

133 ref. N

134 ref. N

Salinity tolerance of Australian snapper - a case study

Conclusions

• The species shows good tolerance to fast changes in salinity

• Serum osmolality for Na+ and Cl- increased (15 -> 45 ppt) and decreased within 24 h and was restored

• Lamellar chloride cells also play a role in homeostasis

135 ref. N

White steenbras: Response to fast and slow changes in salinity

• The design

– Fast and slow change from 35 ppt to 5 ppt and 25ppt

• Gradual decrease: 3 – 9 h (depending on final value) and measurements after 2 days

• Fast decrease: Change was effected within 10 minutes.

– Oxygen consumption using intermittent respirometry

136 ref. G

1 2 3 4 5 6

Measurement

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09O

xyg

en

co

nsu

mp

tio

n (

mg

g-1

h-1

) slow change to 5 ‰

slow change to 25 ‰

fast change to 5 ‰

fast change to 25 ‰

no change; 35 ‰

in 20-min intervals 137 ref. G

Na+, K+-ATPase

• Integral membrane protein

• Assists in translocating Na+ and K+-ions across the cell membrane

• Transport produces a chemical and electrical gradient across the membrane

• Found in chloride cells, intestinal and renal cells

138

Na+, K+-ATPase 1. Keeps intracellular Na+ low and K+ high

Na+, K+, 2Cl- co-transporter 1. Accumulates Cl- into the cell 2. Excess Cl- can exit via anion channel

K+ - correcting channel 1. Channels extra K+ into the blood

Extrusion of chloride via channel

500 mM NaCl 0 mV

150 mM NaCl + 35-40 mV

Accessory cell Chloride cell

Saltwater

139 ref. D

How do freshwater fish take up ions?

• FW: large gradient for both Na+ and Cl-

• Na+ and Cl- are taken up independently of each other

• The enzymes involved are:

– H+-ATPase: Assists in exchange of H+ for Na+ (uptake)

– Na+, K+-ATPase: Assists in moving Na+ into blood

– Carbonic anhydrase: Generates HCO3- to be

exchanged for Cl-

140

Freshwater Fr

esh

wat

er

Bo

dy

flu

ids

H+-ATPase

Na+, K+-ATPase Carbonic anhydrase

Carbonic anhydrase 141 ref. D