Ammonia tolerance in the teleost Misgurnus anguillicaudatus Surviving high environmental ammonia and aerial exposure.

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Joana Catarina Rocha Moreira da Silva

Dissertação de doutoramento em Ciências do Meio Aquático

Ammonia tolerance in the teleost

Surviving high environmental ammonia and aerial exposure

Joana Catarina Rocha Moreira da Silva

doutoramento em Ciências do Meio Aquático

2009

Ammonia tolerance in the teleost Misgurnus anguillicaudatus

Surviving high environmental ammonia and aerial exposure

doutoramento em Ciências do Meio Aquático

Misgurnus anguillicaudatus

Ammonia tolerance in the teleost

Misgurnus anguillicaudatus

Surviving high environmental ammonia

and aerial exposure

Joana Catarina Rocha Moreira da Silva

Dissertação de doutoramento em Ciências do Meio Aquático

2009

Joana Catarina Rocha Moreira da Silva

Tolerância à amónia no teleósteo Misgurnus anguillicaudatus

Sobrevivência a altas concentrações de amónia e exposição aérea

Dissertação de Candidatura ao grau de Doutor em Ciências do Meio Aquático submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto.

Orientador – Professor Doutor João Coimbra Categoria – Professor Catedrático

Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.

Co-orientador – Doutor Jonathan Wilson Categoria – Investigador Auxiliar

Afiliação – Centro Interdisciplinar de Investigação Marinha e Ambiental da Universidade do Porto.

Joana Catarina Rocha Moreira da Silva

Ammonia tolerance in the teleost Misgurnus anguillicaudatus

Surviving high environmental ammonia and aerial exposure

Thesis submitted to the Institute of Biomedical Sciences of Abel Salazar, University of Porto, for the degree of Doctor in Aquatic Sciences.

Supervisor – Professor João Coimbra Category – Full Professor

Affiliation – Institute of Biomedical Sciences of Abel Salazar of University of Porto.

Co-supervisor – Dr. Jonathan Wilson (PhD) Category – Auxiliary Researcher

Affiliation – Centre of Marine and Environmental Research of University of Porto.

Acknowledgments

Várias pessoas tornaram o caminho que tomei até aqui chegar mais fácil de calcorrear, a quem muito sinceramente agradeço:

Ao Professor Coimbra, o meu orientador, porque foi com ele que tudo começou, quando me apoiou no meu estágio curricular no IMC na Sardenha, naquela altura dirigido pela Doutora Silvana Valerga. Até chegar a sua aluna de doutoramento, a décima.

To Jonathan Wilson, my co-supervisor, because he gave me the privilege to learn with him. We always shared the same enthusiasm when achieving good results, but also the same disappointment when things went wrong. He was always there to answer my crazy doubts. And he always gave me the incentive to go abroad and work in other laboratories, getting to know other researchers and techniques. A big part of the work was done only due to his incredible commitment with the loach ammonia excretion story.

A Paolo Domenici, per mi aver ricevuto cosi bene nel suo laboratorio di Biologia degli Organismi in IMC, durante il mio tirocinio.

To Dave Randall, Tommy Tsui, Matt Vijayan, John Steffensen, Peter Scov, because they were very dedicated when having me visiting their laboratories.

À Filipa Gonçalves, pela sua amizade e porque sei que posso contar com ela sempre.

À Inês Páscoa e à Odete Gonçalves, porque ao trazerem a sua alegria para o laboratório, tornaram a minha vida mais feliz.

À Begoña Duran, que me acompanhou durante quase todo este caminho, tenho pena que o tivesse abandonado.

À Claudia Escorcio, pela sua amizade e porque está sempre presente.

À Marta Ferreira, pela sua amizade e belas conversas e por ter sido capaz de ler partes desta tese e sugerir correcções, e ainda por cima de livre vontade.

À Susana Moreira, pela sua amizade e pelo grande apoio que me deu a todos os níveis no final deste caminho.

À Emília Afonso, pela sua amizade e pela grande ajuda nas questões burocráticas.

Ao Filipe Castro, porque enquanto eu tinha uma descrença do tamanho do mundo ele achava os meus qPCRs fantásticos.

To Ralph Urbatzka because he was there when I needed.

À Dolores Resende, à Carla Batista e à Isabel Cunha, pela sua amizade e por estarem presentes quando eu mais precisei.

À Professora Maria Armanda, porque ao permitir que eu utilizasse o seu equipamento e o seu laboratório tornou a execução do meu trabalho bem mais fácil.

À Eliane Bastos, que surgiu no momento em que eu tanto precisava e me ajudou a concluir um trabalho que sem ela não tinha fim à vista.

Ao Hugo Santos, pela sua amizade e apoio constantes, nas matérias mais variadas e particularmente no cuidado que sempre mostrou ao cuidar dos meus bichos.

Ao Carlos Rosa, pela sua dedicação, persistência e amizade na tentativa, ainda que frustada, de construir o mal fadado amplificador.

Ao Pedro Rodrigues, que tão presente está ainda na minha vida.

À Isabel Teixeira, que na recta final me deu um apoio incondicional e umas dicas preciosíssimas.

Aos meus pais e à minha irmã, que são o meu clã, pelo eterno apoio. Especialmente à minha mãe, que sempre me tirou as pedrinhas do caminho e que mais recentemente teve mesmo que tirar grandes pedregulhos.

Ao Luís, pelo seu amor e compreensão infindáveis e porque o seu apoio ilimitado sempre me transmitiu confiança para superar os meus desafios.

À Rita, que nasceu a meio deste caminho e que é o meu sol. Graças a ela não me afundei no arrozal da escrita e consegui sempre subir até à realidade.

The work presented in this dissertation was supported by the Foundation for Science and Technology (FCT) grant POCTI/BSE/47585/2002 and by a PhD fellowship SFRH/BD/16760/2004, integrated in POCI 2010 and in QREN-POPH. Centre of Marine and Environmental Research (CIMAR-UP) and Institute of Biomedical Sciences of Abel Salazar (ICBAS) from University of Porto are acknowledged for providing facilities and logistical support.

Author’s Declaration

The author declares that she gave a major contribution to the conceptual design and technical execution of the experimental work that gave origin to the results presented, as well as in their interpretation and manuscript preparation of the articles under publication included in this dissertation.

The following manuscripts were prepared under the scope of this PhD thesis:

Moreira-Silva, J., Coimbra, J., Wilson, J. M. (2009). Ammonia sensitivity of the glass eel

(Anguilla anguilla L.): Salinity dependence and the role of branchial Sodium/Potassium Adenosine Triphosphatase. Environmental Toxicology and

Chemistry 28, 141-147.

Moreira-Silva, J., Tsui, T. N. K., Coimbra, J., Ip, Y. K., Vijayan, M.M. and Wilson, J. M.

(2009). Branchial ammonia excretion in the Asian weatherloach Misgurnus

anguillicaudatus. Comparative Biochemistry and Physiology C

(doi:10.1016/j.cbpc.2009.08.006).

Moreira-Silva, J., Coimbra, J., Grosell, M., Vijayan, M.M. and Wilson, J. M. Mechanisms

of transepithelial ammonia movement and luminal alkalinisation of Misgurnus

anguillicaudatus gut (in preparation).

Moreira-Silva, J., Coimbra, J., and Wilson, J. M. Intestinal ammonia excretion in the

Asian weatherloach Misgurnus anguillicaudatus: a molecular approach (in preparation).

Moreira-Silva, J., Steffensen, J., Coimbra, J., Bastos, E., and Wilson, J. M. Effect of

Ammonia on weatherloach (Misgurnus anguillicaudatus) metabolic rate under aquatic and aerial conditions (in preparation).

For the sake of consistency the paper on glass eel ammonia sensitivity will not be included in this dissertation.

Dissertation organization

The present dissertation is organized in chapters, starting with General Introduction and finishing with Integration. Chapters 2, 3, 4, and 5 correspond to the experimental work, and are structured as papers. Tables and figures have sequential numbering from one chapter to the next. The bibliography is consolidated at the end of the dissertation. Various appendixes are presented after the bibliography.

Each chapter will give origin to a separate paper and, within this dissertation, they are free standing, which may lead to a certain degree of repetition throughout the thesis. In some chapters Materials and Methods are shortened to make the chapters more concise, but they will be in detail in Appendix A.

Table of Contents

List of Abbreviations ... iii

List of Tables... v

List of Figures ... vii

Abstract ... xv

Resumo ... xix

Résumé... xxiii

Chapter 1 General Introduction... 1

1.1 Weatherloach – Habitat and characteristics ... 1

1.2 Ammonia Origin ... 3

1.3 Ammonia Chemistry... 3

1.4 Ammonia Toxicity... 4

1.5 Defence Strategies Against Ammonia Toxicity ... 6

1.6 Weatherloach dealing with ammonia...10

1.7 Thesis Aims ...12

Chapter 2 Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus ... 15

2.1 Introduction ...15

2.2 Materials and Methods ...17

2.2.1 Animals ...17 2.2.2 Experiments ...18 2.2.3 Analysis...20 2.3 Results...25 2.4 Discussion ...34 Chapter 3 Mechanisms of transepithelial ammonia movement and luminal alkalinisation in the gut of Misgurnus anguillicaudatus ... 39

3.1 Introduction ...39

3.2 Materials and Methods ...43

3.2.1 Animals ...43

3.2.2 Experiments ...43

3.2.3 Analysis...44

3.4 Discussion ... 53

Chapter 4

Intestinal ammonia excretion in the Asian weatherloach Misgurnus

anguillicaudatus: a molecular approach ...57

4.1 Introduction... 57 4.2 Materials and Methods ... 61 4.2.1 Animals... 61 4.2.2 Experiments... 61 4.2.3 Analysis ... 62 4.3 Results ... 69 4.4 Discussion ... 82 Chapter 5

Effect of Ammonia on Weatherloach (Misgurnus anguillicaudatus) Metabolic Rate Under Aquatic and Aerial Conditions ...89

5.1 Introduction... 89 5.2 Materials and Methods ... 91 5.2.1 Animals... 91 5.2.2 Experiments... 92 5.2.3 Analysis ... 94 5.3 Results ... 96 5.3.1 Standard Metabolic Rate and critical oxygen partial pressure (Pcrit) in control

and ammonia conditions ... 96 5.3.2 Aerial O2consumption in control and ammonia exposed fish ... 98

5.3.3 Behavioural responses ... 98 5.4 Discussion ... 99 Chapter 6 Integration...105 References...111 Appendix A Protocols...125 Appendix B

List of antibodies used in immunofluorescence (IF) and Immunoblotting (IB)...161 Appendix C

iii

List of Abbreviations

actb Beta actin gene

APS Ammonium persulfate solution

Atp1a Na+/K+-ATPase gene

Atp6v1b vacuole (V-type) proton ATPase B subunit gene

BSA Bovine serum albumin

BVA Vacuolar (V-type) proton ATPase B subunit

CA Carbonic Anhydrase

CFTR Cystic fibrosis transmembrane regulator

DABCO 1,4-diazabicyclo-[2,2,2]-octane

DIDS 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid

DMSO Dimethyl sulfoxide

DPH 1,6-diphenyl-1,3,5-hexatrienyl-propionic acid

ECL Enhanced chemiluminescence

EDTA Ethylenediaminetetraacetic acid

EIPA 5-(N-ethyl-N-isopropyl)amiloride

ETZ Ethoxzolamide

FAA Free Amino Acids

H+-ATPase Vacuolar (V-type) proton ATPase

HEA High Environmental Ammonia

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

JAcid Net acid flux

JAmm Net ammonia excretion JTA Titratable Acidity Flux MRC Mitochondria rich cell

NADH Reduced nicotinamide adenine dinucleotide

NGS Normal goat serum

NH3 Gaseous or unionized ammonia NH4+ Ionized ammonia or ammonium NHE Na+/H+exchanger

NKA Na+/K+-ATPase

PAT1 Putative Anion Transporter Cl-/HCO3- exchanger PBS Phosphate buffered saline

Pcrit Critical oxygen partial pressure

PFA Paraformaldehyde

Rhcg Non-erythroid Rhesus C glycoprotein

RT-PCR Reverse Transcriptase-Polymerase Chain Reaction

SDS Sodium dodecyl sulfate

SEI Sucrose, EDTA, Imidazole buffer

SEM Standard Error of the Mean

SITS 4-acetomido-4’-isothiocyanatostilbene-2,2’-disulfonic acid

slc26a6 Putative Anion Transporter Cl-/HCO3- exchanger gene

Slc42a3 Rhesus C-glycoprotein gene

Slc9a1 Na+/H+exchanger isoform 1 gene

Slc9a2 Na+/H+exchanger isoform 2 gene

Slc9a3 Na+/H+exchanger isoform 3 gene

SMR Standard Metabolic Rate

TAN Total Ammonia Nitrogen

TEP Transpithelial Potential difference

v

List of Tables

Table 2.1. Conditions of the LC50 test, conducted at pH 7.89 and 26.5 ºC (TAN – total

ammonia nitrogen). ...18

Table 2.2. Conditions of the pH experiment, conducted at pH 7, 8 and 9, at 25.3°C (TAN –

total ammonia nitrogen)...19

Table 2.3. LC50 values and 95 % confidence intervals (in parentheses) to NH4Cl in

weatherloach, at 26.5 ºC and pH 7.89 (TAN – total ammonia nitrogen)...26

Table 2.4. Branchial Na+/K+-ATPase activity (µmol ADP h-1 mg protein-1) and  subunit

expression (as a ratio with -tubulin and relative to the control group) in Misgurnus

anguillicaudatus following acute ammonia exposure and environmental pH variation

with or without ammonia exposure (6.3 mM TAN) (TAN – total ammonia nitrogen). ....30

Table 2.5. Branchial V-type H+-ATPase activity (µmol ADP h-1mg protein-1) and B subunit

expression (as a ratio with -tubulin and relative to the control group) in M.

anguillicaudatus following acute ammonia exposure and environmental pH variation

with or without ammonia exposure (6.3 mM TAN). ...30

Table 3.1. Composition and properties of modified Corland salines (Wolf, 1963) used in

the Ussing chamber experiments. ...45

Table 3.2. Electrophysiological properties of the weatherloach foregut and hindgut under

symmetrical conditions during current clamp and voltage clamp. Values are mean±SEM (n=4) (t-test P values are shown)...47

Table 3.3. Summary of Base Flux data under control (c) and serosal 10 mM TAN (1 h and

2 h) conditions pooled from inhibitor experiments. Data were analyzed by two way repeated measures ANOVA; n=11. Within a given tissue, groups with like characters are not significantly difference. The asterisk (*) indicates a significant difference from foregut...49

Table 4.1. Nucleotide sequences and amplicon sizes of the primers used in the present

study (actb, β-actin; Atp1a, Na+/K+-ATPase; , Putative Anion Transporter Cl-/HCO3

-exchanger; Atp6v1b, H+-ATPase B subunit; Slc9a1, Na+/H+ exchanger isoform 1; Slc9a3, Na+/H+ exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein). Note that RT-PCR primers for Slc9a1 and Slc9a3 are degenerate. ...64

Table 4.2. RT-PCR profiles using DyNAzyme II DNA polymerase (actb, β-actin; Atp1a,

Na+/K+-ATPase; Slc26a6, Putative Anion Transporter Cl-/HCO3- exchanger; Atp6v1b,

H+-ATPase B subunit; Slc9a1, Na+/H+ exchanger isoform 1; Slc9a3, Na+/H+ exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein)...65

Table 4.3. qPCR conditions using iQ SYBR green Supermix (actb,β-actin; Atp1a, Na+/K+

-ATPase; Slc26a6, Putative Anion Transporter Cl-/HCO3- exchanger; Atp6v1b, H+

-ATPase B subunit; Slc9a1, Na+/H+ exchanger isoform 1; Slc9a3, Na+/H+ exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein) ...66

Table 4.4. Reactions efficiencies conditions for qPCR (actb, β-actin; Atp1a, Na+/K+ -ATPase; Slc26a6, Putative Anion Transporter Cl-/HCO3- exchanger; Atp6v1b, H+

-ATPase B subunit; Slc9a1, Na+/H+ exchanger isoform 1; Slc9a3, Na+/H+ exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein). ... 67

Table 4.5. Effect of chronic ammonia exposure (2 months) on mRNA expression of the

studied ion-transporters (Atp1a, Na+/K+-ATPase; Slc26a6, Putative Anion Transporter Cl-/HCO3- exchanger; Atp6v1b, H+-ATPase B subunit; Slc9a1, Na+/H+ exchanger

isoform 1; Slc9a3, Na+/H+ exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein) in foregut and hindgut. Values are mean±SEM (Ctrl n=6; CE=5). Note the higher Atp1a gene expression in the chronic ammonia exposed group (CE) compared to control (Ctrl) and the lower Slc42a3 gene expression in the CE compared to Ctrl. ... 78

vii

List of Figures

Figure 1.1. Un-stained DIC image of M. anguillicaudatus gill sagittal section of a filament

toward the afferent side (scale bar, 50μm). Asterisk (*), cartilage support; black arrow, gill filament; white arrow, lamellae. ... 2

Figure 1.2. Microanatomy of M. anguillicaudatus gut. Micrographs of the foregut

(digestive glandular zone), midgut (spiral transition zone) and hindgut (posterior respiratory zone) at intermediate (a,b,c) and high (d,e,f) magnification (scale bar, 50 µm). The latter series (d,e,f) were higher magnification of a, b and c to show cellular structure. Sections were stained with haematoxylin and eosin. Asterisk (*), mucous cell; arrow, red blood cell. Abbreviation: L, lumen. ... 3

Figure 1.3. Fish defence strategies against exogenous ammonia toxicity (adapted from Ip

et al., 2001b). ... 6

Figure 1.4. NH3excreted to the gill boundary layer will be converted in NH4+, NH3 will be

trapped as ammonium ion, and this will maintain the NH3 gradient across the gills. In

addition the acidification of the water, with the excretions of H+and/or CO2, next to the

gill will enhance ammonia excretion. ... 7

Figure 1.5. Ammonia elimination by NH3volatilization. Ammonia enters the epithelial cells

by either diffusion or facilitated transport (yellow arrow). After entering the cell it will be transported to boundary layer through facilitated transport (green arrow). Boundary layer is alkaline converting the NH4+ into NH3 that being a gas volatilises when in

contact with air. This reaction produces H+, which reacts with HCO3-to form CO2that is

also volatilised. ... 9

Figure 1.6. Weatherloach defence strategies against exogenous ammonia toxicity.

Question marks represent defence mechanisms that have yet to be demonstrated. ....10

Figure 1.7. Proposed model for gut alkalinisation and ammonia volatilisation through the

gut of M. anguillicaudatus. (1) Na+/K+-ATPase, (2) Na+/H+ exchanger or (3) Cl-/HCO3

-anion exchanger, (4) CFTR (Cl- channel), (5) carbonic anhydrase, (6) H+-ATPase and (7) Rhcg. ...13

Figure 2.1. Effect of different ammonia exposure levels (Control, 1 mM, 5 mM and 10 mM

NH4Cl) on the concentration of ammonia in the muscle. Values are mean±SEM

(n=8-11, with the exception of 10 mM group n=2), bars with like characters are not significantly different (P>0.05). Abbreviation: TAN, total ammonia nitrogen. ...26

Figure 2.2. Effect of different pH conditions (pH 7, 8 and 9 Control; with and without

6.3 mM NH4Cl) on muscle ammonia concentrations. Values are mean±SEM (n=7-10),

bars with like characters are not significantly different (P>0.05). The x mark indicates the terminated ammonia group at pH 9. An asterisk (*) indicates significant differences from the control group at the same pH (P<0.05)...27

Figure 2.3. Effects of HEPES buffering (10 mM) at pH 7 and 8 on ammonia (JAmm, net

ammonia flux) and net acid flux (JAcid= JAmm + JTA, being JTA the titratable acidity flux) in M. anguillicaudatus. (A) 3 h Control flux, dechlorinated Porto tap water at pH 7 or 8. (B)

3 h and (C) 24 h Experimental flux, water buffered at pH 7 and 8 with HEPES 10 mM. Values are mean±SEM (n=6) (P>0.05), bars with like characters are not significantly

different (P>0.05), asterisk (*) indicates significant differences in the same flux period between different pH’s (P<0.05)... 28

Figure 2.4. Effects of pharmacological inhibitors on net ammonia fluxes (μmol kg-1 h-1) in

M. anguillicaudatus over a three hour flux period. Fish were exposed to the H+-ATPase

inhibitor bafilomycin A1 (Baf 0.1 μM), sodium transport inhibitor amiloride (AMIL 0.1 mM), or chloride transport inhibitor disulfonic stilbene (SITS 0.1 mM) or kept under control conditions (Ctrl). The inset shows plasma total ammonia levels in control and bafilomycin exposed fish. Values are mean±SEM (n=6), bars with like characters are not significantly different (P>0.05). ... 29

Figure 2.5.a Localization of V-type H+-ATPase B subunit (B2; green) and Na+/K+-ATPase

(5; red) double immunofluorescent labelling of a sagittal section of a M.

anguillicaudatus gill filament toward the afferent side. To provide structural information

nuclei have been counterstained with DAPI (a,d,e; blue) and the DIC image overlaid. A higher magnification view of the separate (b) H+-ATPase, and (c) Na+/K+-ATPase immunoreactive cells as well as merger image are given. Arrowheads indicate Na+/K+ -ATPase immunoreactive cells, while arrows and crossed arrows indicate sharp and diffuse H+-ATPase staining respectively. Asterisks (*) indicate erythrocytes which shown weak fluorescence. Scale bar = 50μm (a) and 10μm (b-e)... 31

Figure 2.6.a Double immunofluorescent localization of Rhcg1(green) and Na+/K+-ATPase

(red) in a sagittal section of the same gill filament seen in Figure 5. A region at higher magnification is shown with the colour channels separate [(b) Rhcg1, (c) Na+/K+ -ATPase, (d) DAPI] and (e) merged with the DIC overlay. Arrowheads indicate Na+/K+ -ATPase immunoreactive cells, while arrows and crossed arrows indicate strong sharp and weak diffuse apical Rhcg1 staining, respectively. Asterisks (*) indicate erythrocytes which shown weak fluorescence. Scale bar = 50μm (a) and 10μm (b-e)... 32

Figure 2.7. Rooted phylogenetic tree of Slc42a3 homolog sequences. Tree was

constructed by the neighbour-joining method with 1 000 bootstrap trials using ClustalX, with the option: exclude positions with gaps. Bootstrap values below 650 are meaningless. Accession numbers are as follows: Tunicate (Ciona savignyi) Slc42a3 (AAY41908), African clawed frog (Xenopus laevis) Slc42a3 (AAH84943), Chicken (Gallus gallus) Slc42a3 (AAP49833), Pig (Sus scrofa) Slc42a3 (ABF69687), Rhesus monkey (Macaca mulatta) Slc42a3 (ABD72472), Chimpanzee (Pan troglodytes) Slc42a3 (AAX39717), Human (Homo sapiens) SLC42A3 (AAG02171), Rabbit (Oryctolagus cuniculus) Slc42a3 (AAK14653), House mouse (Mus musculus) Slc42a3 (AAF19373), Norway rat (Rattus norvegicus) Slc42a3 (NP_898876), Zebrafish (Danio

rerio) Slc42a3-2 (BAF63791), Torafugu (Takifugu rubripes) Slc42a3-2 (AAM48579),

Mangrove rivulus (Rivulus marmoratus) Slc42a3-2 (ABD83662), Stickleback (Gasterosteus aculeatus) Slc42a3 (ABF69690), Rainbow trout (Oncorhynchus mykiss) Slc42a3 (AAU89494), Mangrove rivulus (Rivulus marmoratus) Slc42a3-1 (ABN41463), Green pufferfish (Tetraodon nigroviridis) Slc42a3 (AAY41907), Torafugu (Takifugu

rubripes) Slc42a3-1 (AAM48578), Zebrafish (Danio rerio) Slc42a3-1 (AAM90586),

Weatherloach (Misgurnus anguillicaudatus) Slc42a3 (FJ982777). ... 33

Figure 2.8. Rhcg mRNA expression in control (n=3), ammonia (n=4) and air exposed

(n=4) M. anguillicaudatus determined by semi-quantitative RT-PCR. Data are normalized to β-actin, and expressed relative to the control group. The asterisk (*) indicates significant difference from the control group (P<0.05). ... 34

Figure 3.1. Representative trace of a pH stat titration from a hindgut preparation. An initial

ix was added to the serosal side, and finally by a 2 h experimental flux with 10 mM NH4Cl

on the serosal side and an inhibitor on either mucosal or serosal side. ...48

Figure 3.2. Effects of serosal additional of ouabain (1 mM) on net ammonia flux rates

(µmol cm-2 h-1) and net base flux rates (µEq cm-2 h-1) in the presence of a 10 mM TAN gradient in M. anguillicaudatus foregut (black bars) and hindgut (grey bars). Values are mean±SEM (n=4), bars with like characters are not significantly different (P>0.05). ...49

Figure 3.3. Effect of mucosal addition of DIDS (0.1 and 1.0 mM) on net ammonia flux

rates (µmol cm-2 h-1) and net base flux rates (µEq cm-2 h-1) in the presence of a 10 mM TAN gradient in M. anguillicaudatus foregut (black bars) and hindgut (grey bars). Values are mean±SEM (n=4), bars with like characters are not significantly different (P>0.05). ...50

Figure 3.4. Effect of mucosal addition of EIPA (0.1 mM) on net ammonia flux rates (µmol

cm-2h-1) and net base flux rates (µEq cm-2h-1) in the presence of a 10 mM TAN gradient in M. anguillicaudatus foregut (black bars) and hindgut (grey bars). Note the ammonia flux inhibition by EIPA in both foregut and hindgut. Values are mean±SEM (n=4), bars with like characters are not significantly different (P>0.05). ...50

Figure 3.5. Effect of mucosal addition of EIPA (0.1 mM) (A, B, C and D) and ammonia

(10 mM) (E, F, G and H) on transepithelial potential (TEP, mV) and conductance (G, µSi cm-2) of the weatherloach foregut and hindgut. A, C, E and G show TEP and G measurements made over 5 min. intervals while B, D, F and H present the same data averaged over each hour. Values are mean±SEM (n=4), bars with like characters are not significantly different (P>0.05). ...51

Figure 3.6. Effect of serosal addition of ETZ (0.1 mM) on net base flux rates (µEq cm-2h-1)

in the presence of a 10 mM TAN gradient in M. anguillicaudatus foregut (black bars) and hindgut (grey bars). Values are mean±SEM (n=6), bars with like characters are not significantly different (P>0.05). ...52

Figure 3.7. Effect of ammonia gradient reversal (10 mM TAN serosal-mucosal versus

mucosal-serosal gradients) on net ammonia flux rates (µmol cm-2h-1) in M. anguillicaudatus foregut (black bars) and hindgut (grey bars). Values are mean±SEM

(n=3), bars with like characters are not significantly different (P>0.05). ...53

Figure 4.1. Rooted phylogenetic trees of Atp1a (A); Slc26a6 (B), Atp6v1b (C), Slc9a1,

Slc9a2, and Slc9a3 (D), Slc42a3 (E) homolog sequences. The trees were constructed by the neighbour-joining method with 1 000 bootstrap trials using ClustalX, with the options: correct for multiple substitutions and exclude positions with gaps. Bootstrap values below 650 are meaningless. Accession numbers are as follows: ...71

Figure 4.1A) Atp1a: Human (Homo sapiens) ATP1A4 (AAH94801), House mouse (Mus musculus) Atp1a4 (AAD43813), Norway rat (Rattus norvegicus) Atp1a4 (AAB81285),

Killifish (Fundulus heteroclitus) Atp1a2 (AAL18003), Zebrafish (Danio rerio) Atp1a2a (NP_571758), House mouse (Mus musculus) Atp1a2 (AAH36127), Human (Homo

sapiens) ATP1A2 (AAA51797), Norway rat (Rattus norvegicus) Atp1a2 (AAH85764),

Chicken (Gallus gallus) Atp1a2 (AAA48981), Chicken (Gallus gallus) Atp1a3 (AAA48982), Human (Homo sapiens) ATP1A3 (AAH09282), House mouse (Mus

musculus) Atp1a3 (AAH34645), Norway rat (Rattus norvegicus) Atp1a3 (AAA40777),

Mozambique tilapia (Oreochromis mossambicus) Atp1a3 (AAF75108), Zebrafish (Danio

rerio) Atp1a3a (AAG30275), Chicken (Gallus gallus) Atp1a1 (AAA48607), African

clawed frog (Xenopus laevis) Atp1a1 (AAA19022), Human (Homo sapiens) ATP1A1 (BAA00061), House mouse (Mus musculus) Atp1a1 (AAH21496), Norway rat (Rattus

norvegicus) Atp1a1 (AAA41671), White sucker (Catostomus commersoni) Atp1a1

(CAA41483), Zebrafish (Danio rerio) Atp1a4 (AAG30274), European eel (Anguilla

anguilla) Atp1a1 (CAA53714), Zebrafish (Danio rerio) Atp1a1 (NP_571761),

Mozambique tilapia (Oreochromis mossambicus) Atp1a1 (AAD11455), Killifish (Fundulus heteroclitus) Atp1a1 (AAL18002), Weatherloach (Misgurnus

anguillicaudatus) Atp1a (FJ982782)... 71

Figure 4.1B) Slc26a6: Western clawed frog (Xenopus tropicalis) Slc26a6

(NP_001072916), Cattle (Bos Taurus) Slc26a6 (NP_001070320), Pig (Sus scrofa) Slc26a6 (NP_001012298), Human (Homo sapiens) SLC26A6 (BAC56861), Human (Homo sapiens) SLC26A6-A (AAN07094), House mouse (Mus musculus) Slc26a6-A (AAL13129), House mouse (Mus musculus) Slc26a6-B (AAN07089), Mefugu (Takifugu

obscurus) Slc26a6-B (BAE75797), Zebrafish (Danio rerio) Slc26a6 (ACI05563), Mefugu

(Takifugu obscurus) Slc26a6-C (BAE75798), Japanese eel (Anguilla japonica) Slc26a6 (BAC16761), Weatherloach (Misgurnus anguillicaudatus) Slc26a6 (FJ982783), Gulf toadfish (Opsanus beta) Slc26a6 (ABQ01444), Mefugu (Takifugu obscurus) Slc26a6-A (BAE75796). ... 72

Figure 4.1C) Atp6v1b: Red algae (Cyanidium caldarium) Atp6v1b (AAA85821), Cattle

(Bos taurus) Atp6v1b1 (NP 788827), Human (Homo sapiens) ATP6V1B1 (NP_001683), House mouse (Mus musculus) Atp6v1b1 (AAH62202), Norway rat (Rattus norvegicus) Atp6v1b1 (NP_001101337), African clawed frog (Xenopus laevis) Atp6v1b1 (NP_001090361), European eel (Anguilla Anguilla) Atp6v1b2 (AAC78641), African clawed frog (Xenopus laevis) Atp6v1b2 (NP_001080613), Zebrafish (Danio rerio) Atp6v1b2 (AAL79838), Rainbow trout (Oncorhynchus mykiss) Atp6v1b (AAD33861), Atlantic salmon (Salmo salar) Atp6v1b (CAC15466), Atlantic salmon (Salmo salar) Atp6v1b2 (ACI33405), European eel (Anguilla Anguilla) Atp6v1b1 (AAD55091), Zebrafish (Danio rerio) Atp6v1b1 (AAL79837), European seabass (Dicentrarchus

labrax) Atp6v1b (AAT95863), Weatherloach (Misgurnus anguillicaudatus) Atp6v1b

(FJ982781), Cattle (Bos Taurus) Atp6v1b2 (NP_788844), House mouse (Mus

musculus) Atp6v1b2 (NP_031535), Norway rat (Rattus norvegicus) Atp6v1b2

(NP_476561), Human (Homo sapiens) ATP6V1B2 (NP_001684)... 73

Figure 4.1D) Slc9a: Zebrafish (Danio rerio) Slc9a2 (NP_001107567), Weatherloach

(Misgurnus anguillicaudatus) Slc9a2 (FJ982779), Rainbow trout (Oncorhynchus mykiss) Slc9a2 (ABO32814), Japanese medaka (Oryzias latipes) Slc9a2 (ENSORLP00000015518), Longhorn sculpin (Myoxocephalus octodecemspinosus) Slc9a2 (AAD46576), Atlantic stingray (Dasyatis sabina) Slc9a3 (AAT45738), Rabbit (Oryctolagus cuniculus) Slc9a3 (AAA31420), North American opossum (Didelphis

virginiana) Slc9a3 (AAA98816), Human (Homo sapiens) SLC9A3 (AAB48990), House

mouse (Mus musculus) Slc9a3 (NP_001074529), Norway rat (Rattus norvegicus) Slc9a3 (AAA41702), Weatherloach (Misgurnus anguillicaudatus) Slc9a3 (FJ982780), Rainbow trout (Oncorhynchus mykiss) Slc9a3 (ABO32815), Zebrafish (Danio rerio) Slc9a3 (NP_001106944), Japanese medaka (Oryzias latipes) Slc9a3 (ENSORLP00000011453), Mozambique tilapia (Oreochromis mossambicus) Slc9a3 (BAF80347), Human (Homo sapiens) SLC9A2 (AAD41635), Norway rat (Rattus

norvegicus) Slc9a2 (AAA75406), Rabbit (Oryctolagus cuniculus) Slc9a2 (P50482),

Weatherloch (Misgurnus anguillicaudatus) Slc9a1 (FJ982778), Japanese medaka (Oryzias latipes) Slc9a1 (ENSORLP00000018689), Winter flounder (Pseudopleuronectes americanus) Slc9a1 (AAO32340), European eel (Anguilla

anguilla) Slc9a1 (CAB45085), Common carp (Cyprinus carpio) Slc9a (CAB45232),

Mefugu (Takifugu obscurus) Slc9a1 (BAE75800), Chicken (Gallus gallus) Slc9a1 (ABB82239), House mouse (Mus musculus) Slc9a1 (AAA92976), Human (Homo

sapiens) SLC9A1 (AAF21350), Rabbit (Oryctolagus cuniculus) Slc9a (CAA43721),

xi

Figure 4.1E) Slc42a3: Tunicate (Ciona savignyi) Slc42a3 (AAY41908), African clawed

frog (Xenopus laevis) Slc42a3 (AAH84943), Chicken (Gallus gallus) Slc42a3 (AAP49833), Pig (Sus scrofa) Slc42a3 (ABF69687), Rhesus monkey (Macaca mulatta) Slc42a3 (ABD72472), Chimpanzee (Pan troglodytes) Slc42a3 (AAX39717), Human (Homo sapiens) SLC42A3 (AAG02171), Rabbit (Oryctolagus cuniculus) Slc42a3 (AAK14653), House mouse (Mus musculus) Slc42a3 (AAF19373), Norway rat (Rattus norvegicus) Slc42a3 (NP_898876), Zebrafish (Danio rerio) Slc42a3-2 (BAF63791), Torafugu (Takifugu rubripes) Slc42a3-2 (AAM48579), Mangrove rivulus (Rivulus

marmoratus) Slc42a3-2 (ABD83662), Stickleback (Gasterosteus aculeatus) Slc42a3

(ABF69690), Rainbow trout (Oncorhynchus mykiss) Slc42a3 (AAU89494), Mangrove rivulus (Rivulus marmoratus) Slc42a3-1 (ABN41463), Green pufferfish (Tetraodon

nigroviridis) Slc42a3 (AAY41907), Torafugu (Takifugu rubripes) Slc42a3-1 (AAM48578),

Zebrafish (Danio rerio) Slc42a3-1 (AAM90586), Weatherloach (Misgurnus

anguillicaudatus) Slc42a3 (FJ982777)...75 Figure 4.2. Expression levels of actb, Atp1a, Slc26a6, Atp6v1b, Slc9a1, Slc9a3, and

Slc42a3 mRNA in Misgurnus anguillicaudatus foregut (fg), midgut (mg), hindgut (hg), gill (g), kidney (k), liver (l), and brain (b). (A) qPCR products separated by agarose gel electrophoresis and (B) mRNA expression determined by qPCR (expressed relative to β-actin). (actb, β-actin; Atp1a, Na+_/K+_{-ATPase; Slc26a6, Putative Anion Transporter Cl}

-/HCO3- exchanger; Atp6v1b, H+-ATPase B subunit; Slc9a1, Na+/H+ exchanger isoform

1; Slc9a3, Na+/H+exchanger isoform 3; Slc42a3, Rhesus C-glycoprotein). ...77

Figure 4.3. Expression of Slc9a3 in foregut (black bars) and hindgut (grey bars) of M. anguillicaudatus over time during an acute exposure to HEA. Note the transient

increase in Slc9a3 gene expression at 1h in hindgut (4 fold) and at 6h in foregut (26 fold). (Ctrl n=6; 1 h n=5; 6 h n=5; 24 h n=6)...78

Figure 4.4. Localization of PAT1-like protein (Slc26a6A; green) and Na+/K+-ATPase (5;

red) with double immunofluorescent labelling in M. anguillicaudatus gut. (a) Foregut, (b,c) midgut and (c,d) hindgut. To provide structural information each image was exposed with blue filter and overlaid. Arrowheads indicate PAT1-like protein immunolabelling, while arrows indicate Na+/K+-ATPase staining. Abbreviation: L, lumen. Scale bar = 250μm. ...79

Figure 4.5. Localization of NHE2-like protein (SLC9A2; green) and Na+/K+-ATPase (5;

red) with double immunofluorescent labelling in M. anguillicaudatus gut. (a) Foregut, (b) midgut and hindgut. To provide structural information each image was exposed with blue filter and overlaid. Arrowheads indicate NHE2-like protein immunolabelling, while arrows indicate Na+/K+-ATPase staining. Abbreviation: L, lumen. Scale bar = 250μm. ...80

Figure 4.6. Localization of NHE3-like protein (SLC9A3; green) and Na+/K+-ATPase (5;

red) with double immunofluorescent labelling in M. anguillicaudatus gut. (a) Foregut, (b) midgut and (b,c) hindgut. To provide structural information each image was exposed with blue filter and overlaid. Arrowheads indicate NHE3-like protein immunolabelling, while arrows indicate Na+/K+-ATPase staining. Abbreviation: L, lumen. Scale bar = 250μm...80

Figure 4.7. Effect of ammonia exposure on the concentration of ammonia in the muscle

(A) and in plasma (B) of M. anguillicaudatus over time. Bars with like characters are not significantly different (P>0.05). (Ctrl n=6; 1 h n=5; 6 h n=5; 24 h n=6). ...81

Figure 4.8. Effect of chronic ammonia exposure (2 months) on the concentration of

ammonia in the muscle (A) and in plasma (B) of M. anguillicaudatus. The asterisk (*) indicates a significant difference (P=0.036). (n=5). ... 82

Figure 5.1. Aquatic set up: Resting respirometry with intermittent flow. (1) recirculating

pump, (2) flux pump, (3) O2probe, (4) chamber, (5) water circulation in the tank... 92 Figure 5.2. Aerial set up. (1) chamber, (2) KOH, separated from the fish, (3) pressure

transducer, (4) amplifier, (5) data logger, (6) PC. ... 93

Figure 5.3. Representative metabolic rate (Mo2) measurements of a Misgurnus

anguillicaudatus measured over 24 h under control condition, followed by 10 h under

ammonia exposure conditions, 1.5 mM NH4Cl at pH ~8.5 and at ~25 ºC. (The arrow

indicates time of ammonia addition)... 96

Figure 5.4. Mean standard metabolic rate of M. anguillicaudatus under control, ammonia

and prolonged ammonia exposure (1.5 mM NH4Cl at pH ~8.5) conditions (n=6). Bars

with like characters are not significantly different (P>0.05). (Ctrl, control conditions; Amm, ammonia exposure for 5h; PAE, prolonged ammonia exposure, 1-2 weeks). .... 96

Figure 5.5. Critical oxygen partial pressure (Pcrit) of control (7.520.97 kPa) and ammonia

exposed (1.5 mM NH4Cl at pH ~8.5) (3.160.93 kPa) M. anguillicaudatus. Mo2 vs PO2

graph considering in both control and ammonia the previously obtained control standard metabolic rate (SMR). Control, dotted line; ammonia, dashed line. ... 97

Figure 5.6. Mo2(mg kg-1 h-1) measurements of aerially exposed M. anguillicaudatus. Fish

were previously subjected to different conditions, four were maintained in control conditions (no ammonia, at ~25 ºC) and four were exposed to ammonia (1.5 mM NH4Cl

at pH ~8.5, and ~25 ºC, for more than one week). ... 98

Figure 5.7. Air breathing (gulps h-1) (A) and aquatic breathing (opercular beats min-1) (B)

frequencies in M. anguillicaudatus under control and high environmental ammonia (30 mM at pH ~7.3) conditions. ... 99

Figure 5.8. Air breathing (gulps h-1) (A) and aquatic breathing (opercular beats min-1); (B)

frequencies in M. anguillicaudatus in control and aquatic hypoxia (~2 mg L-1, ~29 % or 6.1 kPa) exposure. Values are mean±SEM, bars with like characters are not significantly different (P>0.05)... 99

Figure 6.1. Mechanisms of gut alkalinization and ammonia excretion through the gut of

Misgurnus anguillicaudatus. The presence of apical NHE3 and 2 as potential NH4+

exchangers has been confirmed and a role in ammonia flux established based on apical EIPA sensitivity. Transcriptional studies indicate that the NHE3 is responsible for NH4+

exchange. Apical Cl-/HCO3- exchanger (Slc26a6) expression and DIDS sensitive base

flux indicate a role for this gene in the alkalinization mechanism although transcriptional changes are not important in the response to ammonia loading conditions. The Rhcg ammonia transporter and carbonic anhydrase do not appear to have important roles in ammonia and base excretion, respectively. Basolateral Na+/K+-ATPase is abundant in foregut and ouabain inhibition of both ammonia and base flux indicates it is involved either directly by transporting NH4+into the cell or indirectly by lowering intracellular Na+

to maintain the inward gradient to drive apical exchange with NH4+ (NHE) and

cotransport across the basolateral membrane with HCO3-(NBC) for ammonia and base

fluxes, respectively. The model remains incomplete because the identification of the apical CFTR anion channel and basolateral Na+: HCO3- cotransporter (NBC) await

xiii (4) CFTR (Cl- channel), (5) carbonic anhydrase, (6) H+-ATPase, (7) Rhcg and (8) NBC, (X) no role on the model, and (?) await confirmation. ...107

xv

Abstract

In the present thesis the mechanisms of ammonia excretion in the weatherloach,

Misgurnus anguillicaudatus, were studied. This fish is a freshwater, non-obligatory

air-breather that lives in streams and rice paddy fields, where it may experience drought, high environmental ammonia (HEA), and hypoxic conditions. It has modified its posterior intestine into a gas exchange organ and has also developed the very unusual ability to get rid of ammonia by volatilization when experiencing ammonia loading and aerial conditions. The weatherloach’s environmental ammonia tolerance was confirmed by its high 96 h LC50of 375μM NH3and the high tissue ammonia tolerance by its capacity of building

up high ammonia levels in the body.

At the gill level we studied the roles of Na+/K+(NH4+)-ATPase (potentially NH4+

transport protein), H+-ATPase (H+ transport) and Rhesus C glycoprotein (Rhcg, an ammonia transport protein) in environmental ammonia tolerance and how the weatherloach copes with ammonia loading conditions and aerial exposure. The importance gill epithelial permeability modulation following exposure to high environmental ammonia (HEA) or to air was also investigated. Branchial Na+/(K+)NH4+-ATPase facilitated

transport does not seem to play an important role in ammonia excretion when animals are subjected to high environmental ammonia (HEA) or to high environmental pH, as no changes in enzyme activity were observed. Boundary layer acidification was confirmed to be important, due to the increased toxic effect of ammonia when the environmental water was buffered with HEPES at pH 8 and the boundary layer disrupted. The acidification of the gill boundary layer could be performed through the proton excretion by the H+ -ATPase, since inhibition with bafilomycin A1 resulted in a decrease on net ammonia flux, an increase in plasma ammonia levels and a decrease in the net acid flux. Apical H+ -ATPase immunoreactivity was also found in the gill. However, no increases in enzyme activity were observed with HEA or high environmental pH. Rhcg1 (Slc42a3) mRNA expression was significantly higher during aerial exposure when compared to control, but no significant changes were observed in response to ammonia loading condition. Rhcg1 was localized apically in the branchial epithelium, by immunofluorescence microscopy, to a population of cells similar to vacuolar (V)-type H+-ATPase. A separate population of cells had strong basolateral Na+/K+-ATPase immunoreactivity, typical of “chloride”-type mitochondrion-rich cells. The colocalization of H+-ATPase and Rhcg1 to a similar cell type supports a role for H+-ATPase in ammonia excretion via Rhcg by NH4+ trapping. Gill

fish exposed to air, which together with the increase in Rhcg expression would most probably enhance NH3permeation through the gill plasma membrane aiding excretion.

The weatherloach possesses the ability to volatilize ammonia through the gut when exposed to air or HEA. The mechanisms involved in ammonia transport to the gut boundary layer and how gut alkalinisation is performed are largely uncharacterized. The Na+/H+ exchanger (NHE) and Na+/K+-ATPase (NKA) are proposed to be involved in NH4+

transport through the gut epithelium. Gut alkalinisation is predicted to be performed by apical HCO3- secretion through a Cl-/HCO3-anion exchanger; carbonic anhydrase (CA) to

provide cytosolic HCO3- for apical excretion and/or convert the gut boundary layer HCO3

-into CO2, removing the H+ left behind following NH3 volatilization. Due to the high pH

found in the gut boundary layer a higher proportion of NH3will be created, consequently,

to avoid NH3 back diffusion, the gut apical membrane is likely relatively impermeable.

Transporters involved in mucosal excretion of HCO3- and ammonia were studied using a

pharmacological approach with a pH-stat technique in Ussing chambers mounted with foregut and hindgut preparations. The inhibitors for: Na+/K+-ATPase (ouabain), Cl-/HCO3

-anion exchanger (DIDS), Na+/H+ exchanger (EIPA), and carbonic anhydrase (ETZ) were applied in the presence of a 10 mM total ammonia serosal-mucosal gradient. Membrane fluidity was measured in the hindgut samples of fish acclimated to different conditions.

The involvement of NHE and NKA in NH4+ transport was confirmed, as their

corresponding inhibitors induced a significant decrease in net ammonia flux. Gut alkalinisation is probably accomplished by a Cl-/HCO3-exchange, since the inhibition of Cl

-/HCO3- anion exchanger induced a significant decrease in net base excretion. Carbonic

anhydrase most probably does not play a role in gut alkalinisation because its inhibition did not induce a decrease in net base flux. Fish exposed to HEA and air exhibited a decrease in gut membrane fluidity (permeability) in hindgut, which appears as a way of decreasing NH3 back diffusion. Consistent with this latter finding was that the ammonia

flux rate in the reversed mucosal to serosal direction was significantly less in hindgut compared to foregut.

The mechanisms of gut ammonia excretion and alkalinisation were further studied using a molecular approach. Gut Na+/K+-ATPase  subunit (NKA), Na+/H+ exchanger isoform 1, 2 and 3 (NHE1, NHE2 and NHE3), putative Cl-/HCO3-anion transporter (PAT1),

and H+-ATPase B subunit (BVA) were partially sequenced. Transcript relative quantification was performed, through real-time PCR, in foregut and hindgut of fish exposed to HEA (6.3 mM NH4Cl at pH 8), in a time course experiment: time 0, 1, 6 and 24

hours and exposed to lower ammonia (2.9 mM NH4Cl at pH 7.6) chronically (more than 2

months). NHE3 involvement in NH4+ transport through the gut was confirmed since its

xvii exposure. NKA is also important in NH4+as its mRNA transcript expression was higher in

fish chronically exposed to ammonia then in control. PAT1 and BVA showed no changes in gene transcript expression in either experiment. Rhcg was down regulated after chronic exposure and no changes were observed in time course exposure.

Immunofluorescence microscopy confirmed the localization that was expected in the studied transporters. NKA presented basolateral localization in foregut and midgut. NHE3 and PAT1 had apical distribution in foregut and midgut. NHE2 was identified apically in mucosal epithelium of foregut. Immunoreactivity was largely absent from the hindgut region.

With the aim of understanding how ammonia and aerial exposure affect the weatherloach’s metabolic rate, respirometry experiments were conducted under aquatic and aerial exposure conditions. Aquatic metabolic rate measurements were performed under control and ammonia (1.5 mM NH4Cl at pH 8.5 and 25 ºC) conditions. Critical

oxygen partial pressure (Pcrit) was also determined in control and ammonia exposed fish.

Aerial metabolic rate was also determined in control and previously ammonia exposed weatherloaches. Breathing behaviour, by means of measuring water breathing frequency and surface air-breathing frequency, was studied in control fish and exposed to HEA or to hypoxia. Ammonia was shown to modulate the weatherloach’s aquatic metabolic rate, as an increase in standard metabolic rate (SMR) was observed when fish were acutely exposed to ammonia. However, prolonged ammonia exposure no longer had an effect on SMR. Critical oxygen partial pressure (Pcrit) determined in control and ammonia exposed

fish, was found to be higher in ammonia exposed than in control fish. The oxygen requirement seems to increase in ammonia pre-exposed fish under aerial conditions, since these fish showed a higher O2consumption than controls after 6 h aerial exposure.

The weatherloach exposed to ammonia or hypoxia showed a decrease in water breathing frequency and an increase in air breathing frequency.

The present thesis allowed the elucidation of the branchial and intestinal mechanisms related with the remarkable ammonia tolerance in Misgurnus anguillicaudatus.

xix

Resumo

Na presente tese os mecanismos de excreção da amónia foram estudados no cobitis, Misgurnus anguillicaudatus. Este peixe de água doce, que possui respiração aérea não obrigatória, habita ribeiros e arrozais, onde pode ser sujeito a diferentes condições como exposição ao ar, a altas concentrações de amónia e hipoxia. Possui o seu intestino posterior transformado num órgão respiratório e desenvolveu uma capacidade invulgar de eliminar amónia por volatilização durante exposição a altas concentrações de amónia ou quando exposto ao ar.

A tolerância do cobitis à amónia ambiental foi confirmada através do seu elevado valor de LC50 às 96 h de 375 µM NH3 e a alta tolerância à amónia nos tecidos pela sua

capacidade de acumular altos níveis de amónia no organismo.

Ao nível da brânquia foram estudados os papeis da Na+/K+(NH4+)-ATPase (proteína

transportadora de NH4+), H+-ATPase (transporte de H+) e Rhesus C glycoproteína (Rhcg,

proteína transportadora de amónia) na tolerância à amónia ambiental e na forma como o cobitis lida com a exposição a altas concentrações de amónia ou ao ar. A importância da modulação da permeabilidade do epitélio branquial após exposição a elevadas concentrações de amónia ambiental ou exposição ao ar foi também investigada. O transporte branquial facilitado através da Na+/(K+)NH4+-ATPase não parece desempenhar

um papel importante na excreção de amónia quando os animais são expostos a elevadas concentrações de amónia ou a elevado pH ambientais, devido a não terem sido observadas variações de actividade enzimática. A importância da acidificação da camada fronteira branquial (gill boundary layer) foi confirmada, devido ao aumento da toxicidade à amónia quando a água envolvente ao peixe foi tamponada com HEPES a pH 8 e a camada fronteira branquial quebrada. A acidificação da camada fronteira branquial poderia ser realizada pela excreção de protões através da H+-ATPase, pois a sua inibição com bafilomicina A1 resultou numa diminuição do fluxo total de amónia, num aumento dos níveis de amónia no plasma e numa diminuição no fluxo ácido total. Imunoreactividade apical da H+-ATPase foi detectada na brânquia. Contudo, não foi observado um aumento da actividade enzimática com exposição a elevadas concentrações de amónia ou a elevado pH ambientais. A expressão de mRNA da Rhcg1 (Slc42a3) elevou-se significativamente durante exposição ao ar quando comparada com o controlo, mas não ocorreram alterações significativas em resposta à exposição a elevadas concentrações de amónia. Rhcg1 foi localizada apicalmente no epitélio branquial, através de microscopia de imunofluorescência, numa população de células similar a H+-ATPase vacuolar. Uma população de células distinta exibiu forte

imunoreactividade basolateral a Na+/K+-ATPase, típica de células-cloro ricas em mitocôndrias. A colocalização da H+-ATPase e da Rhcg1 em células semelhantes apoiam a hipótese de um papel da H+-ATPase na excreção de amónia via Rhcg através do aprisionamento de amónia sob a forma de NH4+. A fluidez membranar da brânquia

apresentou um aumento significativo em peixes expostos ao ar, que em conjunto com a expressão genética aumentada da Rhcg vai muito provavelmente aumentar a permeabilidade da membrana plasmática branquial a NH3aumentando a sua excreção.

O cobitis possui a capacidade de volatilizar amónia através do intestino quando exposto a elevadas concentrações de amónia ou ao ar. Os mecanismos envolvidos no transporte da amónia para a camada fronteira intestinal e na alcalinização do intestino não estão ainda caracterizados. É proposto o envolvimento do trocador Na+/H+ (NHE) e da Na+/K+-ATPase (NKA) no transporte de NH4+através do epitélio do intestino. Prevê-se

que a alcalinização seja realizada pela excreção apical de HCO3- através de trocador

aniónico Cl-/HCO3- e da anidrase carbonica (CA) que providiencia HCO3- citosolico para

ser excretado apicalmente e/ou converte o HCO3- da camada fronteira intestinal em CO2,

removendo o H+ libertado após volatilização de NH3. Devido ao elevado pH encontrado

na camada fronteira intestinal uma grande proporção de NH3 será criada e,

consequentemente, para evitar difusão de NH3de volta ao organismo, a membrana apical

do intestino provavelmente possuirá um certo grau de impermeabilidade. Os transportadores envolvidos na excreção para a mucosa de HCO3- e de amónia foram

estudados utilizando uma abordagem farmacológica através de uma técnica de pH-stat em câmaras de Ussing montadas com preparações de intestino anterior e posterior. Os inibidores para: Na+/K+-ATPase (ouabaína), trocador aniónico Cl-/HCO3- (DIDS), trocador

Na+/H+(EIPA), e anidrase carbonica (ETZ) foram aplicados na presença de um gradiente serosa-mucosa de 10 mM de amónia total. A fluidez membranar foi medida no intestino posterior de peixes aclimatizados a diferentes condições.

O envolvimento de NHE e NKA no transporte de NH4+ foi confirmado, devido aos

seus inibidores correspondentes induzirem uma diminuição significativa no fluxo total de amónia. A alcalinização do intestino é realizada por um trocador aniónico Cl-/HCO3

-porque a sua inibição provocou uma diminuição significativa no fluxo total de excreção de base. A anidrase carbonica muito provavelmente não desempenha um papel na alcalinização do intestino porque a sua inibição não induziu uma diminuição no fluxo total de excreção de base. Peixes expostos a elevadas concentrações de amónia e ao ar exibiram uma diminuição na fluidez membranar (permeabilidade) do intestino posterior, o que parece ser uma forma de diminuir a difusão de NH3 de volta ao organismo.

Consistente com esta diminuição de permeabilidade foi o menor fluxo total de amónia observado no intestino posterior no sentido mucosa-serosa.

xxi Os mecanismos de excreção de amónia através do intestino e alcalinização foram ainda estudados através de uma abordagem molecular. Na+/K+-ATPase (NKA), trocador Na+/H+ isoforma 1, 2 e 3 (NHE1, NHE2 e NHE3), trocador aniónico Cl-/HCO3- putativo

(PAT1), e H+-ATPase subunidade B (BVA) do intestino do cobitis foram parcialmente sequenciados. A quantificação relativa dos transcriptos foi realizada através de real-time PCR, no intestino anterior e posterior de peixes expostos a concentrações elevadas de amónia (6,3 mM NH4Cl a pH 8): tempo 0, 1, 6, e 24 horas e expostos a baixas

concentrações de amónia (2,9 mM NH4Cl a pH 7,6) cronicamente (mais de dois meses).

O envolvimento do NHE3 no transporte de NH4+ através do intestino foi confirmado

através do aumento transiente da sua expressão de mRNA. NKA também é importante no transporte de NH4+ porque a sua expressão aumentou em peixes expostos

cronicamente à amónia comparado com o controlo. PAT1 e BVA não apresentaram alterações na expressão genética em ambas as experiências. Rhcg sofreu supressão de expressão após exposição crónica.

Microscopia de imunofluorescência confirmou a localização esperada para os transportadores estudados. NKA apresentou uma localização basolateral no intestino anterior e médio. NHE3 e PAT1 foram localizados com uma distribuição apical no intestino anterior e médio. O NHE2 foi identificado apicalmente no epitélio da mucosa do intestino anterior. No intestino posterior a imunoreactividade foi geralmente ausente.

Com o objectivo de compreender como a amónia e a exposição ao ar afectam a taxa metabólica, experiências de respirometria foram realizadas em condições de imersão e emersão. Medições da taxa metabólica aquática foram realizadas em condições controlo e de exposição à amónia (1,5 mM NH4Cl at pH 8,5 and 25 ºC). O

máximo estado estável de VO2 (Pcrit) foi determinado em condições controlo e de

exposição à amónia. Medições da taxa metabólica aérea foram realizadas em peixes previamente expostos a amónia e controlo. O comportamento respiratório foi estudado através da medição da frequência respiratória aquática e aérea, em peixes controlo, expostos a amónia e sujeitos a hypoxia. Foi possível demonstrar que a amónia modula a taxa metabólica aquática do cobitis, devido ao aumento observado na taxa metabólica após exposição aguda a amónia. Contudo, exposição prolongada à amónia não afecta a taxa metabólica. O Pcritdeterminado em peixes controlo e expostos à amónia mostrou-se

mais elevado em peixes expostos. As necessidades de oxigénio parecem aumentar em peixes pré-expostos à amónia em condições de emersão, uma vez que estes peixes mostraram um consumo de oxigénio mais elevado após 6 h de emersão. Cobitis expostos a amónia ou a hipoxia mostraram uma diminuição da frequência respiratória aquática e um aumento da frequência respiratória aérea.

A presente tese permitiu a elucidação dos mecanismos branquiais e intestinais relacionados com a impressionante tolerância à amónia no Misgurnus anguillicaudatus.

xxiii

Résumé

Dans la présente thèse, les mécanismes de l'excrétion d'ammoniac chez la loche d’étang, Misgurnus anguillicaudatus, ont été étudiés. Celui-ci est un poisson d'eau douce, reniflard d’air non-obligatoire qui vit dans les ruisseaux et les rizières, où il peut vivre en conditions de sécheresse, de taux d’ammoniac environnemental élevé (HEA), et en conditions hypoxiques. Son intestin postérieur est modifié en un organe d’échanges gazeux et il a également développé la capacité très rare de se débarrasser de l'ammoniac par volatilisation dans des conditions d’exposition à de hautes concentrations en ammoniac ou quand exposé à l’air.

La tolérance de la loche d’étang a l’ammoniac environnemental a été confirmée par sa haute valeur de CL50, à 96h de 375 μM NH3, et la grande tolérance de ses tissus à

l’ammoniac a été mise en évidence par sa capacité à supporter des niveaux élevés d'ammoniac dans l'organisme.

Au niveau des branchies, nous avons étudié le rôle de la Na+/K+-ATPase (NH4+

transport de protéine), de la H+-ATPase (transport de H+) et de la glycoprotéine rhésus C (Rhcg, une protéine de transport d'ammoniac) dans des conditions de tolérance à l'ammoniac environnemental, mais également la façon dont la loche d’étang répond face à des concentrations élevées en ammoniac et en condition d’exposition aérienne. Il était aussi important de savoir si ce poisson a une faible perméabilité de l'épithélium des branchies à l’ammoniac en cas d'exposition à un environemment riche en ammoniac (HEA) ou à l'air. La Na+/NH4+-ATPase branchiale facilite le transport mais ne semble pas

jouer un rôle important dans l'excrétion d'ammoniac lorsque soumis à de forts taux d’ammoniac environnemental (HEA) ou à des pH environnementaux élevés. En effet aucun changement dans l'activité de l'enzyme n’a été observé. L’importance de l'acidification de la couche limite a été confirmée, en raison d’une augmentation de la toxicité d’ammoniac quand l'eau fut tamponné avec de l’HEPES à pH 8 et la couche limite perturbée. L'acidification de la couche limite de la branchie pourrait être réalisée à travers l'excrétion de protons par l´H+-ATPase, puisque l'inhibition avec le bafilomycine A1 entraîne une diminution des flux nets d'ammoniac, une augmentation des niveaux plasmatiques de l'ammoniac et une diminution du flux net d'acide. L’immunoreactivité de l´H+-ATPase apicale a également été constatée dans les branchies. Toutefois, aucune augmentation de l'activité de l'enzyme n’a été observée en conditions d’ HEA ou en condition de pH environnemental élevé. L’expression du ARNm de la Rhcg1 (Slc42a3) est significativement plus élevé pendant l'exposition aérienne par rapport au contrôle, mais aucun changement significatif n’a été observé en réponse à l'état de charge en

ammoniac. La Rhcg1 a été localisée apicalement dans l’épithélium branchial, par microscopie par immunofluorescence, dans une population de cellules similaires à H+ -ATPase. Une population différente de cellules a présenté une forte immunoréactivité à Na+/K+-ATPase, localisée basolateralement, et caractéristique de cellules chlore riches en mitochondries. Cette colocalization de la H+-ATPase et de la Rhcg1 dans le même type de cellules supporte l’hypothèse du rôle de l’H+-ATPase dans l'excrétion de l’ammoniac via Rhcg à travers la capture d’ammoniac sous forme de NH4+. La fluidité membranaire

(perméabilité) de la branchie a montré une augmentation significative chez les poissons exposés à l'air, ce qui, avec l'augmentation de l'expression de la Rhcg, devrait très probablement accroître la perméabilité de la membrane plasmique de la branchie à l’NH3,

augmentant ainsi l’excrétion.

La loche d’étang possède la capacité de faire volatiliser l'ammoniac par le tube digestif en cas d'exposition à l'air ou en HEA. Les mécanismes impliqués dans le transport d'ammoniac à la couche limite de l'intestin et dans l’alcalinisation de l’intestin n’ont pas été caractérisés. Les Na+/H+(NHE) et Na+/K+-ATPase (NKA) pourraient être impliqués dans le transport du NH4+ à travers l'épithélium intestinal. L’alcalinisation de

l’intestin est probablement effectuée par une sécrétion apicale de HCO3- par

l'intermédiaire d'un échangeur d'anions Cl-/HCO3-, et de l'anhydrase carbonique (CA), qui

pourrait fournir le HCO3-cytosolique pour une excrétion apicale et/ou convertir le HCO3-de

la couche limite de l'intestin en CO2, en supprimant le H+ libéré suite à la volatilisation du

NH3. En raison du pH élevé dans la couche limite de l'intestin une proportion élevée de

NH3sera créée, par conséquent, pour éviter une diffusion en retour de NH3, la membrane

apicale de l'intestin est probablement relativement imperméable. Les transporteurs impliqués dans l’excrétion vers la muqueuse de HCO3- et d'ammoniac ont été étudiés en

utilisant une approche pharmacologique à travers une technique de pH -stat dans des chambres Ussing montées avec des préparations d’intestin (antérieur et postérieur). Les inhibiteurs pour: la Na+/K+-ATPase (ouabain), l’échangeur d'anions Cl-/HCO3- (DiDs),

l’échangeur Na+/H+(IEAP) et l'anhydrase carbonique (ETZ) ont été appliqués en présence d’un gradient séreuse-muqueuse de 10 mM de ammoniac total. La fluidité des membranes (indicatif de la perméabilité à NH3) a été mesurée dans les échantillons

d’intestin (postérieur) de poisson acclimatés à différentes conditions.

La participation de NHE et de la NKA dans les transports de NH4+a été confirmée,

leurs inhibiteurs induisant une diminution nette des flux d'ammoniac. L’alcalinisation de l’intestin est probablement faite par un échange de Cl-/HCO3- puisque l'inhibition de

l'échangeur d'anions Cl-/HCO3- induit une diminution significative du flux total d'excrétion

de base. L’anhydrase carbonique ne joue très probablement pas un rôle dans l’alcalinisation de l'intestin, car son inhibition n'induit pas de diminution du flux net de

xxv l’excrétion de base. Les poissons exposés à la HEA et à l'air présentent une diminution de la fluidité des membranes de l’intestin (perméabilité), qui apparaît comme un moyen de réduire le retour de la diffusion de NH3. De la même façon, il apparaît une réduction du

flux total d'ammoniac dans l’intestin postérieur dans le sens muqueuse-séreuse.

Les mécanismes de l'excrétion d'ammoniac dans l'intestin et l’alcalinisation ont également été étudiés en utilisant une approche moléculaire. La Na+/K+-ATPase (NKA) de l’intestin, les isoformes 1, 2 et 3 de l’échangeur Na+/H+ (NHE1, NHE2 et NHE3), l’échangeur d'anions putatif Cl-/HCO3- (PAT1) et le polypeptide B de la H+-ATPase (BVA)

ont été partiellement séquencé. La quantification relative a été effectuée par une réaction de PCR en temps réel dans l’intestin antérieur et postérieur de poissons exposés à des HEA (6,3 mM NH4Cl à un pH de 8), à différents moments (temps 0, 1, 6 et 24 heures), et

exposés à une plus faible concentration d'ammoniac (2,9 mM NH4Cl à un pH de 7,6)

chroniquement (plus de 2 mois). L’importance du NHE3 dans le transport de NH4+ à

travers l'intestin a été confirmée à travers l’augmentation transitoire de l’expression d’ARNm. La NKA joue également un rôle important dans le transport du NH4+,

l’expression de son ARNm transcription étant plus élevée dans les poissons exposés de façon chronique à l'ammoniac que dans le contrôle. Les PAT1 et BVA n’ont montré aucun changement dans l’expression des gènes de transcription dans les deux expériences. Une suppression de l’expression de Rhcg a été observée après une exposition chronique. La microscopie par immunofluorescence a confirmé la localisation qui était attendue pour les transporteurs étudiés. La NKA présente une localisation basolatérale dans l’intestin antérieur et médian. Les NHE3 et PAT1 présentent une distribution apicale dans les mêmes régions. Le NHE2 a été identifié apicalement dans l'épithélium de la muqueuse anterieure. L’immunoreactivité est largement absente de la région postérieure (hindgut).

Dans le but de comprendre comment l'ammoniac et l'exposition aérienne affectent la loche d’étang, les taux métaboliques ont été déterminés dans des conditions d'exposition en milieu aquatique et aérien. Des mesures des taux métaboliques aquatiques ont été effectuées en conditions de contrôle et d'exposition à l’ammoniac (1,5 mM NH4Cl à un pH de 8,5 et 25 ºC). La pression partielle d'oxygène critique (Pcrit) a

également été déterminée en conditions de contrôle et d’exposition à l'ammoniac. Les taux métaboliques aériens ont été également déterminés chez des poissons contrôle et d’autres poissons précédemment exposés à l'ammoniac. Le comportement respiratoire a été étudié chez les poissons exposés à l'HEA ou en hypoxie par des mesures de fréquence respiratoire aquatique et aérienne. Il a ainsi été déterminé que l'ammoniac module le taux métabolique aquatique de la Louche d’étang, suivant une augmentation du taux métabolique standard (SMR) observée lorsque les poissons subissent une exposition

aiguë à l'ammoniac. Cependant, une exposition prolongée à l'ammoniac n'a pas d’effet sur le SMR. La pression partielle d'oxygène critique (Pcrit) est plus élevée pour des

poissons exposés à l'ammoniac qu’en conditions de contrôle. La nécessité d'oxygène semble augmenter chez les poissons précedemment exposés à l'ammoniac dans des conditions d’émersion, car ces poissons ont montré une plus grande consommation d'O2

après 6 h d’exposition aérienne.Les loches d’étang exposés à l'HEA ou l'hypoxie ont démontré une diminution de la fréquence respiratoire dans l’eau et une augmentation de la fréquence respiratoire aérienne.

La présente thèse à permis d’élucider sur les mécanismes des branchies et des intestins relationné avec l’impressionnante tolérance de Misgurnus anguillicaudatus à l’ammoniac.

Appendix A

Protocols

A. Tissue fixation and embedding

Fixation in 3 % paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 12-24h (it can be longer).

Embedding

Before starting verify that the 1:1 Clear Rite:Paraffin, the three paraffins, the new paraffin and the moulds are in the oven (60 ºC). It is important to check the contamination level of the last paraffin; this can be detected by the smell and texture.

a) Label the cassettes with pencil.

b) After fixation in Paraformaldehyde transfer the tissues to 70 % Ethanol and cut the tissue into the desired size and place it in the cassettes. Transfer the cassettes to 70 % Ethanol for 45 min, and then to 95 % Ethanol 45 min.

c) Place the cassettes in:

 100 % EtOH1 45 min.

 100 % EtOH2 45 min.

 100 % EtOH3 45 min.

 1:1 Ethanol:Clear Rite 45 min.

 Clear Rite 1 45 min.

 Clear Rite 2 45 min.

 Clear Rite 3 45 min.

 1:1 Clear Rite:Paraffin 45 min (Oven 60 ºC).

 Paraffin 1 45 min (Oven 60 ºC).

 Paraffin 2 45 min (Oven 60 ºC).

 Paraffin 3 45 min (Oven 60 ºC).

Appendix A

d) Mount:

At this point make sure that the Heating Plate is on (at ± 60 ºC) and that you have one thin curved forceps and a fine tipped metal probe.

 Take 1 mould out of the oven and place it on the Heating Plate.

 Fill the mould with new paraffin. Immediately return the paraffin pot to the oven!!

 With a pair of large forceps take a cassette from Paraffin 3. Transfer the tissues from the cassette to the mould; place the labelled part of the cassette on the hot Plate to prevent paraffin to solidify (the lid is disposed). Arrange the tissue with the mould still in the hot Plate using the fine tipped probe.

 Place the mould on the table, arrange again the tissue and with the help of the fine tipped probe press the tissue down. Place the labelled part of the cassette on top of the mould, making pressure for a couple of seconds and releasing it slowly. The melted paraffin should pass through the slits in the cassette.

 Let the moulds to cool at room temperature and place them in the freezer to ease the release of the blocks from the moulds.

Solutions 1. PBS 10x Reagent Amount of reagent (g) Concentration (1x) NaCl 80 137 mM KCl 2 2.7 mM Na2HPO4 11.1 7.8 mM KH2PO4 2 1.5 mM

Bring the volume to 1 L with destiled water, mix well, and filter with a 0.2 µm filter. Check that pH is approximately 7.4. Store at room temperature.

2. PFA Reagent Amount of reagent Concentration PBS 10x 50 ml 1x dH2O 400 ml -Paraformaldehyde 15 g 3 %

Protocols

127 To a 500 ml blue top bottle add 15 g PFA + 50 ml 10x PBS + 400 ml dH2O; Replace

cap tightly. Shake vigorously. Place in 60 ºC water-bath until PFA is dissolved. Cool to room temperature. Adjust the pH to 7.3, and bring the volume to 500 ml with dH2O.

3. TPBS

Reagent Amount of

reagent (ml) Concentration

PBS 10x 100 1x

Tween 20 0.5 0.05 %

Dissolve in destiled water, adjust the pH to 7.3-7.4 with HCl 1 N (±0.6), and bring the volume to 1 L. Store at room temperature