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Analyses of defense morph forma0on of predator-‐induced polyphenism in
Daphnia pulex捕食者が誘導する表現型可塑性を示すミジンコ(Daphnia pulex)の
防御形態形成プロセスの解析
Department of Natural History Sciences,Faculty of Science
Yuka Naraki
1
Evolu&onary developmental biology(Evo-‐Devo)
How does development influence phenotypic varia&on?How are developmental processes modified in evolu&on?発生過程に生じた変化が形態進化の原動力となる?
PhenotypeGene expression
Animal DevelopmentalChange
Evolu&on
2
Phenotypic plas&city(表現型可塑性)Environmental regula&on of development
PhenotypeGene expression
Animal
Environment A
Environment B
A study model to get insights into evolu&onary processes in Evo-‐Devo.
3
Predator-‐induced polyphenism捕食者に誘導される表現型可塑性
The phenotypic altera&on by modula&ng developmental processes in the presence of predators.
Sco$ F. Gilbert, Developmental Biology 6th Edi=on
Typicalmorph
Predator-‐inducedmorph
Ro?fer Barnacle Bryozoan mollusk Carp
4
Water fleas-‐-‐Daphnia(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata
5
Water fleas-‐-‐Daphnia(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata
predator-‐induced polyphenism
6
Water fleas-‐-‐Daphnia(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata Daphnia carinataDaphnia retrocrva
Daphnia pulexDaphnia ambiguaDaphnia
longicephalaDaphnialumhotzi
predator-‐induced polyphenism
7
Daphnia pulex(ミジンコ)
• Cosmopolitan
• Field ecology
• Breeding in laboratory
• Genome sequenced (2011)
•Molecular techniques 0.5 mm
8
Inducible defenses of Daphnia pulex捕食者によって誘導されるミジンコの防御反応
• Neckteeth (Chaoborus)
• Tail spine (Chaoborus, Notonecta, Lepomis)
• Body width (Chaoborus)
• Body depth (Chaoborus)
• Egg size and number (Chaoborus)
• Behavioral defenses (Fish)
Daphnia pulex(ミジンコ)
9
Predator-‐induced polyphenism in Daphnia pulex
10
Predator-‐induced polyphenism in Daphnia pulex
Predator(Chaoborus larvae)フサカ幼生
11
Predator-‐induced polyphenism in Daphnia pulex
Kairomone
Predator(Chaoborus larvae)フサカ幼生
12
The molecular structure and the ac&on mechanism of the Chaoborus kairomone are unknown.
Kairomone: Chemical or mixture of chemicals, released into the environment by an organism, that induce reac&ons in another species in a way that the recipient rather than the emiOer receives benefit.
13
Predator-‐induced polyphenism in Daphnia pulex
Neckteeth (Necktooth)
14
Predatory ac&vity Chaoborus larvae
Typical morph (1.0-‐1.3 mm)
15
Predatory ac&vity Chaoborus larvae
Typical morph (1.0-‐1.3 mm)
15
Predatory ac&vity Chaoborus larvae
Neckeeth morph (1.0-‐1.3 mm)
16
Predatory ac&vity Chaoborus larvae
Neckeeth morph (1.0-‐1.3 mm)
16
Analysis Daphnia pulex as a model organism in Evo-‐Devo
In this thesis
17
How do environmental cues modify the developmental
process?
In this thesis
18
Contents
CHAPTER1Daphnia pulex as a model organism for the study of predator-‐induced polyphenism
CHAPTER2Iden?fica?on of the kairomone-‐sensi?ve period and the histology of neckteeth forma?on in predator-‐induced polyphenism in Daphnia pulex
19
Contents
CHAPTER1Daphnia pulex as a model organism for the study of predator-‐induced polyphenism
CHAPTER2Iden?fica?on of the kairomone-‐sensi?ve period and the histology of neckteeth forma?on in predator-‐induced polyphenism in Daphnia pulex
20
Animals
From a pond at the Na=onal Ins=tute for Environmental Studies, Tsukuba
From a pond in the Flowering tree garden on Hokkaido University campus
Daphnia pulex Chaoborus flavicans larvae
0.5 mm
5 mm
A single clone was used throughout this study.
21
• Animals were maintained in dechlorinated tap water at 18°C
• Under ar&ficial light condi&ons of 14 h light and 10 h dark
• Concentrated monoculture of the green alga Chlamydomonas reinhard<i
Daphnia pulex rearing
22
Life cycle of Daphnia pulex
Hiruta and Tochinai (2012), Meiosis
23
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Oviposi?on
24
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Oviposi?on
st.1 st.2 st.3 st.4
Ecdysis
25
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Oviposi?on
0 h 10 h 20 h 30 h 40 hst.1 st.2 st.3 st.4
Hatching fromegg chorion(卵膜)
26
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Oviposi?on
0 h 10 h 20 h 30 h 40 hst.1 st.2 st.3 st.4
Hatching fromegg chorion(卵膜)
Exfoliate two-‐layered membrane
27
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Oviposi?on Discharge
0 h 10 h 20 h 30 h 40 hst.1 st.2 st.3 st.4
Hatching fromegg chorion(卵膜)
Exfoliate two-‐layered membrane
28
Time course of development
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
Embryonic stagein the brood chamber(育房)
Postembryonic instar
Oviposi?on Discharge
0 h 10 h 20 h 30 h 40 h 50 h 60 h 70 h 80 hst.1 st.2 st.3 st.4 1st 2nd
Hatching fromegg chorion(卵膜)
Exfoliate two-‐layered membrane
Birth and ecdysis Ecdysis
29
• Does kairomone act directly on embryos to induce neckteeth?
• Do the picking embryos from dissected maternal brood chambers affect normal development?
For the analyses of defense morph formation
Kairomone
?
?
30
Embryonic development outside of brood chamber
Embryos picked frombrood chamber
31
Normal development
Embryos picked frombrood chamber
Embryonic development outside of brood chamber
32
kairomone medium
Direct exposure of embryos to kairomone
33
Kairomone medium
• 1–5 Chaoborus larvae/100 ml for 1 week
• 18°C; 14 h light and 10 h dark
• Daily sufficient D. pulex feeding
• Passed through a 1.2 μm filter
• Dispensed into a 15 ml or 50 ml conical tube and stored at -‐20°C
34
Direct exposure of embryos to kairomone
Neckteeth induc?on
kairomone medium
35
Neckteeth induc?on
Defense morph induc0on system
kairomone medium
Direct exposure of embryos to kairomone
36
Neckteeth: The dorsal carapace ridge between the inser&on points of the first and second antennal muscles transformed into &ny spikes.
Control
Kairomone
1st instar 2nd instar
SEM
37
1st 2nd 3rd 4th0
20
40
60
80
100
0
1
2
3
4
ControlKairomoneNumber of neckteeth
Induction(%)
Number of neckteeth
Instar
*
**
**
(152) (147) (145) (145) (115) (129) (40) (78)
Neckteeth: The second-‐instar stage showed the highest frequency, and neckteeth was completely absent in adults.
38
Predatory ac&vity Chaoborus larvaeNeckteeth morph has the greater escape efficiency in the 1.0-‐1.3 mm body size.
Typical morph Neckteeth morphBody lengthExperimentalgroup
0.8 mm
0.9 mm
1.0-1.3 mm
1
2
3
4
5
6
7
8
9
10
11
preyescape
39
Concentration effect of kairomone mediumst.1 st.2 st.3 st.4 1st 2nd
Kairomonetreatment
Indu
ctio
n (%
)
Strength of kairomone medium (%)
0/18
3/26
10/13
10/15
12/15
16/16
22/28
10/1037/40
37/37
0 10 20 25 30 35 40 45 50 1000
20
40
100
60
80
40
Conclusion of CHAPTER1
• Neckteeth offer protec=ve effects against Chaoborus because of neckteeth interfere with the predator’s ability to handle and manipulate the prey.
• Kairomone directed directly at the D.pulex embryos to induce neckteeth.
• There is the concentra=on dependency of kairomone.
41
Contents
CHAPTER1Daphnia pulex as a model organism for the study of predator-‐induced polyphenism
CHAPTER2Iden?fica?on of the kairomone-‐sensi?ve period and the histology of neckteeth forma?on in predator-‐induced polyphenism in Daphnia pulex
42
Hypothe&cal process of defense morph forma&on
Kairomone recep?on
physiological change
Alteration of gene
expression
Cytological change
Neckteeth forma?on
Developmental fate determina?on Morphogenesis
CHAPTER1
43
Hypothe&cal process of defense morph forma&on
Kairomone recep?on
physiological change
Alteration of gene
expression
Cytological change
Neckteeth forma?on
Developmental fate determina?on Morphogenesis
CHAPTER1
44
0 10 20 30 40 50 60 70 0 20 40 60 80 100
Time (h) Induction (%)
72 68 64 62 61 56 48 44 40 40 37 36 36 32 32 30 28 28 24 24 16 14 14 12 10 10 8 6.5 4 4 4 4 4 2 2 2 2 2 2 0
Total exposuretime (h)
Experimentalgroup [N]
Control
ABCDEFGHIJKLMNOPQRSTUVPAPBPCPDPEPFPGPHP IPJPKPLPMPNPOPPPQ
[55][ 9 ][ 9 ][ 5 ][ 9 ][16][15][12][23][16][ 9 ][30][15][51][51][14][ 3 ][17][23][12][33][10][33][10][38][22][17][12][ 6 ][ 7 ][ 8 ][ 7 ][ 8 ][10][16][19][12][ 9 ][ 3 ][65]
Proportions of individuals with neckteeth in 2nd instar
st.1 st.2 st.3 st.4 1st 2nd
Embryonic stage Postembryonic instar
45
0 10 20 30 40 50 60 70 0 20 40 60 80 100
Time (h) Induction (%)
72 68 64 62 61 56 48 44 40 40 37 36 36 32 32 30 28 28 24 24 16 14 14 12 10 10 8 6.5 4 4 4 4 4 2 2 2 2 2 2 0
Total exposuretime (h)
Experimentalgroup [N]
Control
ABCDEFGHIJKLMNOPQRSTUVPAPBPCPDPEPFPGPHP IPJPKPLPMPNPOPPPQ
[55][ 9 ][ 9 ][ 5 ][ 9 ][16][15][12][23][16][ 9 ][30][15][51][51][14][ 3 ][17][23][12][33][10][33][10][38][22][17][12][ 6 ][ 7 ][ 8 ][ 7 ][ 8 ][10][16][19][12][ 9 ][ 3 ][65]
Proportions of individuals with neckteeth in 2nd instar
st.1 st.2 st.3 st.4 1st 2nd
Embryonic stage Postembryonic instar
46
0 10 20 30 40 50 60 70 0 20 40 60 80 100
Time (h) Induction (%)
72 68 64 62 61 56 48 44 40 40 37 36 36 32 32 30 28 28 24 24 16 14 14 12 10 10 8 6.5 4 4 4 4 4 2 2 2 2 2 2 0
Total exposuretime (h)
Experimentalgroup [N]
Control
ABCDEFGHIJKLMNOPQRSTUVPAPBPCPDPEPFPGPHP IPJPKPLPMPNPOPPPQ
[55][ 9 ][ 9 ][ 5 ][ 9 ][16][15][12][23][16][ 9 ][30][15][51][51][14][ 3 ][17][23][12][33][10][33][10][38][22][17][12][ 6 ][ 7 ][ 8 ][ 7 ][ 8 ][10][16][19][12][ 9 ][ 3 ][65]
Proportions of individuals with neckteeth in 2nd instar
st.1 st.2 st.3 st.4 1st 2nd
Embryonic stage Postembryonic instar
47
0 10 20 30 40 50 60 70 0 20 40 60 80 100
Time (h) Induction (%)
72 68 64 62 61 56 48 44 40 40 37 36 36 32 32 30 28 28 24 24 16 14 14 12 10 10 8 6.5 4 4 4 4 4 2 2 2 2 2 2 0
Total exposuretime (h)
Experimentalgroup [N]
Control
ABCDEFGHIJKLMNOPQRSTUVPAPBPCPDPEPFPGPHP IPJPKPLPMPNPOPPPQ
[55][ 9 ][ 9 ][ 5 ][ 9 ][16][15][12][23][16][ 9 ][30][15][51][51][14][ 3 ][17][23][12][33][10][33][10][38][22][17][12][ 6 ][ 7 ][ 8 ][ 7 ][ 8 ][10][16][19][12][ 9 ][ 3 ][65]
Proportions of individuals with neckteeth in 2nd instar
st.1 st.2 st.3 st.4 1st 2nd
Embryonic stage Postembryonic instar
48
0 h 10 h 20 h 30 h 40 h 50 h 60 h 70 h 80 hst.1 st.2 st.3 st.4 1st 2nd
“Developmental window”Kairomone-‐sensi&ve period of D. pulex was embryonic stage 4 to first instar.
Hatching fromegg chorion
Exfoliate two-‐layered membrane
Birth and ecdysis Ecdysis
49
“Developmental window”Afer the third embryonic molt, the influx of various chemicals in the water appears to have increased.
FAINT
DODO
FAINTA B C
DextranSoaking
Dextran
st.3 st.4AB C
1 h2 h 30 min
A B C
Dextran tetramethylrhodamine: 10,000MW
50
Kairomone ac&on in Daphnia pulex
st.4 Neckteethinduc?on
Kairo
mon
e treatm
ent
51
Kairomone-‐sensi&ve period of D. pulex
• It was rela=vely short, extending from embryonic stage 4 to postembryonic first instar.
• If kairomone disappears from the environment, D. pulex seems promptly to lose the kairomone s=mulus from the body.
• It was hypothesized that the propor=on of individuals that form neckteeth depends on the total amount of the s=mulus received or accumulated at the end of embryogenesis.
52
Hypothe&cal process of defense morph forma&on
Kairomone recep?on
physiological change
Alteration of gene
expression
Cytological change
Neckteeth forma?on
Developmental fate determina?on Morphogenesis
CHAPTER1
53
Neckteeth: crest & spikes
Crest Spikes
54
Cell prolifera&on in neckteeth forma&on
0 h 10 h 20 h 40 h 50 h 60 h 70 h
Embryonic stage Postembryonic instarst. 2 st. 3 st. 4 1st 2nd
80 h 90 h
ⅠⅡ
ⅢⅣ
Ⅴ
30 h
BrdU soaking
55
Ⅴ
Ⅴ
Ⅲ
Ⅲ
Ⅰ
Ⅰ
Cell prolifera&on in neckteeth forma&onKa
iromon
eCo
ntrol
0 h 10 h 20 h 40 h 50 h 60 h 70 h
Embryonic stage Postembryonic instarst. 2 st. 3 st. 4 1st 2nd
80 h 90 h
ⅠⅡ
ⅢⅣ
Ⅴ
30 h
BrdU soaking
Ⅱ(32 h-52 h)
kairomonecontrol
Number of BrdU-positive cells
*
** **
**
5
10
15
20
25
0
(14)(12)
(12)
(10)
(25)
(23)
(10)(12)
(8)
(8)
Ⅰ(20 h-40 h)
Ⅲ(44 h-64 h)
Ⅳ(56 h-76 h)
Ⅴ(68 h-88 h)
56
Histology of neckteeth
The epidermal cells lining the cu&cle beneath the necktooth were of high density and single-‐layered.The crest consisted of loose connec&ve &ssue.
Neckteeth2nd instar
Control2nd instar
crest
57
Hypothe&cal model for cellular changes during neckteeth forma&on
The superficial cells secrete thecu&cle of spikes, and the cells underlining them enlarge as a loose connec&ve &ssue, leading to thickening of the crest.
58
Hypothe&cal process of defense morph forma&on
Kairomone recep?on
physiological change
Alteration of gene
expression
Cytological change
Neckteeth forma?on
Developmental fate determina?on Morphogenesis
CHAPTER1
59
LiCl treatment effect on crest forma&on
Wnt
Frizzled
Disheveled
GSK-‐3β
β-‐catenin
Transcription
LiCl
(0 / 18)
(0 / 20)
LiCl free
3 mM LiCl
(0 / 18) (5 / 12)
(8 / 11)(0 / 20)
KairomoneControl1st instar
Kairomone1st instar
60
Future direc&on:Comparison between Daphnia species
Various head shapes might be formed by same cellular mechanism as crest of D. pulex.
61
Normalst.1
2nd-‐3rd instar
st.2
st.3
st.4
4th-‐5th instar
2nd-‐3rd instar
4th-‐5th instar
Developmental windowRapid cell prolifera?on
EscapeNeckteeth
Normal Normal
Neckteeth induc?on
Typical development
kairomone
WntSignaling?
In the presence of the predator In the absence of the predator
Adult female
62
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