This document describes research analyzing the predator-induced polyphenism in Daphnia pulex. Daphnia pulex is used as a model organism to study how environmental cues modify developmental processes. The study identifies the sensitive period when Daphnia embryos can respond to chemical cues from predators by developing neckteeth defenses. Experiments show that when embryos are directly exposed to kairomones from the predator Chaoborus larvae, they develop neckteeth upon hatching, demonstrating the cues can act directly on embryos to induce morphological defenses. This defense morph induction system provides insights into evolutionary developmental processes.

1. 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

2. Evolu&onary developmental biology
(Evo-­‐Devo)
Gene expression
Animal
Developmental
Change
Phenotype
Evolu&on
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？
2

3. Phenotypic plas&city（表現型可塑性）
Environmental regula&on of development
Gene expression
Phenotype
Environment A
Animal
Environment B
A study model to get insights into evolu&onary processes
in Evo-­‐Devo.
3

4. Predator-­‐induced polyphenism
捕食者に誘導される表現型可塑性
Ro?fer
Barnacle
Bryozoan
mollusk
Carp
Typical
morph
Predator-­‐
induced
morph
Sco$ F. Gilbert, Developmental Biology 6th Edi=on
The phenotypic altera&on by modula&ng
developmental processes in the presence of predators.
4

5. Water fleas-­‐-­‐Daphnia
(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata
5

6. Water fleas-­‐-­‐Daphnia
(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata
predator-­‐induced polyphenism
6

7. Water fleas-­‐-­‐Daphnia
(Crustacea, Cladocera, Anomopoda)
Daphnia cucullata
Daphnia retrocrva
Daphnia carinata
predator-­‐induced polyphenism
Daphnia
lumhotzi
Daphnia
longicephala
Daphnia ambigua
Daphnia pulex
7

8. Daphnia pulex（ミジンコ）
• Cosmopolitan
• Field ecology
• Breeding in laboratory
• Genome sequenced
(2011)
• Molecular techniques
0.5 mm
8

9. 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
• Tail spine
• Body width
• Body depth
• Egg size and number
• Behavioral defenses
（Chaoborus）
（Chaoborus, Notonecta, Lepomis）
（Chaoborus）
Daphnia pulex
（ミジンコ）
（Chaoborus）
（Chaoborus）
（Fish）
9

10. Predator-­‐induced polyphenism in
Daphnia pulex
10

11. Predator-­‐induced polyphenism in
Daphnia pulex
Predator
(Chaoborus larvae)
フサカ幼生
11

12. Predator-­‐induced polyphenism in
Daphnia pulex
Kairomone
Predator
(Chaoborus larvae)
フサカ幼生
12

13. 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.
The molecular structure and the ac&on mechanism of
the Chaoborus kairomone are unknown.
13

14. Predator-­‐induced polyphenism in
Daphnia pulex
Neckteeth
(Necktooth)
14

15. Predatory ac&vity Chaoborus larvae
Typical morph (1.0-­‐1.3 mm)
15

16. Predatory ac&vity Chaoborus larvae
Typical morph (1.0-­‐1.3 mm)
15

17. Predatory ac&vity Chaoborus larvae
Neckeeth morph (1.0-­‐1.3 mm)
16

18. Predatory ac&vity Chaoborus larvae
Neckeeth morph (1.0-­‐1.3 mm)
16

19. In this thesis
Analysis Daphnia pulex as a
model organism in Evo-­‐Devo
17

20. In this thesis
How do environmental cues
modify the developmental
process?
18

21. Contents
CHAPTER1
Daphnia pulex as a model organism for the study of
predator-­‐induced polyphenism
CHAPTER2
Iden?fica?on of the kairomone-­‐sensi?ve period and
the histology of neckteeth forma?on in predator-­‐
induced polyphenism in Daphnia pulex
19

22. Contents
CHAPTER1
Daphnia pulex as a model organism for the study of
predator-­‐induced polyphenism
CHAPTER2
Iden?fica?on of the kairomone-­‐sensi?ve period and
the histology of neckteeth forma?on in predator-­‐
induced polyphenism in Daphnia pulex
20

23. Animals
Daphnia pulex
Chaoborus flavicans larvae
5 mm
0.5 mm
From a pond in the Flowering tree garden
on Hokkaido University campus
A single clone was used
throughout this study.
From a pond at the Na=onal Ins=tute for
Environmental Studies, Tsukuba
21

24. Daphnia pulex rearing
• 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
22

25. Life cycle of Daphnia pulex
Hiruta and Tochinai (2012), Meiosis
23

26. Time course of development
Embryonic stage
in the brood chamber（育房）
Oviposi?on
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
24

27. Time course of development
st.1
st.2
st.3 st.4
Ecdysis
Embryonic stage
in the brood chamber（育房）
Oviposi?on
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
25

28. Time course of development
st.1
0 h 10 h 20 h 30 h 40 h
st.2
st.3 st.4
Hatching from
egg chorion（卵膜）
Embryonic stage
in the brood chamber（育房）
Oviposi?on
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
26

29. Time course of development
st.1
0 h 10 h 20 h 30 h 40 h
st.2
st.3 st.4
Hatching from
egg chorion（卵膜）
Exfoliate two-­‐
layered membrane
Embryonic stage
in the brood chamber（育房）
Oviposi?on
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
27

30. Time course of development
st.1
0 h 10 h 20 h 30 h 40 h
st.2
st.3 st.4
Hatching from
egg chorion（卵膜）
Exfoliate two-­‐
layered membrane
Embryonic stage
in the brood chamber（育房）
Oviposi?on
Discharge
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
28

31. Time course of development
st.1
0 h 10 h 20 h 30 h 40 h 50 h 60 h 70 h 80 h
st.2
st.3 st.4
1st
2nd
Hatching from
egg chorion（卵膜）
Exfoliate two-­‐
layered membrane
Birth and
ecdysis
Embryonic stage
Ecdysis
Postembryonic instar
in the brood chamber（育房）
Oviposi?on
Discharge
Maintained at 18°C under ar=ficial light condi=ons of 14 h light and 10 h dark
29

32. For the analyses of defense morph formation
Kairomone
?
• Does kairomone act
directly on embryos
to induce neckteeth?
• Do the picking
?
embryos from
dissected maternal
brood chambers
affect normal
development?
30

33. Embryonic development outside of brood
chamber
Embryos picked from
brood chamber
31

34. Embryonic development outside of brood
chamber
Embryos picked from
brood chamber
Normal development
32

35. Direct exposure of embryos to kairomone
kairomone
medium
33

36. 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

37. Direct exposure of embryos to kairomone
kairomone
medium
Neckteeth induc?on
35

38. Direct exposure of embryos to kairomone
kairomone
medium
Neckteeth induc?on
Defense morph induc0on system
36

39. Neckteeth: The dorsal carapace ridge between
the inser&on points of the first and second antennal
muscles transformed into &ny spikes.
1st instar
Control
2nd instar
SEM
Kairomone
37

40. Neckteeth: The second-­‐instar stage showed the
highest frequency, and neckteeth was completely
absent in adults.
4
Control
**
80
Kairomone
Number of neckteeth
3
60
40
2
*
20
0
1
**
(152) (147)
(145) (145)
(115) (129)
(40) (78)
1st
2nd
3rd
4th
Number of neckteeth
Induction（%）
100
0
Instar
38

41. Predatory ac&vity Chaoborus larvae
Neckteeth morph has the greater escape efficiency in
the 1.0-­‐1.3 mm body size.
Experimental
group
1
2
Body length
Typical morph
Neckteeth morph
0.8 mm
3
4
0.9 mm
5
6
7
8
9
1.0-1.3 mm
10
11
escape
prey
39

42. Concentration effect of kairomone medium
st.1
st.2
st.3
st.4
1st
2nd
Kairomone
treatment
16/16
100
37/37
37/40
80
Induction (%)
10/10
10/13
12/15
22/28
10/15
60
40
20
0 0/18
0
3/26
10
20 25 30 35 40 45 50
100
Strength of kairomone medium (%)
40

43. 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

44. Contents
CHAPTER1
Daphnia pulex as a model organism for the study of
predator-­‐induced polyphenism
CHAPTER2
Iden?fica?on of the kairomone-­‐sensi?ve period and
the histology of neckteeth forma?on in predator-­‐
induced polyphenism in Daphnia pulex
42

45. Hypothe&cal process of defense
morph forma&on
CHAPTER1
Kairomone
recep?on
physiological
change
Developmental fate
determina?on
Alteration of
gene
expression
Cytological
change
Neckteeth
forma?on
Morphogenesis
43

46. Hypothe&cal process of defense
morph forma&on
CHAPTER1
Kairomone
recep?on
physiological
change
Developmental fate
determina?on
Alteration of
gene
expression
Cytological
change
Neckteeth
forma?on
Morphogenesis
44

47. Embryonic stage
st.1
st.2
Postembryonic instar
st.3 st.4
1st
2nd
Total
exposure
time (h)
Experimental
group [N]
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
PA
PB
PC
PD
PE
PF
PG
PH
PI
PJ
PK
PL
PM
PN
PO
PP
PQ
Control
Proportions of individuals
with neckteeth in 2nd instar
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
[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]
0
10
20
30
40
Time (h)
50
60
70
0
20
40
60
80
100
Induction (％)
45

48. Embryonic stage
st.1
st.2
Postembryonic instar
st.3 st.4
1st
2nd
Total
exposure
time (h)
Experimental
group [N]
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
PA
PB
PC
PD
PE
PF
PG
PH
PI
PJ
PK
PL
PM
PN
PO
PP
PQ
Control
Proportions of individuals
with neckteeth in 2nd instar
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
[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]
0
10
20
30
40
Time (h)
50
60
70
0
20
40
60
80
100
Induction (％)
46

49. Embryonic stage
st.1
st.2
Postembryonic instar
st.3 st.4
1st
2nd
Total
exposure
time (h)
Experimental
group [N]
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
PA
PB
PC
PD
PE
PF
PG
PH
PI
PJ
PK
PL
PM
PN
PO
PP
PQ
Control
Proportions of individuals
with neckteeth in 2nd instar
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
[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]
0
10
20
30
40
Time (h)
50
60
70
0
20
40
60
80
100
Induction (％)
47

50. Embryonic stage
st.1
st.2
Postembryonic instar
st.3 st.4
1st
2nd
Total
exposure
time (h)
Experimental
group [N]
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
PA
PB
PC
PD
PE
PF
PG
PH
PI
PJ
PK
PL
PM
PN
PO
PP
PQ
Control
Proportions of individuals
with neckteeth in 2nd instar
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
[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]
0
10
20
30
40
Time (h)
50
60
70
0
20
40
60
80
100
Induction (％)
48

51. “Developmental window”
Kairomone-­‐sensi&ve period of D. pulex was
embryonic stage 4 to first instar.
st.1
0 h 10 h 20 h 30 h 40 h 50 h 60 h 70 h 80 h
st.2
st.3 st.4
1st
2nd
Hatching from
egg chorion
Exfoliate two-­‐
layered membrane
Birth and
ecdysis
Ecdysis
49

52. “Developmental window”
Afer the third embryonic molt, the influx of various
chemicals in the water appears to have increased.
st.3
Dextran
Soaking
A
st.4
A
B
A
1 h
2 h
B
B
FA
Dextran
C
C
INT
30 min
C
INT
FA
DO
DO
Dextran tetramethylrhodamine: 10,000MW
50

53. Kairomone ac&on in Daphnia pulex
Kairomone treatment
st.4
Neckteeth
induc?on
51

54. 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

55. Hypothe&cal process of defense
morph forma&on
CHAPTER1
Kairomone
recep?on
physiological
change
Developmental fate
determina?on
Alteration of
gene
expression
Cytological
change
Neckteeth
forma?on
Morphogenesis
53

56. Neckteeth: crest & spikes
Crest
Spikes
54

57. Cell prolifera&on in neckteeth forma&on
Embryonic stage
st. 2
0h
10 h
st. 3
20 h
30 h
Postembryonic instar
st. 4
1st
40 h
Ⅰ
BrdU soaking
50 h
60 h
Ⅱ
2nd
70 h
Ⅲ
80 h
Ⅳ
90 h
Ⅴ
55

58. Cell prolifera&on in neckteeth forma&on
st. 2
0h
10 h
st. 3
20 h
Postembryonic instar
st. 4
30 h
1st
40 h
Ⅰ
50 h
60 h
Ⅱ
Kairomone
BrdU soaking
Ⅰ
Control
Ⅰ
Ⅲ
Ⅲ
25
2nd
70 h
Ⅲ
80 h
**
90 h
Ⅳ
Ⅴ
Ⅴ
Ⅴ
Number of BrdU-positive cells
Embryonic stage
20
control
kairomone
15
**
(8)
**
10
(12)
(25)
5
(23)
(14)
(12)
(10)
(10)
0
Ⅰ
Ⅱ
* (12)
Ⅲ
Ⅳ
(8)
Ⅴ
（20 h-40 h）
（32 h-52 h）
（44 h-64 h）
（56 h-76 h）
（68 h-88 h）
56

59. Histology of neckteeth
Control
2nd instar
Neckteeth
2nd instar
crest
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.
57

60. Hypothe&cal model for cellular
changes during neckteeth forma&on
The superficial cells secrete the
cu&cle of spikes, and the cells underlining them enlarge as
a loose connec&ve &ssue, leading to thickening of the crest.
58

61. Hypothe&cal process of defense
morph forma&on
CHAPTER1
Kairomone
recep?on
physiological
change
Developmental fate
determina?on
Alteration of
gene
expression
Cytological
change
Neckteeth
forma?on
Morphogenesis
59

62. LiCl treatment effect on crest forma&on
Control
1st instar
Wnt
(5 / 12)
LiCl free
(0 / 18)(0 / 18)
Kairomone
Kairomone
1st instar
Frizzled
Disheveled
3 mM LiCl
LiCl
(0 / 20)(0 / 20)
(8 / 11)
GSK-­‐3β
β-­‐catenin
Transcription
60

63. Future direc&on:
Comparison between Daphnia species
Various head shapes might be formed by same
cellular mechanism as crest of D. pulex.
61

64. In the presence of the predator
In the absence of the predator
Adult female
Normal
Normal
4th-­‐5th
instar
4th-­‐5th
instar
st.1
Neckteeth
Normal
Escape
st.2
2nd-­‐3rd
instar
kairomone
Wnt
Signaling?
2nd-­‐3rd
instar
st.3
Neckteeth
induc?on
Typical
development
st.4
Rapid cell prolifera?on
Developmental
window
62