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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