defense morph formation of daphnia pulex

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


Daphnia cucullata

predator-‐induced polyphenism

6

http://plankt.oxfordjournals.org/content/8/2/289.abstract



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(Chaoborus larvae)フサカ幼生

11


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


Neckteeth (Necktooth)

14

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

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



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




Oviposi?on

st.1 st.2 st.3 st.4

Ecdysis

25




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




Oviposi?on

0 h 10 h 20 h 30 h 40 hst.1 st.2 st.3 st.4


Exfoliate two-‐layered membrane

27




Oviposi?on Discharge

0 h 10 h 20 h 30 h 40 hst.1 st.2 st.3 st.4



28




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



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


Neckteeth induc?on

kairomone medium

35

Neckteeth induc?on

Defense morph induc0on system

kairomone medium


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



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


Kairomone recep?on


Alteration of gene

expression

Cytological change

Neckteeth forma?on


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


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



Control


[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]




46

0 10 20 30 40 50 60 70 0 20 40 60 80 100


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



Control


[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]




47

0 10 20 30 40 50 60 70 0 20 40 60 80 100


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



Control


[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]




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


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


Kairomone recep?on


Alteration of gene

expression

Cytological change

Neckteeth forma?on


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


Kairomone recep?on


Alteration of gene

expression

Cytological change

Neckteeth forma?on


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

defense morph formation of daphnia pulex

Education