ZebrafishZebrafish ((Danio Danio reriorerio): ): FeedingFeeding and and NutritionTiziano Verri

Department of Biological and Environmental Sciences and TechnologiesUniversity of Salento

(Lecce, Italy)

ZF-MED - ZEBRAFISH AND OTHER AQUATIC MODELS IN MEDITERRANEAN LABSAQUATIC

Giornate studio sull’impiego dei Modelli Acquatici a fini scientificiGiornate studio sull’impiego dei Modelli Acquatici a fini scientificiOctober

Giornate studio sull’impiego dei Modelli Acquatici a fini scientificiGiornate studio sull’impiego dei Modelli Acquatici a fini scientificiOctoberOctober 16, 2018

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ -- Teramo

1. Redundancy of transporters 1. Redundancy of transporters involved in amino acid/peptide involved in amino acid/peptide

absorption

ProteinProtein digestiondigestion, ProteinProtein digestiondigestiondigestion, terminal (membrane) terminal (membrane)

digestionterminal (membrane) terminal (membrane)

digestiondigestion and digestiondigestion and and absorption

KEGG KEGG pathways

SLC34 family SLC34 family -- NaNa+Na+/+/phosphatephosphate transporters

EvolutionaryEvolutionaryrelationships

EvolutionaryEvolutionaryrelationshipsrelationships amongrelationships

Pept1relationships

Pept1Pept1-relationshipsrelationshipsrelationships

Pept1Pept1--type relationshipsrelationships amongamongrelationships

type type transportersPept1Pept1Pept1in

type type transporterstransporterstype type type in in vertebrates

Zebrafish Zebrafish Slc15a1 (Pept1) kinetic Slc15a1 (Pept1) kinetic analysis by TEVC

zfPepT1a

zfPepT1b

7.6 6.5ZebrafishZebrafish Slc15a1a Zebrafish

(Pept1a) ZebrafishZebrafish

(Pept1a) (Pept1a) isSlc15a1a Zebrafish Slc15a1a Slc15a1a Zebrafish

isisisis functional

zfPepT1a zfPepT1b

5 oo PepT1a, 4 oo PepT1b

-160 -140 -120 -100 -80 -60 -40 -20 0 20 40

-160

-140

-120

-100

-80

-60

-40

-20

0-160 -140 -120 -100 -80 -60 -40 -20 0 20 40

-800

-700

-600

-500

-400

-300

-200

-100

0

nA

mV

7.6

6.5

zfPepT1a zfPepT1b

sub GQ 3mM

Vh -60mV

nA

mV

-260

-240

-220

-200

-180

-160

-140

-120

-100

-80

-60

-40

zfP

epT

1a

zfP

epT

1b

7.6

6.5

GQ 3mM

zf PepT1a zfPepT1b

-60 mV

EvolutionaryEvolutionaryrelationshipsrelationships

amongrelationshipsrelationships

amongamong peptide amongamong peptide peptide transporters

peptide peptide transporterstransporters in transporterstransporters in in

salmonids

Spatial distribution of Spatial distribution of slc15a1aslc15a1a ((pept1apept1a) and ) and slc15a1bslc15a1b ((pept1bpept1b) Spatial distribution of mRNA

Spatial distribution of Spatial distribution of mRNA mRNA in the

Spatial distribution of Spatial distribution of (pept1apept1apept1apept1a) and ) and Spatial distribution of in the in the Atlantic salmon digestive

) and ) and Atlantic salmon digestive Atlantic salmon digestive tract (RTtract (RTtract (RT-

(pept1bpept1bpept1btract (RTtract (RT-PCR)

Atlantic Atlantic salmon

ssPepT1a

GQPGKGGKGQ

sec

40 nA

ssPepT1b

GQKKKKMMKGQ

GQPGKGGKGQ GQKKKKMMKGQ

sec

10 nA

-2,5

-2,0

-1,5

-1,0

-0,5

0,0GQ GK KG PG MK KM KKK

ssPepT1b

ssPepT1a

pH 7.6 subs 1mM

Vh -60mV

I (nA

)/I (

nA)

GQ

GQ GK KG PG MK KM KKK

Fig. 4. Transport currents elicited by different Lysine containing substrates. A, representative currents recorded in the presence of the indicatedsubstrates (1 mM) at pH 7.6 in ssPepT1b (upper traces) and PepT1a, the oocytes were clamped at -60 mV. B, Mean of transport associated current at -60 mV in the presence of indicated substrates and normalized to the current in Gly-Gln. C, current-voltage relationship of transport-associated currentin ssPepT1b (top) and in ssPepT1a (bottom) recorded from -140 to +20 mV at two pH conditions: 6.5 (gray symbols) and 7.6 (empty symbols). Thecurrent value reported in I/V relationship and in bar histograms are obtained by subtracting from the current in the presence of indicated substrate, thecurrent in its absence.

-140 -120 -100 -80 -60 -40 -20 0 20

-800

-600

-400

-200

0-140 -120 -100 -80 -60 -40 -20 0 20

-150

-100

-50

0

KG (mM) KG (mM)

GQ 3mMGQ 3mM10 3310.30.1 GQ 3mM

60 sec

40 nA

GQ 3mM

Vm (mV)

301010.30.11

40 sec

10 nA

CVm (mV)

0.1 mM

0.3 mM

1 mM

3 mM

10 mM

B

D

I (nA)

0.1 mM

0.3 mM

1 mM

3 mM

10 mM

30 mM

A

ssPept1b ssPepT1a

2. Pept12. Pept1-2. Pept1-type transporters, Lystype transporters, Lys-type transporters, Lys-GlyGly-2. Pept12. Pept12. Pept1 type transporters, Lystype transporters, Lystype transporters, Lyscontaining peptides &

type transporters, Lystype transporters, Lystype transporters, Lystype transporters, Lyscontaining peptides & containing peptides & peptide(Lyspeptide(Lyspeptide(Lys-

GlyGlyGlyGlyGlyGlypeptide(Lyspeptide(Lys--GlyGlyGly)GlyGly))-containing peptides & containing peptides & peptide(Lyspeptide(Lys

containing diets

growthgrowth rate PepT1 PepT1 PepT1 expression

Pept1 Pept1 and peptide(and peptide(LysLys-Lys-GlyGly)Gly)-)-containingcontaining dietsdiets ((rainbowrainbow trouttrout)

PP, wheat gluten protein-based dietplus Lys-GlyAA, wheat gluten protein-based dietplus Lys and GlyCON, wheat gluten protein-basedcontrol diet with no LysAF, commercial starter (Aller Futura)

Adapted from:Ostaszewska et al., Comp Biochem Physiol A Mol Integr Physiol. 155:107, 2010

Pept1 Pept1 and peptide(and peptide(LysLys-Lys-GlyGly)Gly)-)-containingcontaining dietsdiets (common (common carpcarp)

PepT1 PepT1 PepT1 expressiongrowthgrowth rate

PP; wheat gluten protein-based diet plus Lys-GlyAA, wheat gluten protein-based diet plus Lys and GlyCON, wheat gluten protein-based control diet with no LysZ, frozen zooplanktonAN, commercial starter food Aglo Norse

Adapted from:Ostaszewska et al., Comp Biochem Physiol A

Mol Integr Physiol. 157:158, 2010

growthgrowth rate PepT1 PepT1 expression

Pept1 Pept1 and peptide(and peptide(LysLys-Lys-GlyGly)Gly)-)-containingcontaining dietsdiets ((yellowyellow perchperch)

LG, wheat gluten protein‐based diets plus Lys‐GlyFL, wheat gluten protein-based diets plus Lys and GlyC, control diet with no LysBO; commercial starter Bio Oregon

Adapted from:Ostaszewska et al., Aquaculture Nutr. 19:100,

2013

Conclusions

•• Pept1a and Pept1b operate Pept1a and Pept1b operate in in in fishfish intestine

•• Pept1a and Pept1b are Pept1a and Pept1b are bothboth expressedexpressed in in fishfish postpost-post-gastricgastric canal

•• A A A functionalfunctional relationshiprelationship betweenbetween Pept1 Pept1 transporterstransporters and

dipeptide(dipeptide(LysLys-Lys-GLyGLy)GLy)-)-containingcontaining dietsdiets can can be be assessedassessed under under certain

conditions

3. 3. Pept1 and compensatory Pept1 and compensatory (‘catch(‘catch-(‘catch-up’) Pept1 and compensatory growth

Pept1 Pept1 and and compensatorycompensatory (‘catch(‘catch-(‘catch-up’) up’) growthgrowth and the ‘and the ‘seasea bassbass’ model)

EuropeanEuropean seasea bass

Terova et al., Aquaculture 294:288 (2009)Verri et al., Biol. Lett. 7:597 (2011)

Mean body weight

day 0,

before

fastin

g

35 day

s fas

ted

21 day

s refe

d0

20406080

100120140160180 fed ad libitum

fasted and refed

*

Fish

wei

ght

(g)

Condition factor (K)

day 0,

before

fastin

g

35 day

s fas

ted

21 day

s refe

d0.00.20.40.60.81.01.21.41.61.82.02.22.4 fed ad libitum

fasted and refed

*

K(b

ody

wei

ght x

100

/sta

ndar

d bo

dy le

ngth

3 ) Mean daily food intake

day 0,

before

fastin

g

4 day

s fas

ted

35 day

s fas

ted

4 day

s refe

d

14 day

s refe

d

21 day

s refe

d-5

0

5

10

15

20

25 fed ad libitumfasted and refed

Food

inta

ke(g

/Kg

body

wei

ght/d

ay)

Ghrelin

day 0,

before

fastin

g

4 day

s fas

ted

35 day

s fas

ted

4 day

s refe

d

14 day

s refe

d

21 day

s refe

d0.00.20.40.60.81.01.21.41.61.82.02.22.4 fed ad libitum

fasted and refed

**

*

Gen

e ex

pres

sion

(x 1

06 )(m

RN

A c

opy

num

ber/

100

ng to

tal R

NA

)

Gastricsin

day 0,

before

fastin

g

4 day

s fas

ted

35 day

s fas

ted

4 day

s refe

d

14 day

s refe

d

21 day

s refe

d0

1

2

3

4

5

6

7 fed ad libitumfasted and refed

*

**

Gen

e ex

pres

sion

(x 1

06 )(m

RN

A c

opy

num

ber/

100

ng to

tal R

NA

)

Peptide Transporter 1

day 0,

before

fastin

g

4 day

s fas

ted

35 day

s fas

ted

4 day

s refe

d

14 day

s refe

d

21 day

s refe

d0.00.51.01.52.02.53.03.54.0 fed ad libitum

fasted and refed

*

**

Gen

e ex

pres

sion

(x 1

06 )(m

RN

A c

opy

num

ber/

100

ng to

tal R

NA

)

PEPT1 and PEPT1 and compensatorycompensatory (‘catch(‘catch-(‘catch-up’) up’) growthgrowth (the (the ‘(the ‘seasea bassbass’bass’ model)

Peptide Transporter 1

day 0,

before

fastin

g

4 day

s fas

ted

35 day

s fas

ted

4 day

s refe

d

14 day

s refe

d

21 day

s refe

d0.00.51.01.52.02.53.03.54.0 fed ad libitum

fasted and refed

*

**

Gen

e ex

pres

sion

(x 1

06 )(m

RN

A c

opy

num

ber/

100

ng to

tal R

NA

)

Pept1 Pept1 and and compensatorycompensatory (‘catch(‘catch-(‘catch-up’) up’) growthgrowth (the (the ‘(the ‘seasea bassbass’bass’ model)

European sea bass

TerovaTerova et al., Aquaculture 294:288 (2009)et al., Aquaculture 294:288 (2009)et al., Aquaculture 294:288 (2009)Verri

et al., Aquaculture 294:288 (2009)et al., Aquaculture 294:288 (2009)VerriVerri et al., Biol.

et al., Aquaculture 294:288 (2009)et al., Aquaculture 294:288 (2009)et al., Biol. et al., Biol. Lett

et al., Aquaculture 294:288 (2009)et al., Aquaculture 294:288 (2009)LettLett. 7:597 (2011)

Pept1 Pept1 and and compensatorycompensatory (‘catch(‘catch-(‘catch-up’) up’) growthgrowth ((zebrafishzebrafish)

KovenKoven and Schulte, Fish. Physiol. and Schulte, Fish. Physiol. BiochemBiochem. 38:1565 (2013)

zebrafish

Not only Pept1

but also Slc6a19a

responds to

fasting and

refeeding

Conclusions

•• Pept1a and Pept1b operate Pept1a and Pept1b operate in in in fishfish intestine

•• Pept1a and Pept1b are Pept1a and Pept1b are expressedexpressed in in fishfish postpost-post-gastricgastric canal

•• A functional relationship between A functional relationship between Pept1’s Pept1’s and compensatory and compensatory (‘catch(‘catch-

up’) up’) growth exists in fish (… focusing on growth exists in fish (… focusing on Pept1 Pept1 function in mammals

during their growth, humans included)

4. Automatic feeding 4. Automatic feeding systems

TRITONE: TRITONE: AutomaticAutomatic feedingfeeding system

Automated diet delivery system for zebrafish labsIt reduces:

• Fish feeding costs• High variability associated to ‘more than one person’ feeding

It improves:• Hygiene at tank level

• Ergonomics during diet distribution• System water quality

DottoratoDottorato di di ricercaricerca industrialeindustriale (2017)Distribuzione automatizzata di cibo funzionale per zebrafish: verso una migliore alimentazione dei modelli animali acquatici in laboratorio (ZFeedPlus)

• Sviluppo di un sistema per la distribuzione automatizzata di cibi funzionali, compatibile con un sistema di allevamento a ricircolo usato per mantenere zebrafish in condizioni di stabulazione.

• Per standardizzare le condizioni di mantenimento di zebrafish in laboratorio migliorando il benessere degli animali, nella prospettiva di rendere più ‘certificabile’ il dato sperimentale, il progetto coniuga la necessità di sviluppare nuovi cibi nel rispetto delle esigenze alimentari di zebrafish e di rendere la somministrazione dell’alimento un fatto sempre più controllabile, riproducibile e indipendente dal fattore umano.

• Collaborazione tra un centro universitario del Sud Italia, che si occupa di fisiologia dei pesci, un’azienda del Nord Italia, leader indiscusso a livello mondiale nel settore della stabulazione, e un centro universitario in Norvegia, dove produrre pesce di interesse commerciale è settore industriale strategico nazionale, che è oggi uno dei più importanti centri mondiali di biologia e biotecnologie marine.

DottoratoDottorato di di ricercaricerca industrialeindustriale (2018)Distribuzione automatizzata di cibo funzionale per zebrafish: verso una migliore alimentazione dei modelli animali acquatici in laboratorio (ZFeedPlus)

• In 3 pacchetti di lavoro (WP), il progetto vuole:

• 1. Approfondire l’analisi molecolare e morfo-funzionale del sistema digerente di zebrafish

(WP1)

• 2. Mettere a punto diete speciali (WP2)

• 3. Analizzare gli effetti delle diete su crescita e, in generale, benessere dei pesci allevati

(WP3)

DottoratoDottorato di di ricercaricerca industrialeindustriale (2018)Distribuzione automatizzata di cibo funzionale per zebrafish: verso una migliore alimentazione dei modelli animali acquatici in laboratorio (ZFeedPlus)

Metodologie (procedure in silico, sperimentali e/o di sviluppo tecnologico)

Per l’analisi dei geni espressi nel sistema digerente in zebrafish (WP1):• Banche dati (*)• Genomica e trascrittomica funzionale comparata (*)• Bioinformatica (*)• Isolamento di acidi nucleici e proteine (*)• Real-time PCR semi-quantitativa e quantitativa (*)• In situ hybridization• Western blot (*)• Whole-mount in situ hybridization (*)• Analisi ed elaborazione dei dati (*)

• (*) Parte della ricerca sarà svolta presso l’Università di Bergen

DottoratoDottorato di di ricercaricerca industrialeindustriale (2018)Distribuzione automatizzata di cibo funzionale per zebrafish: verso una migliore alimentazione dei modelli animali acquatici in laboratorio (ZFeedPlus)

Metodologie (procedure in silico, sperimentali e/o di sviluppo tecnologico)

Per la messa a punto delle diete speciali (WP2):• Banche dati• Chemical libraries• Screening di molecole mediante incubazione con embrioni/larve di zebrafish (vedi ad es.

http://www.oecd.org/chemicalsafety/testing/36817242.pdf) (*) • Preparazione di mangimi contenenti molecole selezionate (***)• Analisi ed elaborazione dei dati

• (*) Parte della ricerca sarà svolta presso l’Università di Bergen• (***) I mangimi saranno preparati da SPAROS LDA (vedi www.sparos.pt)

DottoratoDottorato di di ricercaricerca industrialeindustriale (2018)Distribuzione automatizzata di cibo funzionale per zebrafish: verso una migliore alimentazione dei modelli animali acquatici in laboratorio (ZFeedPlus)

Metodologie (procedure in silico, sperimentali e/o di sviluppo tecnologico)

Per l’analisi degli effetti delle diete speciali (WP3):• Ricerca e sviluppo su Tritone (**)• Implementazione di nuova tecnologia su Tritone (**)• Standardizzazione di protocolli sperimentali (**)• Analisi biometriche, fisiologiche e comportamentali (vedi ad es.

https://www.ncbi.nlm.nih.gov/books/NBK5216/) • Analisi ed elaborazione dei dati

• (**) La ricerca tecnologica sarà svolta presso Tecniplast S.p.A.